Iraqi J Pharm Sci, Vol.25(1) 2016 Effect of metformin treatment in women with endometriosis 28 Effect of Metformin Treatment on some Blood Biomarkers in Women with Endometriosis. Noor A.Omer *,1 , Muhammad A.Taher ** and Henan Dh Skheel Aljebory *** * Abu-Ghraib General Hospital, Ministry of Health , Baghdad, Iraq. ** Department of Clinical Laboratory Sciences College of Pharmacy, University of Baghdad, Baghdad, Iraq. *** College of Medicine ,Al Mustansyria University, Baghdad, Iraq. Abstract Endometriosis is a common women health disorder that occurs when Endometrial-like tissue grows outside the uterus. This may lead to irregular bleeding , pelvic pain, infertility and other complications. Metformin, because of its activity to improve insulin sensitivity, it is used for the treatment of diabetes; it also has a modulatory effect on ovarian steroid production and has anti- inflammatory properties, all may suggest its possible effect in treatment of endometriosis. This study was planned to determine the effect of metformin on serum levels of interleukin- eight(IL- 8), Tumor necrosis factor-alpha (TNF –α) and estradiol (E2) production , and related symptomatic changes that accompany with endometriosis (pelvic pain, dysmenorrhea and menorrheaga) after three months of study. Blood samples were obtained from those taking metformin and measure the serum levels of (IL-8) , TNF –α and (E2) were measured before and after three months of taking a metformin .Metformin therapy resulted in a significant reduction in the clinical symptoms of endometriosis (pelvic pain and dysmenorrhea) and insignificant changes in menorrhagia. Metformin therapy resulted in a significant reduction in the serum levels of IL-8, TNF- α while insignificant reduction in estradiol E 2 in the study group after 3 months of treatment .In conclusion the results of this study, demonstrated that metformin may be a well-tolerated treatment for endometriosis that relieved pain and reduces menstrual disorders and serum levels of the inflammatory markers (IL- 8 and TNF-α) are decreased in study group treated with metformin after 3 months due to its anti- inflammatory effects. Keywords: Endometriosis, Metformin, TNF – a ,IL-8, E 2. فعالٍَ دّاء الوٍتفْرهٍي فً بعض الوؤشرات الحٌٍَْ فً الدم على الٌساء الالتً ٌعاًٍي هي داء بطاًَ الرحن الِاجرٍ ًْر اركاى عور *‘1 ، هحود عباس طاُر ** الجبْري ّحٌاى ضاٌع *** * .ٔصاسج انصحح ، تغذاد ، انعشاق ،انعاو يستشفٗ اتٙ غشٚة ** اد ، انعشاق .فشع انعهٕو انًختثٛشٚح انسشٚشٚح ، كهٛح انصٛذنح ، رايعح تغذاد ، تغذ *** . ، تغذاد ، انعشاق حزايعّ انًستسصشٚ،انكهّٛ انطة الخالصــــة انشحى خاسد تًُٕ تطاَح انشحى تشثّ أَسزحَتٛزّ انز٘ ٚحذث ٔ انٓارشِ ْٕ يٍ اكخش االيشاض شٕٛعا نذٖ انُساء تطاَح انشحى . اخشٖ يعاعفاخٔانعقى ٔآالو انحٕض اظطشاب انذٔسِ انشٓشّٚ , ْزا قذ ٚؤد٘ إنٗٔ عهٗ تأحٛش نالنتٓاتاخ ٔ خصائص يعادجٔ نذّٚ أٚعا انحساسٛح نالَسٕنٍٛ انز٘ ٚحسٍ يشض انسكش٘ نعالدٚستخذو انًٛتفٕسيٍٛ انثطاَح انشحى انٓارشِ . احتًال اٌ ٚكٌٕ نّ دٔس فعال فٙ عالد، نٓزا اثٛطانً انستٛشٔٚذ اإلَتاد يهي ٕٚيٛيا نًيذِ 0011 انًٛتفيٕسيٍٛ اسيتخذاو تقٛيٛىل تطاَح انشحى انٓارشِ يٍ خال عهٗ فعاال انًٛتفٕسيٍٛكاٌ استخذاو نذساسّ يا إرا انتيٙ تحيذث فيٙ اععيشاضانتغٛٛيشاخ ٔانذو فٙ انستٛشٔٚذ ٔاَتاد أنفا َخش انٕسو عايمٔ 8 –االَتشنٕكٍٛيستٕٚاخ حالث اشٓش عهٗ .أشٓش يٍ اخز انعالد عسش ٔ غضاسِ انطًج (تعذ حالحح، )أنى انحٕض يع تطاَح انشحى ٚعاٍَٛ يٍ داء تطاَّ انشحى انٓارشِ ٔ ُٔٚقسًٌٕ انٗ: 01تشًم رًع عُٛاخ يٍ َساء يٍ يختهف االعًاس ٔعذدْى ,Danazol عقاس انذاَاصٔل, حثٕب يُع انحًم, ْشيٌٕ انثشٔرٛستشٌٔتانًشض ٔٚستخذيٍ انعالد انتقهٛذ٘)َساء يصاتٍٛ -0 ( .Gn-RH-Agonists يخٛم يحشس ْشيَٕاخ انتُاسم يه يٍ انًٛتفٕسيٍٛ. 0011َساء يصاتٍٛ تانًشض ٚستخذيٍ انعالد انتقهٛذ٘ اظافّ انٗ -2 شٓش يٍ اخز انًٛتفٕسيٍٛ ٔارشاء تعط انتحانٛم انًختثشّٚ عهٗ انعُٛاخ انتٙ تشًم تشًم اخز عُٛاخ يٍ انذو قثم ٔتعذ حالحّ أ ٔيقاسَح E2االستشٔرٍٛ ( ٔ tumor necrosis factor alpha(, عايم َخش انٕسو )interleukin-8)8يستٕٖ االَتشنٕكٍٛ انُتائذ . 