Iraqi J Pharm Sci, Vol.26(2) 2017 Blood. levels of trace elements and heavy metals in PCO 85 A comparative Study of Blood Levels of Manganese, Some Macroelements and Heavy Metals in Obese and Non-Obese Polycystic Ovary Syndrome Patients Sarah H. Mhaibes*,1 , Mohammed A. Taher* and Ala H. Badr** *Department of Clinical Laboratory Science, College of Pharmacy, University of Baghdad, Baghdad, Iraq. ** Kamal Samarrai Hospital , Ministry of Health/Environment , Baghdad, Iraq. Abstract Polycystic ovary syndrome (PCOS) is a prevalent condition in women of reproductive age. It is characterized by androgen excess and chronic anovulation. Some trace elements, macroelements, and heavy metals have been linked to pathophysiological mechanisms of PCOS . To study the alterations in the serum levels of the trace element manganese (Mn), some macroelements, magnesium(Mg) and calcium (Ca), and the heavy metals cadmium (Cd) and lead (Pb), in obese and non-obese PCOS patients; and the association of these alterations with some of the hormonal changes occurring in PCOS. The study was carried out at Kamal Al-Samarrai Hospital (Center for Infertility treatment and in vitro Fertilization "IVF") Baghdad- Iraq. Eighty-two women were enrolled in the study. Fifty-four of them were diagnosed by a specialist gynecologist as PCOS patients; they were subdivided into two subgroups according to their body mass index (BMI); twenty-seven obese PCOS patients with BMI > 30 kg/m2, and another twenty seven non obese patients PCOS with BMI <30 kg/m2. Whereas, twenty- eight apparently healthy women with regular menstruation and of comparable age, were selected to serve as control groups; they were subdivided into, fourteen obese women with BMI > 30kg/m2, and fourteen non obese women with BMI <30 kg/m2. Blood lead and cadmium levels were significantly higher in both of the obese and the non- obese PCOS groups, than in their corresponding control groups. While, serum magnesium, calcium and manganese levels were significantly lower in both of the obese and the non-obese PCOS groups, as compared to their corresponding control groups. The results revealed no significant difference in the levels of the measured elements, between the obese PCOS group and the non-obese PCOS group. The serum FSH levels was significantly lower in obese PCOS patients than in the obese and non-obese control groups. There was a positive correlation between blood lead and serum TSH levels in non- obese PCOS women; and between serum total testosterone and cadmium levels in obese PCOS women. Finally, there was negative correlation between serum magnesium and serum LH levels in non-obese PCOS women. the study has demonstrated higher blood levels of lead and cadmium; and lower serum levels of magnesium, calcium and manganese in PCOS groups than control subject. There were no significant differences between obese PCOS women and non-obese PCOS women in the levels of the studied hormones, elements and heavy metals. Keywords: PCOS, Trace elements, Macroelements, Heavy metals, infertility. فيالعناصر الرئيسية والفلزات الثقيلة بعض و من المنغنيزمستويات الدم ل ةدراسة مقارن المريضات المصابات بمتالزمة المبيض متعدد االكياس البدينات وغير البدينات من **و االء حازم بدر *حمد عباس طاهر، م 1 *,سارة هاشم محيبس بغداد ، بغداد ، العراق .فرع العلوم المختبرية السريرية ، كلية الصيدلة ، جامعة * ، بغداد ، العراق . والبيئة مستشفى كمال السامرائي ، وزارة الصحة ** الخالصة عدم االباضة و بزيادة االندروجينهي حالة سائدة لدى النساء في سن اإلنجاب. وتتميز متعدد االكياسمتالزمة المبيض آلليات المرضية الفيزيولوجية لمتالزمة المبيض قد ترتبط با الثقيلة فلزاتوال، العناصر الرئيسية بعض العناصر النزرة ان. المزمن ،بعض العناصر الرئيسية من لعنصر المنغنيزالتغيرات في مستوى المصل مع دراسة ارتباط التغيرات الهرمونية .