Study participants and inclusion criteria
This study included 150 fertile women with a diagnosed polycystic ovary syndrome (PCOS). The diagnosis was determined by ESHERE/ASRM criteria . Participants were divided into two groups of 75 participants. The first group (group I, G1) is a control group with de novo diagnosed PCOS, without therapy involved. The second group (group II, G2) consists of patients with PCOS therapy. Metformin 500 mg (2x1) and myoinositol 1000 mg (2x1) are recommended. In the case of HOMA-IR 2.9, the recommended dose of metformin is 3x1. During therapy, patients underwent regular check-ups every 4 months. At the end of the fourth month of therapy (group II), patients were included in the study. Diagnosed PCOS and informed consent of all participants are criteria for inclusion in the study.
Database pre-screening and polycystic ovary syndrome diagnostic criteria
Demographic and clinical parameters for patients in these studies were taken from the database of the Agram Polyclinic in Sarajevo (Bosnia and Herzegovina). Completed blood tests to measure hormone levels, transvaginal ultrasound to imaging of the ovaries, and a thorough personal and family history were required to confirm the PCOS diagnosis.
PCOS was diagnosed using ESHERE/ASRM criteria that require the presence of two out of three criteria (according to the Rotterdam consensus): ovulatory dysfunction, hyperandrogenism, and morphological PCOS detected by ultrasound diagnostics .
Body mass index measurements
Body mass index (BMI) was defined as the ratio of body weight to body height squared, expressed in kg/m2. BMI was determined with a standard BMI calculator (Española). After the measurement, BMI categories were determined according to the manufacturer: underweight ≤ 18.5, normal weight 18.5–24.9, overweight 25–29.9, and obesity = BMI of 30 or greater.
Insulin resistance measurements
A homeostatic model (HOMA-IR) was used to assess insulin resistance. HOMA-IR is calculated as HOMA-IR = insulin (mU/L) × glucose (mg/dL). The homeostatic model calculates beta-cell function (% B) and insulin sensitivity (% S) as a percentage. A healthy range is 0.5–1.4. Less than 1.0 means the patient is insulin-sensitive, above 1.9 indicates early insulin resistance, and above 2.9 indicates significant insulin resistance.
Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and LH/FSH ratio were measured when considering a PCOS diagnosis. Electrochemiluminescence immunoassay (ECLIA) was used for FSH analysis. The Elecsys LH assay employs two monoclonal antibodies specifically directed against human LH . The results were measured on a calibration curve using a Cobas e 411 analyzer (Roche Diagnostics, Germany).
Elevated LH/FSH ratio
The luteinizing hormone/follicle-stimulating hormone (LH/FSH) ratio is often requested to help diagnose PCOS despite a recent consensus recommending against its use. In healthy women, the ratio between LH and FSH usually lies between 1 and 2, and in PCOS women, this ratio becomes reversed, and it might reach as high as 2 or 3 .
Glucose levels and insulin profile
Serum glucose levels were measured using the enzymatic UV method with hexokinase. Glucose-6-phosphate formed from glucose and ATP by hexokinase is oxidized by NAD in a reaction catalyzed by glucose-6-phosphate dehydrogenase to create NADH, which is quantitated spectrophotometrically at 340 nm. An Olympus AU400 automated chemistry analyzer was used to measure serum glucose levels and commercial kits (Roche, Manheim, Germany)
Serum insulin level was measured using commercial kits and an electrochemiluminiscence device (Elicsys, Roche Diagnostics). The method is based on the binding of insulin to a specific monoclonal antibody labeled with ruthenium with the addition of streptavidin-coated microparticles. The application of voltage to the electrodes induces chemiluminescent emission measured by a photomultiplier.
Glucose and insulin levels were measured in venous blood on an empty stomach and the second evaluation 120 min after taking 75 g of glucose.
HDL and LDL cholesterol measurements
Women with PCOS have a greater tendency to have high cholesterol (lower levels of high-density lipoproteins or HDL and higher levels of low-density lipoproteins or LDL) . Besides, triglyceride levels, another component of cholesterol, tend to be high in women with PCOS. Cholesterol esterase is mainly used in clinical studies to determine the level of cholesterol in human blood. The determination is based on the method of Allain et al.  that monitors generated free cholesterol in the reaction catalyzed by cholesterol oxidase. Color intensity is directly proportional to the amount of cholesterol, with maximum absorption at the wavelength of 500 nm.
Enzymatic hydrolysis of serum triglycerides by lipoprotein lipase generates free fatty acids and glycerol, with an absorbance maximum at 540 nm.
HDL and LDL cholesterol are measured directly in the serum. Sulfated alpha-cyclodextrin in the presence of Mg2+, which forms complexes with apoB. This complex contains lipoproteins and polyethylene glycol-coupled cholesterol esterase and cholesterol oxidase to measure HDL-cholesterol. LDL-cholesterol is calculated from measured values of total cholesterol, triglycerides, and HDL-cholesterol according to the relationship:
LDL-chol = total chol − HDL-chol − [TG]/5
Where [TG]/5 is an estimate of VLDL-cholesterol, and all values are expressed in mmol/L.
Cholesterol components and triglycerides were analyzed using an Olympus AU400 analyzer.
Statistical analysis of serum constituents was performed by variance analysis (ANOVA) using the IBM SPSS (Version 20.0, SPSS, Inc., Chicago, IL, USA). Differences between means were determined by a range test (p<0.01). The Pearson correlation coefficient is a measure of the strength of the linear association between two variables.