1 Corresponding author E-mail:noor23.arkan@yahoo.com Received: 4/2/2015 Accepted: 26 /1/2016 Iraqi J Pharm Sci, Vol.25(1) 2016 Effect of metformin treatment in women with endometriosis 29 انحٕض ٔآالو عسش انطًج اَخفاض كثٛش فٙ فٙ انتٓاب تطاَح انشحى انًشظٗ انزٍٚ ٚعإٌَ يٍ فٙ انًٛتفٕسيٍٛ انعالد أدٖ يًا ٚشٛش إنٗ .,انعالد أشٓش يٍ 0تعذ نًزًٕعح انذساسح - نًستٕٚاخ يصم IL-8 ٔ TNF-a فٙ ل اَخفاض كثٛش أسفشخ أٚعا عٍ .انثطاَٙ انشحًٙ عقاس نالنتٓاب كًانهًٛتفٕسيٍٛ اإليكاَاخ انعالرٛح قذ ٚكٌٕ نٓا أَٓا .TNF-a، IL-8 ، E2،الوٍتفْرهٍي ،داء بطاًَ الرحن الِاجرٍ الكلوات الوفتاحٍة : Introduction Endometriosis is a common health problem in women .It got its name from the word endometrium, which is the tissue lining the uterus (1) .During a woman’s menstrual period, this tissue thickens in preparation for a fertilized egg (pregnancy). If there is no fertilization, the tissue breaks down and bleeds with each menstrual period, allowing it to exit the body. In endometriosis, the tissue that normally lines the uterus grows outside the uterus. Most commonly, it is found on the ovaries, fallopian tubes, tissue that holds the uterus in place, outer surface of the uterus, or the lining of the pelvic cavity. Other sites for growths can include the bladder, bowel, cervix, rectum and vagina, or vulva (2) . The different theories involved in the pathogenesis of endometriosis indicate that the etiology of endometriosis is complex and multifactorial, involving hormonal,genetic immune and environmental components (3) Although the etiology of disease is undetermined, four main hypotheses have been circulated as understandable causes (4) : A. Sampson’s theory of retrograde menstruation. B. Ceolomic metaplasia and induction theories (an extension of the ceolomic metaplasia theory). C. The embryonic rest theory. D. Lymphatic and vascular metastasis theories. Several theories considering the pathogenesis of endometriosis are shown in Table-1 , retrograde menstruation may be one of the initiating steps in the pathogenesis of superficial endometriosis, genetic and micro environmental factors that prevent clearance of ectopic lesions and allow remodeling of peritoneum are essential for the propagation of endometriotic lesions (3,5) . Table (1): Mechanism of the different theories in the pathogenesis of endometriosis (6) Mechanism Theory Flow of endometrial content into pelvis, allowing implantation of endometrial lesions Retrograde menstruation Transformation of peritoneal tissue or cells into endometrial tissue through hormonal and/ or immunological factors Metaplasia Estrogen-driven proliferation of endometrial lesions. Resistance to progesterone-mediated control of endometrial proliferation Hormones Recruitment of immune cells and their production of cytokines that promote endometrial Growth Oxidative stress and Inflammation Prevention of eliminating menstrual debris andpromotion of implantation and growth of endometrial lesions Immune Dysfunction Promoting survival of endometrial cells and Down regulation of apoptotic pathways Apoptosis suppression Alteration of cellular function that increases attachment of endometrial cells and evasion of these cells from immune clearance Genetic Initiation of endometriotic deposits by undifferentiated cells with natural ability to regenerate Stem cells Iraqi J Pharm Sci, Vol.25(1) 2016 Effect of metformin treatment in women with endometriosis 30 Tumor necrosis factor-alpha TNF-α is the main pro-inflammatory cytokine known to impair glutathione (GSH) production by several ways, making an environment conducive to the development of oxidative stress(OS). This pathogenic cycle of GSH disturbances and enhanced TNF-α production may be active in the female reproductive tract in endometriosis. An in vitro study investigating endometriosis- associated infertility showed that the quality of spermatozoa may be decreased following incubation with TNF-α in a dose- and time- dependent manner (7). TNF-α secretion is stimulated by IL-1 and bacterial endotoxin. When mediated by IL-8, TNF-α has been known to promote the growth of endometriotic cells (8) .The mechanisms connecting endometriosis and infertility involved:  Distorted pelvic anatomy, including adhesions resulting from endometriosis, which can impair oocyte release or prevent ovum pickup and transport, (9) as well as damaged or plugged fallopian tubes or acquired or congenital uterine defects. (10) .  