متعدد االكياس بين النساء البدينات وغير البدينات المصاباتالكادميوم والرصاص الفلزات الثقيلة ومستوى الدم الكلي منالمغنيسوم والكالسيوم -أجريت الدراسة في مستشفى كمال السامرائي )مركز عالج العقم والتخصيب في المختبر( بغداد . كياسمتعدد اال متالزمة المبيضب ةتحت إشراف أخصائي مبيض متعدد االكياسالمتالزمة ب يضاتمنهن مر ٤٥من النساء، تم اختيار ة متطوع ٢٨العراق. شملت الدراسة لواتيال المبيض متعدد االكياسمتالزمة ب ةمريض ٨٧: الىإلى مجموعات فرعية وفقا لمؤشر كتلة الجسم تم تقسيمهن و نسائية وتوليد ، جيدة صحيةبحالة امرأة على ما يبدو ٨٢ن من السمنة المفرطة في حين تم اختيار ييعان ال اخريات٨٧ن من السمنة المفرطة و ييعان . طيف االمتصاص الذري هو طريقة تستخدم مع انتظام الدورة الشهريةالسمنة ال يعانين٤٥ويعانين من السمنة ٤٥إلى هنتم تقسيم العينات. كلالثقيلة في فلزاتالنزرة وال العناصرلقياس كمية من 1Corresponding author E-mail: phsarahhashim@gmail.com Received: 9/9/ 2017 Accepted:27 /11/2017 mailto:phsarahhashim@gmail.com Iraqi J Pharm Sci, Vol.26(2) 2017 Blood. levels of trace elements and heavy metals in PCO 86 المبيض متعدد ن من متالزمة ييعان لواتيال يضاتالمر ةالرصاص والكادميوم أعلى بكثير في مجموع اظهرت الدراسة ان المبيض متعدد متالزمة ب يضاتالمغنيسيوم والكالسيوم والمنغنيز في مر انخفاض ، في حين الطبيعياتالنساء ةمقارنة بمجموع االكياس على التوالي. لم تكشف النتائج النساء الطبيعيات البدينات وغير البدينات على حد سواء بالمقارنة مع البدينات وغير البدينات االكياس في مستوى العناصر البدينات وغير البدينات متعدد االكياس المبيض عانين من متالزمةعن وجود فرق معنوي بين النساء اللواتي ي المقاسة. يضاتالمر ةمجموعهناك عالقة طردية بين مستويات الدم من الرصاص مع مستويات المصل من الهرمون المحفز للغدة الدرقية في ينات باال ضافة الى ذلك كان هناك ارتباط طردي بين مستويات الدم من المبيض متعدد االكياس غير البدن من متالزمة ييعان لواتيال المبيض متعدد االكياس ن من متالزمة ييعان لواتيال يضاتالمر ةمجموعالكادميوم ومستويات مصل الدم من التيستوستيرون في لواتيفي النساء الLH من هرمون بين المغنيسيوم في الدم ومستويات المصل عكسيوعالوة على ذلك، كان هناك ارتباط البدينات. المبيض متعدد االكياس غير الديناتمن متالزمة ينيعان في ارتفاع مستويات الدم من الرصاص والكادميوم في حين انخفاض مستويات المصل من المغنيسيوم والكالسيوم والمنغنيز . لم تكن هناك فروق ذات النساء الطبيعيات ةمقارنة بمجموع المبيض متعدد االكياسن من متالزمة ييعان لواتيال يضاتالمر ةمجموع في المعلمات المدروسة البدينات وغير البدينات المبيض متعدد االكياسداللة إحصائية بين النساء اللواتي يعانين من متالزمة العناصر والفلزات الثقيلة.للهرمونات، . ، العقميسيةئالعناصر الر، العناصر النزرة ، المعادن الثقيلة، االكياسمتعدد ة: متالزمة المبيضالكلمات الرئيسي Introduction Citsycylop ovary syndrome (PCOS) is symptomatic nearly 6% to 10% of women of reproductive age (12–45 years old) (1). It is a principal cause of female subfertility and the most frequent endocrine problem in women of reproductive age, although up to 70% of women with PCOS may be undiagnosed (2). Polycystic ovary syndrome is characterized by anovulation, insulin resistance and hyperandrogenism. Anovulation causes irregular menstruation, amenorrhea, ovulation- related infertility and polycystic ovaries. Hyperandrogenism results in acne and hirsutism. Insulin resistance is often correlated with obesity, Type 2 diabetes, and high cholesterol level (3). The etiology of PCOS is not yet entirely known. It is believed that it is a complex, multifaceted disease involving abnormalities in the hypothalamic-pituitary axis, uncontrolled ovarian steroidogenesis, excessive oxidative stress, aberrant insulin signaling and genetic/environmental factors (4,5). Many studies have indicated increased oxidative stress in the patients with PCOS (6,7). Several characteristics and associations of PCOS, including androgen excess, abdominal adiposity, insulin resistance and obesity, may contribute to the development of local and systemic oxidative stress which may reciprocally worsen these metabolic abnormalities (8). The whole dollb of heavy metals and serum levels of trace elements were altered in patients with PCOS (9). Environmentally relevant levels of metals are also associated with modest changes in reproductive hormone levels (10). Cadmium is a heavy metal and may have a role in the formation of reactive oxygen species (ROS) (11). Lead may also lead to the production of ROS by depleting glutathione (GSH) and protein-bound sulfhydryl groups and enhancing lipid peroxidation (12). Calcium ia sn essential macroelement to body organization and structure and is the material of many indispensable substances, such as 25- hydroxyvitamin D (25(OH)D) and high- density lipoprotein (HDL) cholesterol, which contribute to the cardiovascular system (13). Magnesium, one of the abundant cations in the human body categorized as macro element, participates in enzymatic reactions and insulin secretion. Magnesium is closely related to diabetes mellitus type 2 and other metabolic diseases (14). Magnesium deficiency is believed to indirectly enhance the oxidative damage of biomolecules by inducing a stress response. It is possible that a magnesium deficiency stimulates catecholamine release from the adrenal glands. However, catecholamine increases the production of ROS. This creates a vicious positive feedback cycle where, for example, elevated blood epinephrine levels result in a further reduction of the magnesium concentration. Contrastingly, Magnesium deficiency leads to the activation of the renin- angiotensin system that also induces oxidative stress (15). Manganese is an essential micronutrient incorporated into many metalloenzymes and proteins involved in cell metabolism and regulatory pathways controlling oxidative stress (16). Subjects and Methods This study was carried out at Kamal Al-Samarrai Hospital (Center for Infertility treatment and in vitro Fertilization "IVF"), from October /2016 to April/2017. Eighty-two women were enrolled in the study. Fifty-four of them were patients with PCOS, with the age (obese 29.04± 5.64, non-obese 26.56±4.97) and twenty-eight apparently healthy women were selected as controls, their age (obese 32.07±5.86 and non-obese 24.64±4.25). Every participant woman was interviewed and requested to answer a specially designed questioning format Iraqi J Pharm Sci, Vol.26(2) 2017 Blood. levels of trace elements and heavy metals in PCO 87 including; demographic data, menstrual, obstetric, medical and family histories. The diagnosis of PCOS in this study was based on the revised Rotterdam criteria, which require, two of the following three manifestations: (1) clinical and/or biochemical hyperandrogenism (2) oligo-and/or anovulation (cycle length >35 days), and (3) polycystic ovaries on ultrasound (polycystic ovary was defined as the appearance of more than 11 follicles in each ovary, each measuring 2-9 mm in diameter, and/or increased ovarian volume > 10 ml) (17). With the exclusion of other etiologies (androgen-secreting tumors, thyroid disorder, Cushing syndrome, hyperprolactinemia, congenital adrenal hyperplasia). All of the patients were selected under the supervision of a specialist gynecologist. The studied women were divided into: A- Fifty-four PCOS