In this prospective study, women presenting between January 2017 to July 2018 with symptoms suggestive of PCOS such as oligomenorrhea and/or clinical hyperandrogenism and/or ultrasonography indicative of PCOM were informed about the study. Those who expressed desire and signed an informed consent were enrolled in the study. A total of 188 pre-menopausal women belonging to the age group of 18 to 35 years were recruited. The study was conducted as per Helsinki Declaration, and was approved by Institutional Ethics Committee.
Study subjects
Cases: A total of 113 cases (aged 18-35 years) with PCOS were enrolled in the current study. PCOS was defined when at least two of the three components were present, according to the Rotterdam criteria. Clinical hyperandrogenism (HA) was defined by a modified Ferriman and Gallwey (FG) score of greater than 8 with or without presence of acne and androgenic alopecia. Biochemical HA was defined as an elevation of serum total testosterone levels above 97.5 percentile limits, (i.e. >0.45 ng/ mL or 2.1 nmol/L).
Controls: Age-matched healthy women (n=75) with regular menstrual cycles (cycle length of 25-35 days) with no evidence of hirsutism, acne, alopecia or endocrine dysfunction and PCOM on trans-abdominal ultrasonography were taken as controls. Subjects were recruited as a part of community outreach programs conducted by the department of Clinical Research periodically.
Clinical assessment and anthropometric evaluation
A pre-validated questionnaire was administered to all the subjects that included recording of medical facts such as menstrual cyclicity (age of menarche, cycle interval, cycle length), weight trajectory, symptoms of hyperandrogenism (unwanted male pattern hair growth, moderate to severe or treatment resistant acne vulgaris, androgenic alopecia), infertility etc. Oligomenorrhea was defined as inter-menstrual interval of >35 days or less than 8 cycles per year while as no menstrual cycle for >6 months was labeled as amenorrhea and >12 cycles per year was defined as polymenorrhea. Details of medications if any, including those of various health events such as clinically significant cardiovascular, liver, lung, neurologic, renal, musculoskeletal dysfunction etc. were recorded and women with these conditions were excluded. Other exclusions included non-classical adrenal hyperplasia (NCAH), Cushing's syndrome, androgen secreting tumors, hyperprolactinemia and thyroid dysfunction.
Besides these, women with known medication history in last six months that included use of one or more drugs like insulin sensitizers, steroids, anti-androgens, oral contraceptives, anti-depressants, lipid-lowering agents, or other drugs known to effect glucose tolerance, insulin sensitivity etc. were also excluded from the study.
All the enrolled women underwent a brief general physical and systemic examination followed by detailed anthropometry -measurement of height (cms), weight (kgs), waist circumference (cms) with calculation of BMI (Kg/m2) using standardized scales (SECA-18, Hamburg, Germany) as a mean of three readings. Waist circumference was measured at the narrowest portion of the torso, approximately midway between the lower costal margin and iliac crest. Blood pressure (systolic and diastolic) mm of Hg was calculated from the mean of three readings using standard calibrated instrument (Omron 714, Kyoto Japan). Evaluation of hirsutism was done by using modified Ferriman-Gallwey (mFG) score by a single observer. The densities of terminal hair over 9 specific sites were scored from 0 to 4 and a total score was calculated. Acne vulgaris was graded as per standard guideline (0= no acne; 1= minor acne; 2= minor acne on face only and 3= severe acne on face, back and or chest) and androgenic alopecia was graded from I-III as per Ludwig scale as was acanthosis nigricans (1-3 as per severity).
Laboratory Evaluation
All the women slated for testing were asked to report after an overnight (10-12hr) fast at 8:00 hours on a day arranged in such a way that the samples for T4, TSH, LH, FSH, prolactin (PRL), serum total testosterone, cortisol (morning), 17-hydroxyprogesterone (17-OHP) and serum AMH were collected from days 2nd to 7th of spontaneous or medroxyprogesterone-induced menstrual cycle. Oral glucose tolerance test (OGTT) was performed using venous blood samples obtained at baseline (0 min), 60 minutes and 120 minutes after an oral load of 75gm anhydrous glucose dissolved in 200-300 ml of water. Aliquots for hemogram, LFT, KFT, calcium, phosphorous, uric acid and lipid profile were collected from the basal sample. ACTH-stimulated 17-OHP or overnight dexamethasone-suppressed cortisol test was done to rule out NCAH or Cushing’s syndrome after the baseline, wherever necessary. Trans-abdominal and transvaginal ultrasonography (wherever appropriate) was performed using GE LOGIQP5 PRO USG (Chicago, USA) equipment to demonstrate any evidence of PCOM described in literature[31, 32] i.e. presence of 10 or more peripheral follicles each measuring 2-8mm in size with echogenic ovarian stroma and/or increased (>10 ml) ovarian volume in all subjects by a single observer following a common SOP.
Assays
Sample for PRL, TSH, T3, T4, LH, FSH and total testosterone were assayed using a commercial electrochemiluminescent immunoassay using (COBAS e-411, Roche Diagnostics, Indiana, USA). Serum AMH levels were assayed using the ELISA kits (Ansh Labs 445 Medical Centre Blvd, U.S.A). Plasma glucose and other parameters-uric acid, lipids, calcium, phosphorous, LFT, KFT etc. were estimated on a fully automated biochemistry analyzer (Respons®910, DiaSys, Holzheim, Germany). The inter and intra assay coefficients of variation wee according to manufacturer’s protocol.
Statistical Analysis
SPSS version 26.0 was used to analyze the date and its was expressed as Mean±SD. Group differences were compared using the Chi-square test for categorical variables and the’t’ test for continuous variables. For comparing continuous variables ANOVA with f-test was used. Cut-off values of AMH levels as a predictor of the diagnosis of PCOS were analyzed using a Receiver Operating Characteristic (ROC) procedure. Pearson correlation was used to analyze the association of serum AMH levels with various clinical, hormonal and ultrasonographic parameters. The p-value < 0.05 was considered statistically significant.