Altered peritoneal function, including increases in fluid volume; concentration of activated microphages; prostaglandins; IL-1, IL-6, TNF-alpha, IgG, and IgA antibodies; lymphocytes; an ovum capture inhibitor preventing cumulus -fimbria interaction; (11).  Endocrine and an ovulatory disorders, including luteinized unruptured follicle syndrome (LUF), luteal phase defect, abnormal follicular growth, and premature as well as multiple luteinized hormone surges. It has been hypothesized that LUF may not be a consequence of endometriosis, but, in fact, may be a cause or cofactor in the development of the disease (9) .  Impaired implantation, with evidence suggesting that endometriosis may be responsible for reduced expression of the (alpha(v)beta(3) integrin)αvβ3 cell adhesion molecule during the time of implantation (11) . The diagnosis of endometriosis can be substantiated only by direct visualization during laparoscopy or laparotomy confirmed by tissue biopsy (12) Larger lesions may be seen within the ovaries as ovarian endometriomas or "chocolate cysts" as they contain a thick brownish fluid, mostly old blood. However, smaller endometriosis implants cannot be visualized with ultrasound technique (12) . Surgically, endometriosis can be staged I–IV according to the (Revised Classification of the American Society of Reproductive Medicine) (13) . In principle the various stages show these findings: Stage I (Minimal): Endometriosis restricted to only superficial lesions and possibly a few filmy adhesions, there are isolated incidents of endometrial tissue growth outside the uterus (14) . Stage II (Mild): This diagnosis occurs when there are several small implants and a few small areas of scar tissue or adhesions and some deep lesions are present in the cul-de- sac. (15) . Stage III (Moderate): As in stage II, plus presence of endometriomas on the ovary and more adhesions. The implants in stage three must be superficial and deep. Stage IV (Severe): This is the most severe stage of endometriosis. Patients with stage IV endometriosis will have many superficial and deep implants as well as large adhesions found. The purpose of medical management is to minimize proliferation/reduce pain, inhibit inflammation, minimize menstrual volume, frequency and oppose E2 action (16) . Medical treatment included :Initial therapy should include a non-steroidal anti- inflammatory drugs (NSAIDs) such as : a- Naproxen 500 mg at first then followed by 250 mg orally three times daily, or b-Ibuprofen 800 mg as single dose, then 400 mg orally every 6 to 8 hours or c- Mefenamic acid 500 mg orally then followed by 250 mg every 6 hours. (17) . Surgical treatment is highly effective for the alleviation of symptom, pain reduction and can increase fertility in sub-fertile women (18) .Medical management is based on hormonal suppression of endometriotic lesions and is particularly effective when amenorrhea occurs via down -regulation of the hypothalamic -pituitary-ovarian axis (19) .As shown in table-2. Iraqi J Pharm Sci, Vol.25(1) 2016 Effect of metformin treatment in women with endometriosis 31 Table (2): Hormonal Treatment for Endometriosis (20) . Medication Indication Dosing Comment Depot MDPA (Depo- Provera) R Pain relief 150 mg intramuscularly every three months Common usage in primary care MDPA (Provera) R Pain relief 30 to 100 mg daily, given orally Common usage