patients, they were subdivided into two subgroups based on their body mass index (BMI), which was calculated as the ratio of the body weight in kilograms to the square of its height in meters (18). The two subgroups were as follows; twenty-seven obese PCOS patients with BMI ≥ 30 kg/m2 (34.81±0.820), and another twenty-seven non-obese PCOS patients with BMI <30 kg/m2 (26.41± 0.401). B- Twenty-eight apparently healthy control women were subdivided into two subgroups; fourteen obese control subjects with BMI ≥ 30kg/m2 (32.81±0.720), and fourteen non- obese control subjects with BMI <30 kg/m2 (23.90±0.678). Venous blood samples (10cc) was withdrawn hcle msrf woman between days two and four of menstrual cycle after overnight fasting. Blood samples were divided into two parts, one part (2.5cc) were collected in an anticoagulant containing tubes for direct measurement of lead (Pb), and cadmium (Cd) blood levels and the other part (7.5cc) were collected in gel and clot activator tubes without anticoagulant then samples left for 30 minutes to clot. After complete clotting, the serum was separated by centrifugation (centrifuged for 10 minutes at 3500 to 4000 rpm to obtain serum). The collected serum was divided in 10 Eppendorf tubes [9 tubes kept frozen (-80˚C) until their assay, and the rest of them used for immediate measurement of fasting glucose level]. Assay The whole blood levels of cadmium, lead and serum levels of magnesium, calcium, manganese were measured by atomic absorption spectrophotometer in Poisoning Consultation Center in Medical City-Baghdad. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH) and total testosterone (TES) were tested by Enzyme- linked final fluorescent assay (ELFA) (19,20). Serum prolactin (PRL) and serum thyroid stimulating hormone (TSH) were measured by a two-site sandwich immunoassay using direct chemiluminometric technology (21,22). Fasting blood glucose was evaluated according to the method of Barham and Trindoer (23). Statistical analysis The results were expressed as a mean ± standard error of the mean (SEM) or percent changes. LEVENE test was used to test the homogeneity of variances for equality of variances of the several independent groups. One-way analysis of variance (ANOVA) and least significant tests were used for comparisons between the studied groups, and to examine the degree of significance. Pearson’s correlation coefficient (r) was used to study the correlation of trace element (manganese), some macroelements and heavy metals with the measured parameters of hormones. The statistical analysis was performed using SPSS, version 16 (24). Results The anthropometric and demographic characteristics of Polycystic Ovary Syndrome (PCOS) patients and controls are summarized in table 1. As illustrated in table 2, the serum FSH level was significantly lower in obese PCOS patients than in the obese and non-obese control groups (P<0.01, P<0.05 respectively); there was no significant difference between obese and non-obese PCOS (p˃0.05). Serum LH, LH/FSH ratio, prolactin and total testosterone levels were significantly higher in both obese and non-obese PCOS patients as compared to their corresponding control groups. Yet, there was no significant difference between obese and non-obese PCOS groups as well as between the control groups. TSH levels were significantly higher in the obese PCOS than in the obese control (P<0.05). Serum glucose levels were significantly higher in the obese PCOS than in the obese and non-obese control groups (P<0.05, P<0.01 respectively), but there was no significant