The study was approved by the Institutional Review Board of the Department of Obstetrics and Gynecology, University "Magna Graecia" of Catanzaro, Italy. The purpose of the protocol was explained carefully to all the patients and written consent was obtained before the study began.
Twenty young normal weight patients with PCOS who had received metformin treatment to induce ovulation and, then, scheduled for laparoscopy were enrolled at our Academic Centre of Reproductive Medicine and Surgery between October 2001 and February 2010, and studied as cases. The majority of the subjects had participated in our earlier studies [19, 23].
All patients with PCOS had received the same metformin regimen (two 850 mg tablets daily) for one year. On the basis of the response to treatment received, cases were distinguished according to ovarian response to metfomin into two groups (Met-anov and Met-ov groups). Specifically, Met-anov group (n. 10) was composed of PCOS patients who remained anovulatory despite treatment, and Met-ov group (n. 10) included PCOS women who resulted normally cycled under metformin treatment (for at least six cycles) but had failed to conceive.
According to our Institutional guidelines, subjects from the Met-anov group were scheduled for ovarian drilling procedure, whereas subjects from the Met-ov group were scheduled for diagnostic laparoscopy in order to exclude potential infertility/subfertility factors.
Other 20 patients were enrolled as controls. Of them, 10 were untreated patients with PCOS [24, 25], affected by uterine fibroids and scheduled for laparoscopic myomectomy (PCOS controls), whereas other 10 normally cycled women were scheduled for diagnostic laparoscopy because they referred chronic pelvic pain (non-PCOS controls).
In PCOS patients, PCOS diagnosis was based initially on the presence of both chronic anovulation and clinical and/or biochemical hyperandrogenism [25], even if all patients with PCOS originally had bilateral polycystic ovaries (PCO) [24]. In healthy controls, ovulatory cycles were confirmed by biochemistry, and clinical and/or biochemical hyperandrogenism and PCO were systematically excluded.
Were considered exclusion criteria for all subjects: an age less than 18 or greater than 35 years; a body mass index (BMI, kg/m2) less than 18 or greater than 25; major medical disorders and/or current or previous use of hormonal and/or metabolic drugs; tubal or male factor infertility or sub-fertility investigated with hysterosalpingography and standard semen analysis, respectively (Male Infertility Best Practice Policy Committee of the American Urological Association, 2006; Practice Committee of the American Society for Reproductive Medicine 2006); any organic pelvic diseases at laparoscopy or diseases potentially affecting the ovarian environment and/or function (including endometriosis, leiomyomas, and so on); and the intention to start a diet or a specific programme of physical activity. In addition, subjects with dominant follicle(s) (follicles with a diameter equal or higher than 10 mm) and/or with persistent corpora lutea and/or functional cysts at transvaginal ultrasound performed before surgery were excluded. Clinical, biochemical, and ultrasonographic parameters at baseline or before metformin administration were acquired retrospectively, whereas all other data were evaluated prospectively at the hospital admission.
Clinical evaluation, blood sampling, transvaginal ultrasonography, and laparoscopy were performed in each subject. Clinical evaluation consisted of gynecological examination, anthropometric measurements and Ferriman-Gallwey score calculation. Biochemical assessment consisted of complete hormonal, including evaluation of serum follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), prolactin (PRL), estradiol (E2), P, 17-OH-progesterone (17-OHP), total testosterone (T), androstenedione (A), dehydroepiandrosterone sulfate (DHEAS), and sex-hormone binding globulin (SHBG)], and metabolic evaluation, including evaluation of fasting glucose and insulin levels. Insulin resistance was evaluated using the homeostasis model analysis (HOMA) [fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5] and the fasting glucose-to-insulin ratio (GIR, mg/10-4U). The free androgen index (FAI) [T (nmol/l)/SHBG × 100] was also calculated for each participant.
Serum and follicular fluid AMH levels were assessed by using a second generation enzyme immunoassay (AMH-EIA kit; Immunotech A Beckman Coulter Company, Marseilles, France), according to the supplier's instructions. The intra-assay and inter-assay coefficients of variation (CV) for each biochemical or hormonal parameter were evaluated, and the values of the CVs ranged from 1.2 to 5.8%.
Finally, the ovarian dimensions, volume and morphology and the number of antral follicles (follicular diameter ranged from 2 to 9 mm) were evaluated bilaterally by transvaginal ultrasonography. The antral follicle number per ovary, defined as the average for the total number of antral follicles counted from both ovaries, was also calculated.
All laparoscopic interventions were performed by the same experienced operator (F.Z.) during the early follicular phase for ovulatory subjects and randomly in anovulatory patients. Firstly, the antral follicles on the ovarian surface were visualized and each one was aspirated with a 1 mL syringe and a 26 gauge needle. Follicular fluid of antral follicles was collected from both ovaries in each patient, it was transferred to the laboratory on dry ice, and purified from the granulosa cells. Thereafter, the remaining follicular fluid was centrifuged, and the supernatant was stored at -20°C until it underwent biochemical analysis.
As scheduled, ovarian diathermy and myomectomy were performed in Met-anov and PCOS control group, respectively.
Statistical analysis
Continuous variables were tested for normality using the Kolmogrov-Smirnov test resulting normally distributed and were expressed as the mean ± standard deviation (SD).
Data were analyzed with one-way analysis of variance (ANOVA) and ANOVA for repeated measures, and the Bonferroni test was used for post-hoc analysis.
For categorical variables, the Pearson chi-square test was performed; Fisher's exact test was used for the frequency tables when more than 20% of the expected values were lower than five.
A simple linear regression analysis was used to establish the relationships between the AMH in the follicular fluid, and the variation (Δ) in plasma T levels (ΔT), HOMA (ΔHOMA), and AMH (ΔAMH). A bivariate two-tailed correlation analysis was performed by calculating the Spearman's coefficient (Spearman's rho, r), and the significance of the correlation was set at the 0.05 level.
The level of statistical significance was set at p < 0.05 for all statistical analyses. The Statistics Package for Social Sciences (SPSS 14.0.1, 18 Nov 2005; SPSS Inc., Chicago, IL) was used for all calculations.