in primary care Oral contraceptives Pain relief 0.02 to 0.03 mg ethinyl estradiol and 0.15 mg desogestrel daily (cyclically) for six months Common usage in primary care Levonorgestrel intrauterine system (Mirena) R Pain relief Intrauterine system Can be placed easily Common usage in primary care Gonadotropin-releasing hormone analogues (e.g. goserelin [Zoladex], Pain relief 3.75 mg of leuprolide injected every four weeks or 3.6 mg of goserelin implanted subcutaneously for six months Expensive; significant side effects (hypoestrogenic symptoms) Nafarelin (Synarel) R Pain relief 200 mcg intranasally twice daily for six months Expensive; significant side effects Danazol Pain relief 200 mg given orally three times Daily 400 mg given orally twice daily for six months Significant androgenic side effects Gestrinone Pain relief 2.5 mg given orally twice weekly for six months Hot flashes This study was conducted to:- 1- Investigate the effect of metformin on symptoms of endometriosis and biochemical markers. 2-Investigate the effects of metforminon serum interleukin- 8 (IL-8), tumor necrosis factor alpha (TNF-α )and estradiol (E2)levels at baseline and after 3 months of treatment, compared to baseline values. Patients and methods Thirty women were included in this study from AL-Elwiyah Maternity Teaching Hospital and Al-Yarmok Teaching Hospital for the period from January 2014 to June-2014. Verbal consent was obtained from all women prior to enrollment in the study. Inclusion criteria 1- Women undergo diagnostic laparoscopy for pelvic pain or treated for some causes of infertility. 2- Women undergo diagnostic laprotomy for ovarian cyst or acute abdominal pain who were found to have endometriosis. 3-Women came for follow up for endometriosis. Grouping of patients A-Control group Fifteen patients diagnosed by laparoscopy to have different stages of endometriosis. These patients were complaining of one or more symptoms such as dysmenorrhea, pelvic pain or menorrhagia as seen in table -3. They received classical drugs such as danazol capsules (isoxazolic derivative of a synthetic steroids,17 α- ethinyl testosterone) , some women treated with zoladex (goserelin acetate implant) ,and some women treated with oral contraceptive pills as seen in table - 4. Iraqi J Pharm Sci, Vol.25(1) 2016 Effect of metformin treatment in women with endometriosis 32 Table (3): Patient’s characteristics of women with endometriosis. P value (control versus study group) Study group Control group Patient’s characteristics 0.5404 32.93±7.488 34.69±7.476 Age in years (mean ± S.D) 0.9415 4.100±1.955 4.045±1.387 Duration of infertility in years (mean ± S.D). 0.02 12/15(80.00% ) 6/15(40.00% ) Dysmenorrhea 0.14 9/15(60%) 5/15(33.33%) Pelvic pain 0.19 2/15(13.33%) 5/15(33.33%) Menorrhagia Table (4): Number of patients treated with Danazol , goserelin acetate (zoledax )R , and oral contraceptive for control groups. Drugs No. of Patients Danazol (4)26.66% (goserelin acetate) Zoledax R (10)66.66% oral contraceptive pill (1)6.66% B. Study Group Fifteen patients with stages (I-IV) endometriosis diagnosed by laparoscopy or laprotomy and were complaining of one or more symptoms such as pelvic pain or menorrhagia and received metformin at 500 mg every 8 hour in addition to classical drugs as shown in table -5. Table (5): Numbers of patients treated with Danazol, zoledax R , and oral contraceptive pills plus metformin(study group). Drugs No. of Patients Danazol and metformin R (6)40% Zoledax R and metformin R (5)33.33% oral contraceptive and metformin R (4)26.66% Methods Sampling Blood samples were obtained for the estimation of the cytokines as (IL-8 (21) and TNF-α (22) )in addition to estradiol E2 (23 levels at the start of the study from the fasting patients in morning and also at the follow up visits after three months.Estimation of the cytokines IL-8, TNF-α level