difference between the non- obese PCOS and the non-obese control (P> 0.05). Table 3 showed that, the whole blood concentrations of lead (Pb) and cadmium (Cd) were significantly higher in the PCOS groups than in the control groups; yet, there was no significant difference between the obese and non-obese PCOS groups (P> 0.05), as well as between the obese and non-obese control Iraqi J Pharm Sci, Vol.26(2) 2017 Blood. levels of trace elements and heavy metals in PCO 88 groups. Finally, serum concentrations of magnesium(Mg), calcium(Ca) and manganese (Mn) were significantly lower in both of the obese and non-obese PCOS groups as compared to their corresponding control groups; and there was no significant difference between the obese and non- obese PCOS group. Table (1): The anthropometric and demographic Data of PCOS patients and controls P-value Controls PCOS Groups Variables non-obese (14) obese (14) non-obese (27) obese (27) 0.204 24.64 ± 1.14 32.07 ± 1.57 26.56 ± 0.96 29.04 ± 1.09 Age(years) 0.000** 23.90 ± 0.67 32.81 ± 0.72 26.41 ± 0.40 34.81 ± 0.82 BMI (kg/m2) 0.000** 76.64 ± 2.04 100.00 ± 1.96 87.76 ±1.68 103.48 ± 2.81 Waist (cm) 0.000** 94.00 ± 0.70 119.4 ± 1.58 103.6 ± 1.64 116.9 ± 2.73 Hip (cm) 0.000** 0.782 ± 0.02 0.838 ± 0.012 0.847 ±0.012 0.885 ± 0.011 WHR 0.214 0 0 5 (18.5%) 9 (33.3%) F.Hx of PCOS 0.001** 0 2(14.2%) 2 (7.4%) 13 (48.1%) F.Hx of obesity - - - + + U/S for PCOS 0.190 0 0 4 (14.8%) 8 (29.6%) A.N. 1.000 0 0 24 (88.9%) 24 (88.9%) Hirsutism 0.564 0 0 10 (37%) 8 (29.6%) Acne 0.776 0 0 9 (33.3%) 10 (37%) Male pattern baldness 0.379 0 0 17 (63%) 20 (74.1%) OM 0.535 0 0 8 (29.6%) 6 (22.2%) AM 1.000 14 (100%) ٤٥ (100%) 2 (7.4%) 1 (3.7%) Regular cycle The data are expressed as the numbers (percentage) or mean ± standard error (SE). **P < 0.01 are highly significantly different. F.Hx: family history, U/S: ultrasound, A.N: Acanthosis nigricans, OM: Oligomenorrhea, AM: Amenorrhea Table (2): Descriptive statistics of studied sex hormones with comparative significant studies between groups (A) and groups (B) in serum levels of sex hormones. saliairaV sluorV Numbers Mean ±SE Groups( A) Groups(B) P-value* FSH (mIU/ml) Obese PCOS (27) 4.592 ± 0.194 Obese PCOS Obese Control 0.004 (HS) Non-Obese PCOS 0.348(NS) Obese control (14) 6.499 ± 0.790 Non-Obese Control 0.030 (S) Non-obese PCOS (27) 5.092 ± 0.332 Obese Control Non-Obese PCOS 0.031(S) Non-obese Control (14) 6.010 ± 0.657 Non-Obese Control 1.508 (NS) Non- Obese PCOS Non-Obese Control 0.156 (NS) LH (mIU/ml) Obese PCOS (27) 5.006 ± 0.389 Obese PCOS Obese Control 0.001 (HS) Non-Obese PCOS 1.000 (NS) Obese control (14) 2.840 ± 0.315 Non-Obese Control 0.000 (HS) Non-obese PCOS (27) 5.000 ± 0.428 Obese Control Non-Obese PCOS 0.001(HS) Non-obese Control (14) 2.541 ± 0.259 Non-Obese Control 0.882 (NS) Non- Obese PCOS Non-Obese Control 0.000 (HS) Iraqi J Pharm Sci, Vol.26(2) 2017 Blood. levels of trace elements and heavy metals in PCO 89 Continued table (2) Variables Groups Numbers Mean ±SE Groups(A) Groups(B) P-value* LH/FSH Ratio Obese PCOS (27) 1.152 ± 0.114 Obese PCOS Obese Control 0.000 (HS) Non-Obese PCOS 0.985 (NS) Obese control (14) 0.448 ± 0.068 Non-Obese Control 0.000 (HS) Non-obese PCOS (27) 1.095 ± 0.112 Obese Control Non-Obese PCOS 0.000 (HS) Non-obese Control (14) 0.465 ± 0.055 Non-Obese Control 0.993 (NS) Non-Obese PCOS Non-Obese Control 0.000 (HS) Prolactin (ng/ml) Obese PCOS (27) 27.54 ± 2.44 Obese PCOS Obese Control 0.000 (HS) Non-Obese PCOS 0.796(NS) Obese control (14) 11.30 ± 2.02 Non-Obese Control 0.000 (HS) Non-obese PCOS (27) 24.06 ± 2.91 Obese