2.4 Measurement of circulating hormonal, metabolic and inflammatory biomarkers

Serum hormones levels, lipid profile and fasting glucose were analyzed using enzymatic colorimetric methods. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, total cholesterol (TC), high-density lipoprotein (HDL-c), triglycerides (TG) and high sensitivity C-reactive protein (hs-CRP) were measured using a COBAS 6000 automated analyzer (Roche Diagnostics, Mannheim–Germany), following the manufacture instructions. The concentrations of low-density (LDL-c) and very low-density lipoproteins (VLDL-c) were calculated through the Friedwald formula (VLDL = TG/ 5; LDL-c = TC–HDL-c–VLDL-c) for TG values of up to 4.5 mmol/L [16–17]. Circulating leptin and iterleukin(IL)-6 levels were estimated using enzyme-linked immunosorbent assay (Quantikine^® ELISA kits, R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacture instructions. All experiments were conducted in duplicate.