was performed using a commercially available enzyme -linked immunosorbent assay (ELISA) kits. Estimation of the estradiol E2 was performed using Radioimmunoassay. Five ml of venous blood was withdrawn aseptically into dry plastic tubes. Then the collected blood samples were centrifuged at 3000g for 15 min and the separated serum were stored at −20 °C until time of assay. Statistical analysis The collected data were tabulated, compared and proper statistical analyses were performed. A t-test is any statistical hypothesis test in which the test statistic follows a Student's t distribution if the null hypothesis is supported. It can be used to determine if two sets of data are significantly different from each other. .When the scaling term is unknown and is replaced by an estimate based on the data, the test statistic (under certain conditions) follows a Student's t distribution (24) .Frequency, mean and standard deviation (SD) were used to describe data .P value was considered positive and significant if less than 0.05. Results and Discussion The study sample included (30) patients comprising of 15 female as control groups and 15 female as study groups; endometriosis was diagnosed by laparotomy or laparoscopy as shown in table - 6. Iraqi J Pharm Sci, Vol.25(1) 2016 Effect of metformin treatment in women with endometriosis 33 Table(6): Endometriosis diagnosis by laparotomy or laparoscopy Diagnostic routes Causes No. of patients Laparoscopy pelvic pain 3 Laparoscopy Treated some causes of infertility 7 Laparotomy ovarian cyst 8 Laparotomy acute abdominal pain 6 follow up Endometriosis 6 Endometriosis classified into stages according to the revised criteria of the American Society of Reproductive Medicine (ASRM) (13) Table -7 represents the % of stages in endomtrosis were I, II, III and IV in 3(10%), 14(46%), 8(27%) and 5(17%) patients. Endometriosis can occur in teenagers and adult women of all ages, but most typically it occurs in women ages 23 – 45 for control groups 34.69±7.476.and for study groups are 32.93±7.488 as shown in Table-3. Table(7): Stages of endometriosis. Stages of endometriosis Frequency Minimal (I) 3/30 (10%) Mild (II) 14/30(46%) Moderate (III) 8/30(27%) Sever (IV) 5/30(17%) Dysmenorrhoea is the most common reported symptom in endometriosis, it occurs in six women in control groups (34.69±7.476.and twelve women in study groups are 32.93±7.488 as shown in table -3. Endometriosis is estimated to occur in 5/15(33.33%) of women presenting with pelvic pain for control groups , 9/15(60%) in study groups also showed that 5/15(33.33%) of women presenting menorrhagia in control groups and 2/15(13.33%) in study groups as shown table-3. The serum levels of TNF- α , IL-8 and E2 in the control and study groups levels in the endometriosis before treatment and after 3 months of treatment. As shown in Table -8. Table (8): Tumor necrosis factor-alpha (TNF- α), Interlukin-8(IL-8) and Estradiol (E2) levels in the control and study groups: Control group(mean ± S.D) Study group (mean ± S.D) P value Before(1 st visit) After(2 nd visit) Before(1 st visit) After(2 nd visit) TNF-α (pg/ml) 83.90±9.23 78.05±8.88 89.90±11.83 68.39±10.91 P 0.15 0.004 P1 0.23 P2 0.05 IL-8 (pg/ml) 41.31±6.376 39.86±6.239 52.70±8.646 44.54 ± 13.94 P 0.09 0.04 P1 0.16 P2 0.01 E2 (pg/ml) 133.2±32.33 114.8±30.43 89.90±11.83 68.39±10.91 P 0.37 0.25 P1 0.92 P2 0.34 P< 0.05 is significant &P> 0.05 is not significant. P: levels at start of the study versus after 3 months. P1: levels at 1start of the study for Control versus study group. P2: levels after 3 months of the study for Control versus study group. http://www.mefsjournal.org/article/S1110-5690(12)00095-7/fulltext#t0015 Iraqi J Pharm Sci, Vol.25(1) 2016 Effect of metformin treatment in women with endometriosis 34 in the present study there was a significant reduction in the number of cases with