Control Non-Obese PCOS 0.005 (HS) Non-obese Control (14) 9.99 ± 1.61 Non-Obese Control 0.956(NS) Non-Obese PCOS Non-Obese Control 0.001 (HS) uaar aaVauVaalute (ng/ml) Obese PCOS (27) 3.078 ± 0.363 Obese PCOS Obese Control 0.000 (HS) Non-Obese PCOS 0.882(NS) Obese control (14) 1.229 ± 0.104 Non-Obese Control 0.000(HS) Non-obese PCOS (27) 2.667 ± 0.424 Obese Control Non-Obese PCOS 0.031(S) Non-obese Control (14) 1.175 ± 0.163 Non-Obese Control 0.992 (NS) Non-Obese PCOS Non-Obese Control 0.012 (S) ST (mIU/L) Obese PCOS (27) 3.064 ± 0.868 Obese PCOS Obese Control 0.018 (S) Non-Obese PCOS 0.054(NS) Obese control (14) 0.935 ± 0.109 Non-Obese Control 0.075 (NS) Non-obese PCOS (27) 1.639 ± 0.156 Obese Control Non-Obese PCOS 0.428 (NS) Non-obese Control (14) 1.472± 0.259 Non-Obese Control 0.598 (NS) Non-Obese PCOS Non-Obese Control 0.850 (NS) FSG (mmol/l) Obese PCOS (27) 6.530 ± 0.387 Obese PCOS Obese Control 0.012 (S) Non-Obese PCOS 0.122(NS) Obese control (14) 5.229 ± 0.149 Non-Obese Control 0.002 (HS) Non-obese PCOS (27) 5.874 ± 0.312 Obese Control Non-Obese PCOS 0.207 (NS) Non-obese Control (14) 4.936± 0.193 Non-Obese Control 0.616 (NS) Non-Obese PCOS Non-Obese Control 0.068 (NS) (*) NS: Non Significant (P> 0.05); HS: Highly Significant (P<0.01); S: Significant (P<0.05) Iraqi J Pharm Sci, Vol.26(2) 2017 Blood. levels of trace elements and heavy metals in PCO 90 Table (3): Descriptive statistics of trace elements and heavy metals with significant comparative studies between groups (A) and groups (B) in blood levels of studied elements saliairaV sluorV Mean ±SE Groups(A) Groups(B) P-value* Lead (µg/dl) Obese PCOS (27) 22.48 ± 0.42 Obese PCOS Obese Control 0.000 (HS) Non-Obese PCOS 0.360 (NS) Obese control (14) 14.29 ± 0.42 Non-Obese Control 0.000 (HS) Non-obese PCOS (27) 23.00 ± 0.45 Obese Control Non-Obese Morbid 0.000 (HS) Non-obese Control (14) 14.29 ± 0.44 Non-Obese Control 1.000 (NS) Non-Obese PCOS Non-Obese Control 0.000 (HS) Cadmium (µg/dl) Obese PCOS (27) 0.317 ± 0.009 Obese PCOS Obese Control 0.000 (HS) Non-Obese PCOS 1.000 (NS) Obese control (14) 0.144 ± 0.006 Non-Obese Control 0.000 (HS) Non-obese PCOS (27) 0.318 ± 0.011 Obese Control Non-Obese PCOS 0.000 (HS) Non-obese Control (14) 0.139 ± 0.008 Non-Obese Control 0.974 (NS) Non-Obese PCOS Non-Obese Control 0.000 (HS) Magnesium (mg/dl) Obese PCOS (27) 0.979 ± 0.025 Obese PCOS Obese Control 0.000 (HS) Non-Obese PCOS 0.721 (NS) Obese control (14) 1.410 ± 0.038 Non-Obese Control 0.000 (HS) Non-obese PCOS (27) 0.963 ± 0.030 Obese Control Non-Obese PCOS 0.000 (HS) Non-obese Control (14) 1.378 ± 0.060 Non-Obese Control 0.706 (NS) Non-Obese PCOS Non-Obese Control 0.000 (HS) Calcium (mg/dl) Obese PCOS (27) 7.319 ± 0.111 Obese PCOS Obese Control 0.000 (HS) Non-Obese PCOS 0.228(NS ) Obese control (14) 9.107 ± 0.174 Non-Obese Control 0.000 (HS) Non-obese PCOS (27) 7.082 ± 0.153 Obese Control Non-Obese PCOS 0.000 (HS) Non-obese Control (14) 8.979 ± 0.228 Non-Obese Control 0.6 (NS) Non-Obese PCOS Non-Obese Control 0.000 (HS) Iraqi J Pharm Sci, Vol.26(2) 2017 Blood. levels of trace elements and heavy metals in PCO 91 Continued table (3) Variables Groups Mean ±SE Groups(A) Groups(B) P- value* Manganes e (µg/dl) Obese PCOS (27) 0.086 ± 0.002 Obese PCOS Obese Control 0.000 (HS) Non-Obese PCOS 0.931(N S) Obese control (14) 0.225 ± 0.009 Non-Obese Control 0.000 (HS) Non-obese PCOS (27) 0.084 ± 0.002 Obese Control Non-Obese PCOS 0.000 (HS) Non-obese Control (14) 0.236 ± 0.009 Non-Obese Control 0.837 (NS) Non-Obese PCOS Non-Obese Control 0.000 (HS) (*) NS: Non Significant (P> 0.05); HS: