This study was approved by the Institutional Review Board of Inje University Haeundae Paik Hospital (IRB No. 129792-2014-035), and patient’s informed consent in this study was waived by the IRB. All patients were newly diagnosed with PCOS at 18–35 years of age from January 2010 to December 2013 at the above-mentioned university hospital. The patients were diagnosed with PCOS on the basis of the 2003 Rotterdam criteria. All patients who met at least two of the three criteria, including (1) oligo-anovulation, (2) biochemical and/or clinical signs of hyperandrogenism (e.g., hirsutism, acne, androgenic alopecia), and (3) ultrasonographically identified polycystic ovarian morphology (PCOM) of at least 1 ovary, were diagnosed with PCOS after excluding other diseases or etiologies [31]. Using the transvaginal or transrectal ultrasound approach, PCOM was defined as an ovarian volume of over 10 cm³ and/or the presence of over 12 follicles (2–9 mm in size). Pelvic ultrasonography (through the vagina or rectum) for assessing PCOM was conducted in the early follicular phase using a Voluson LOGIQ S7 (GE Ultrasound Korea, Seongnam, Korea) equipped with a transvaginal probe with a frequency range of 3.6–9 MHz, and all ultrasound examinations were conducted by the same reproductive endocrinologist. Oligo-anovulation was estimated on the basis of menstrual history and the presence of amenorrhea or oligomenorrhea. Amenorrhea was defined as a menstrual cycle interval of over 90 days without menstruation, and oligomenorrhea was defined as an interval of over 35 days. The most common clinical sign of hyperandrogenism is the presence of hirsutism, which was identified using a modified Ferriman-Gallwey score >6, based on a previous study of hirsutism in Korean women [32]. Biochemical hyperandrogenism was confirmed by elevated serum androgen concentration beyond the 95% confidence limits in the control group of a previous study (total testosterone >0.68 ng/mL and/or free testosterone >1.72 pg/mL) [9,32]. Patients with a previous history of diagnosed diabetes, thyroid disease, hyperprolactinemia, or ovarian surgery were excluded. Patients who were taking oral contraceptives with or without prescriptions within the last 6 months and anti-diabetic drugs, including insulin sensitizers, were also excluded. All patients were divided into two groups based on the serum E1/E2 ratio: group 1 (E1/E2 ratio <2.0) and group 2 (E1/E2 ratio ≥2.0).

All clinical variables of the study participants were assessed when they first visited the outpatient department. Blood samples for biochemical laboratory analyses were taken from all subjects in the early follicular phase after overnight fasting. Serum E1 was measured using the Dsl-8700 Estrone ELISA kit (Beckman Coulter, Brea, CA, USA) and serum E2 was measured using Elecsys Estradiol II (Roche, Indianapolis, IN, USA) [23]. Serum insulin and glucose levels were analyzed using an Elecsys Insulin assay (Roche) and an L-Type GluI device (Wako, Osaka, Japan), respectively. Cholesterol and triglyceride levels were measured using Pureauto S (Sekisui, Tokyo, Japan), and serum high-density lipoprotein and low-density lipoprotein levels were measured using Cholestest (Sekisui) [30]. Both intra- and inter-assay coefficients of variation for all assays were below 8%.

After overnight fasting, serum glucose and insulin levels were checked. Glucose levels at 60 minutes and 120 minutes after glucose ingestion during a 2-hour 75-g OGTT were measured. The fasting glucose level and postprandial glucose level at 2 hours (PPG2) were analyzed using an L-Type GluI device (Wako, Osaka, Japan). The insulin resistance parameters included HOMA-IR, QUICKI, and GIR. The GIR was calculated by dividing the glucose value (mg/dL) by the insulin value (μU/mL); HOMA-IR was calculated as fasting glucose (mg/dL)×fasting insulin (µU/mL)/405; and QUICKI was calculated as 1/{log[insulin value (μU/mL)]+log[glucose value (mg/dL)]}.

All values are expressed as mean±standard deviation. All statistical analyses were performed using SPSS ver. 18.0 (SPSS Inc., Chicago, IL, USA). The paired t-test was used to compare clinical and biochemical parameters, including hormonal and glucose and insulin metabolism-related parameters, between the two groups categorized by the E1/E2 ratio. The correlations between the serum E1/E2 ratio and insulin resistance-related parameters were analyzed with Pearson correlation coefficients, and partial correlation coefficients were used after adjusting for body mass index (BMI) and the waist-to-hip ratio (WHR). In all analyses, p-values <0.05 were considered to indicate statistical significance.