dysmenorrhea and pelvic pain (P< 0.05) after three months of metformin therapy. as seen in table -9. Metformin significantly decreased pain and the level of C-reactive protein after 6 months of treatment in patient with endometriosis associated chronic pelvic pain (25) Rannou F et al 2007 , study demonstrated that IL-6, may be considered as a major regulator of C-reactive protein gene (25) tumor necrosis factor alpha (26) , nuclear factor ĸɃ (27) and transforming growth factor beta- one (TGFβ-1) (28) .The inflammation associated with endometriosis, through increased levels of peritoneal fluid Vascular Endometriosis growth factor (VEGF), may promote angiogenesis for progressive growth of endometriosis (29) . Table(9): Changes in clinical symptoms in endometriosis control and study group before and after three months of treatment . P value Study group [N(%)] Control group [N(%)] After2 nd visit Before1 st visit After2 nd visit Before1 st visit After2 nd visit Before1 st visit 0.02 0.02 12/15 80.00% 0.02 6/15 40 % 6/15 40.00% 0.46 7/13 53.8% Dysmenorrhea P 0.61 0.19 2/15 13.33% 0.09 6/15 40 % 5/15 33.33% 0.88 4/13 30.76% Menorrhagia P 0.51 0.14 9/15 60% 0.02 3/15 20% 5/15 33.33% 0.88 4/13 30.76% Pelvic pain P N: number of positive cases/total number. Thus , endometriosis had a direct effect on adhesion formation (30) . Moravek et al (2009) ,provided a preliminary data about the effectiveness of rosiglitazone, an insulin sensitizer, in treating endometriosis -related pain in six patients and concluded that it was effective and promising for usage in endometriosis patients desiring the chance to conceive (31) . The result of this study showed that the level of IL-8 was significantly decreased after 3 months of metformin therapy (P< 0.05). IL-8 level was significantly higher in the endometriosis groups than in the control groups (p=0.01). Table 8. Arici et al 1998. reported" that IL-8 is produced in the human endometrium in vivo, especially in glandular cells" (32) .IL-8 raise the proliferation of endometrial stromal cells as a potential autocrine growth factor (36) . A previous study Y. Takemura et al (33) has shown that metformin in non decidualized ovarian endometriotic stromal cells could reduce IL-1b-induced IL-8 production, aromatase activation and proliferation. So far only a reduction of aromatase activation and a reduction of proliferation has been demonstrated in vitro in ovarian endometriotic stromal cells after treatment with metformin (Y. Takemura et al 2007) (33) .The results of Iwabe et al (8) clearly demonstrated that "IL-8 is a growth- promoting factor in normal endometrium as well as in endometriotic cells". The study of Baracz et al 2012. (34) excluded the primary role of IL-8 in the formation of endometriosis -associated peritoneal adhesions and confirmed the role of cytokines in the pathogenesis and progression of endometriosis.The result of the present study showed that there was significant reduction in TNF-α levels in women with endometriosis after 3 months of metformin treatment.The result of Iwabe et al 2001 (8) ,proved that it through induction of IL-8 gene and protein expression in endometriotic stromal cells ,TNF-α stimulated proliferation of endometriotic stromal cells in a dose - dependent fashion. The addition of anti- TNF-α antibody or anti-IL-8 antibody, lead to neutralized the proliferated effect of the endometriotic stromal cells, and the stimulatory effects of TNF-α (34) Bedaiwy et al (2002) (35) demonstrated that serum TNF-α had a high degree of sensitivity and specificity so it could be surprisingly used to differentiate between patients with and without endometriosis. The result of the current study showed that there was no significant reduction in level Iraqi J Pharm Sci, Vol.25(1) 2016 Effect of metformin treatment in women with endometriosis 35 of estradiol E2 in women with endomterosis