Highly Significant (P<0.01); S: Significant (P<0.05) Correlation studies Pearson´s correlation was conducted to study the association of the alterations in the levels of the measured elements, with some of the hormonal changes occurring in PCOS. The results are shown in Table 4. Table (4): Pearson´s correlation of the measured elements and heavy metals with the hormone levels in the obese PCOS and non-obese PCOS groups Groups Hormones Correlation and Significant Elements and heavy metals Pb Cd Mg Ca Mn Non-obese PCOS FSH r -0.145 0.271 -0.180 0.089 0.093 p 0.471 0.171 0.368 0.660 0.643 LH r 0.344 0.066 -0.43* -0.012 0.267 p 0.079 0.742 0.027 0.954 0.642 LH\FSH r 0.366 -0.202 -0.078 -0.065 0.310 p 0.061 0.313 0.698 0.748 0.116 TSH r 0.384* -0.138 0.262 -0.028 0.245 p 0.048 0.494 0.187 0.891 0.218 Prolactin r -0.005 -0.202 0.053 0.066 -0.080 p 0.981 0.312 0.846 0.744 0.690 Testosterone r 0.109 0.064 0.203 0.168 0.045 p 0.589 0.751 0.309 0.402 0.824 FBS r 0.061 0.280 -0.141 0.057 -0.070 p 0.761 0.157 0.482 0.780 0.728 Obese PCOS FSH r 0.084 -0.118 0.141 -0.233 -0.130 p 0.677 0.559 0.483 0.241 0.518 LH r 0.012 -0.063 0.107 -0.014 0.202 p 0.953 0.756 0.597 0.945 0.313 LH\FSH r -0.009 -0.046 0.008 0.152 0.171 p 0.966 0.821 0.970 0.448 0.393 TSH r 0.122 -0.130 -0.143 -0.044 0.090 p 0.544 0.519 0.478 0.826 0.654 Prolactin r 0.254 0.090 0.023 0.632 -0.371 p 0.201 0.657 0.908 0.743 -0.130 Testosterone r -0.070 0.465 * -0.123 0.330 0.004 p 0.727 0.015 0.540 0.093 0.986 FBS r -0.207 -0.260 0.212 -0.060 0.078 p 0.299 0.191 0.288 0.767 0.699 *Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level. Iraqi J Pharm Sci, Vol.26(2) 2017 Blood. levels of trace elements and heavy metals in PCO 92 There was positive correlation between blood lead and serum TSH levels (r 0.384, p 0.048) in non-obese PCOS women (figure 1). In addition, there was positive correlation between serum total testosterone and cadmium levels (r 0.465, p 0.015) in Obese PCOS Women (figure 2). Furthermore, there was negative correlation between serum magnesium and serum LH levels (r - 0.43, p 0.027) in non-obese PCOS women (figure 3). Figure (1): Correlation between blood Lead (µg/dl) and serum TSH (L/ Im) (r 0.384, p 0.048) in non-obese PCOS women. Figure (2): Correlation between Serum total testosterone (ng/ml) and Cadmium(Cd) (µg/dl) (r 0.465, p 0.015) in Obese PCOS Women. Figure (3): Correlation between serum magnesium (mg/dl) and serum LH (mIU /ml) (r - 0.425, p 0.027) in non-obese PCOS women. Discussion The diagnosis of PCOS in this study was based on Rotterdam criteria that are confirmed by the results obtained from analysis data related to the measured hormones (25). Trace elements and heavy metals might be involved in the development of PCOS (9). The present study was designed to investigate serum heavy metal, manganese and some macroelement concentrations in relation to hormone levels in PCOS. In table 3, lead (Pb) levels were significantly higher in PCOS patients than in the control groups. And there was a positive correlation between blood lead concentration and serum TSH level in non- obese PCOS patients (r 0.384, p 0.048). This occurs in accordance with Singh et al. who has declared that, an increase in serum TSH levels was associated with exposure to lead, without alteration in serum T3 and T4; and proposed that lead may enhance pituitary TSH release (26). Chang et al. has investigated the relationship between lead exposure and the risk of infertility in Taiwan women, reporting mean blood lead levels of 35.5 and 27.8 μg/L in infertile women and