treated with metformin for periods of 3 months and this disagree with the study of Mansfield et al 2003 (36) . Also table 8 showed that the significant reduction in estradiol E2 ,FSH ,insulin -stimulated progesterone production in granulosa cells Thus, metformin may be effective in treatment endometriosis through suppression both ovarian and local production of estrogen (36) . So the result from this study about serum estradiol E2 may be attributed to the small sample size and short time of study or the dose of metformin was not enough to lower of estradiol E2 level. Conclusion From the results of this study, it was found that metformin may be well tolerated treatment for endometriosis that relieved pain and reduced menstrual disorders.Serum levels of the inflammatory markers (IL-8 and TNF-α) are decreased in study groups with metformin after 3 months indicating that metformin may be have anti-inflammatory effect. References 1. U.S. Department of Health and Human Services, Office of Women’s Health 2009. 2. Gynaecology Treatment - Adelaide Obstetrics & Fertility 2011 3. J. Gilabert-Estelles, L. A. Ramon, F. Espa˜ na, J. Gilabert, R. Castello, and A. Estelles, “Expression of fibrinolytic components in endometriosis,”Pathophysiology of Haemostasis and Throm-bosis, 2006;35,( 1-2):136–140. 4. Morin-Papunen L, Rautio K, Ruokonen A, Hedberg P, Puukka M, Tap-anainen J SMetformin reduces serum C-reactive protein levels in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2003, 88:4649 – 4654. 5. H.Du and H. S .Taylor , “Reviews :stemcells and female reproduction ,”Reproductive Sciences,vol.16,no.2, 2009. pp.126–139. 6. Samer Sourial, Nicola Tempest, and Dharani K. Hapangama International Journal of Reproductive Medicine Volume 2014, Article ID p.179515, 9 . 7. Said TM, Agarwal A, Falcone T, Sharma RK, Bedaiwy MA Li L.nfliximab may reverse the toxic effects induced by tumor necrosis factor alpha in human spermatozoa: an in vitro model. Fertil Steril. 2005;83:1665-73. 8. Iwabe T, Harada T, Tsudo T, Nagano Y, Yoshida S,Tanikawa M, et al. Tumor necrosis factor-alpha promotes proliferation of endometriotic stromal cells by inducing nterleukin-8 gene and protein expression. J Clin Endocrinol Metab. 2000;85:824-9. 9. Practice Committee of the ASRM. Endometriosis and infertility . Fertil Steril.2006; 86(4):S156-S160. 10. Giudice L, Evers JLH, Healy DL. Endometriosis: Science and Practice.Chichester, West Sussex: Wiley- Blackwell; 2012 Buyalos RP, Agarwal SK ."Endometriosis-associated infertility". Current Opinion in Obstetrics and Gynecology .October 2000, 12(5):377–81 11. Hughes EG, Fedorkow DM, Collins JA: A quantitative overview of controlled trials in endometriosis-associated infertility. Fertil Steril , 1993,59: 963. 12. American College of Obstetrics and Gynecology. Chronic pelvic pain. ACOG technical bulletin no. 223. Washington, D.C.:, 1996:3 . 13. American Society For Reproductive M"Revised American Society for Reproductive Medicine classification of endometriosis: 1996". Fertility and Sterility May 1997; 67 (5): 817–21. 14. Vercellini P,L Trespidi,O De Georgi,I Cortesi, F Parazzini,PG Crosignnani ,.Endometriosis and pelvic pain :Relation to disease stage and location on fertile 1996 ;65:299. 15. Schenken RS, Guzick DS: Revised endometriosis classification: 1996. Fertil Steril .1997; 67: 815 16. Paul D. Chan, M.D.Christ opher R. Winkle, M.D Current Clinical Strategies/Gynecology and Obstetrics,.2000.35. 17. Annel. mounsey,alex wilgaus, M.D M.D, david. SLAWSON, M.D. Diagnosis and Management of Endometriosis; 2006;,74(4) August 15. 18. Royal College of Obstetricians and Gynaecologists (RCOG). The investigation and management of endometriosis. RCOG, UK; 2008. 19. Streuli I, De Ziegler D, Santulli P, et al. An update on the pharmacological management of endometriosis. Expert Opin Pharmacother 2013;14(3):291–305. 