pregnant women, respectively. The blood lead level was significantly higher in infertile women (27). The pathogenic effect of lead is multifactorial since it directly hinders the activity of enzymes, competitively inhibits absorption of important trace minerals and deactivates antioxidant sulfhydryl pools. Free radical-induced damage by lead is accomplished by two independent, although related mechanisms. The first involves the direct formation of ROS including singlet oxygen, hydrogen peroxides, and hydro peroxides, and the second mechanism is achieved by depletion of the cellular antioxidant pool. Interrelations between these two mechanisms exist so that the increase in ROS on one side simultaneously leads to depletion of antioxidant pools on the other (28). Table 3 also showed that, there was significantly higher blood concentration of cadmium in PCOS patients than in controls, and there was a positive correlation between blood concentration of cadmium and serum total testosterone levels, which is one of the main characteristics of PCOS, in obese PCOS patients (r 0.465, p 0.015). The toxic mechanisms of cadmium are not well understood, but it is known to act intracellularly, mainly via free radical-induced damage, particularly to reproductive organs (28). As with lead, the higher cadmium levels in PCOS may contributes to the creation of ROS. The possible mechanism explaining the role of cadmium in free radical generation may involve displacement of copper and iron by Iraqi J Pharm Sci, Vol.26(2) 2017 Blood. levels of trace elements and heavy metals in PCO 93 cadmium from binding protein, the displaced copper can catalyze breakdown of hydrogen peroxide via the Fenton reaction (28). Excessive generation of ROS is documented in patients with PCOS (6). Therefore, lead (Pb) and cadmium (Cd) may have a significant role in the pathogenesis of PCOS linked to the oxidative stress. Calcium, is a basic element in the human skeletal system, maintains body growth and is involved in muscle activity and neurotransmitter release. Both of the obese and non-obese PCOS patients in this study, had significantly lower levels of serum calcium than in the control subjects as can be seen in the table 3. Similar findings were shown by Li M et al (29). Another study has showed that serum calcium levels are low in PCOS patients, and these low levels can cause atherosclerosis (30). Mazloomi et al. has reported that calcium positively affects PCOS (31). In addition, a clinical study has found that calcium and vitamin D supplements are effective medicines for PCOS (32). Concerning magnesium, the results indicated a significantly lower levels, in both of the obese and non-obese PCOS patients, than the levels in the control groups, as can be seen in table 3. Usharani et al. has revealed a lower level of serum magnesium in PCOS patients (33). Hypomagnesemia alters glucose entry into the cells leading to insulin resistance, thus, dysregulation of the hypothalamus-pituitary- gonadal axis (34). The serum LH level was inversely correlated with magnesium levels in non-obese PCOS patients. Fantidis et al. agrees with our result, by showing that low magnesium level in rats was associated with increased release of LH hormone (34). Table 3 showed that, serum manganese levels in both of the obese and non-obese PCOS patients, were significantly lower than that in the corresponding control subjects. Manganese is a cofactor for some metalloenzymes, like manganese-containing superoxide dismutase SOD (MnSOD). 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