20. Annel. mounsey,alex wilgaus, M.D M.D, david. SLAWSON, M.D.Diagnosis and Management of Endometriosis . , 2006, 74, ( 4 )August 15. 21. (Reagents for assay supplied by BioMereux – LYON – FrancThe instrument used was Mini VIDAS – BioMereux), http://www.google.com.iq/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAUQjhw&url=http%3A%2F%2Fwww.adelobs.com.au%2Fgynaecology-fertility-treatment%2F&ei=fpQFVfvEMsfa7Aab84CYAQ&bvm=bv.88198703,d.ZGU&psig=AFQjCNE4ep_4xtJVc7JVMUPQDoWIXJGuWw&ust=1426515133426657 http://www.google.com.iq/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAUQjhw&url=http%3A%2F%2Fwww.adelobs.com.au%2Fgynaecology-fertility-treatment%2F&ei=fpQFVfvEMsfa7Aab84CYAQ&bvm=bv.88198703,d.ZGU&psig=AFQjCNE4ep_4xtJVc7JVMUPQDoWIXJGuWw&ust=1426515133426657 http://meta.wkhealth.com/pt/pt-core/template-journal/lwwgateway/media/landingpage.htm?issn=1040-872X&volume=12&issue=5&spage=377 Iraqi J Pharm Sci, Vol.25(1) 2016 Effect of metformin treatment in women with endometriosis 36 22. (Human dendritic cells engineered to express alpha tumor necrosis factor maintain cellular maturation and T -cell stimulation capacity.Ye Z, Chen Z, Sami A, El-Gayed A, Xiang J, Cancer Biother Radiopharm .2006; 21:613-622. 23. ABBIOTEC 7985 Dunbrook Road, Suite A San Diego, CA 92126) 24. Fadem, Barbara High-Yield Behavioral Science (High-Yield Series). Hagerstwon, MD: Lippincott Williams and Wilkins 2008. 25. Rannou F, Ouanes W, Boutron I, Lovisi B, Fayad F, Mace Y. High-sensitivity C- reactive protein in chronic low back pain with vertebral end-plate modic signal changes. Arthritis and Rheumatism (Arthritis Care and Research), 2007;57(7); 1311-131. 26. Arai M, Uchiba M, Komura H, MizuochI Y, Harada N, Okajima K.Metformin, an antidiabetic drug, suppresses the production of tumor necrosis factor and tissue factor by inhibiting early growth response factor-1 expression in human monocytes in vitro. JPET. 2010;334(1): 206-213. 27. Hattori Y, Suzuki K, Hattori S, Kikuo S. Metformin inhibits cytokine-induced nuclear factor-ĸɃ activation via AMP- activated protein kinase activation in vascular endothelial cells. Hypertension. 2006;47:1183-1188 28. Xiao H, Ma X, Feng W, Fu Y, Lu Z, Xu M, Shen Q, Zhu Y, Zhang Y. Metformin attenuates cardiac fibrosis by inhibiting the TGF beta I –Smad 3 signalling pathway. Cardiovasc. Res .2010;87(3):504-13. 29. Mohammad zadeh A, Heidari M, Ghoraii HS, Zarnani AH, Ghaffari Novin M, Akhondi MM, et al.. Induction of endometriosis by implantation of endometrial fragments in female rats. Iran J Reprod Med 2006;4:63–7. 30. Bullimore DW. Endometriosis is sustained by tumor necrosis factor-alpha. Medical Hypotheses. 2003;60(1): 84-88. 31. Moravek MB, Ward EA, Lebovic DI. Thiazolidinediones as therapy for endometriosis: a case series. Gynecol Obstet Invest 2009;68(3):167–70. 32. Arici A, Seli E, Zeyneloglu HB, Senturk LM, Oral E, Olive DL. Interleukin-8 induces proliferation of endometrial stromal cells: a potential autocrine growth factor. The Journal of clinical endocrinology and metabolism. 1998;83:1201-5. 33. Y. Takemura, Y. Osuga, O. Yoshino, A. Hasegawa, T. Hirata, Y. Hirota, et al. Metformin suppresses interleukin (IL)-1β- induced IL-8 production, aromatase activation, and proliferation of endometriotic stromal cells Clin Endocrinol Metab. 2007;92 (8): 3213– 3218 . 34. Barcz E, Milewski L, Dziunycz P, Kaminski P, Ploski R, Malejczyk J. Peritoneal cytokines and adhesion formation in endometriosis: an inverse association with vascular endothelial growth factor concentration. Fertility and sterility. 2012;97:1380-6 e1. 35. Bedaiwy MA, Falcone T, Sharma RK, Goldberg JM, Attaran M, Nelson DR, et al. Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. Hum Reprod. 2002;17:426-31. 36. Mansfield R, Galea R, Brincat M, Hole D, Mason H. Metformin has direct effects on human ovarian steroidogenesis.Fertil Steril.2003;79:956– 96.