In this study, we studied 98 PCOS patients and 93 healthy controls. All researchs with human samples were done with written informed consent of the patients and with approval of the ethical committee of the Ege Universty Hospital. The patients had been referred to the Endocrinology and Metabolism Disease outpatient clinic at the Ege University Hospital. PCOS was defined by the Rotterdam PCOS consensus criteria [¹⁴]. Patients who had DM, hyperprolactinemia, congenital adrenal hyperplasia (diagnosed with the adrenocorticotropic hormone stimulation test), thyroid disorders, Cushing's syndrome, hypertension, hepatic or renal dysfunction were excluded from the study. Confounding medications, including oral contraceptive agents, hypertensive medications and insulin-sensitizing drugs, and those which may affect the metabolic criteria were questioned. Another 93 healthy young volunteer females matched for age, body mass index (BMI), and allele frequency were included from the study, and considered as the control group. Their health state was determined by medical history, physical and pelvic examination, and complete blood chemistry. The patients with PCOS and the control group were genetically unrelated.

At study entry, all subjects underwent venous blood drawing for complete hormonal assays, lipid profile, glucose, insulin and ABCA genetic study. All blood samples were obtained in the morning between 08.00 and 09.00 hours after an overnight fasting, and resting in bed during early follicular phase of the spontaneous or P-induced menstrual cycle. During the same visit, all subjects underwent anthropometric measurements including BMI and detail history, systolic and diastolic blood pressure. In present study, data related to the serum malondialdehyde, nitric oxide and disulfide levels, homocystein and fibrinogen levels and the genetic evaluation of ABCA will be shown and discussed.

Serum total cholesterol, LDL and HDL cholesterol were measured by Olympus AU 2700 automated analyzer. Plasma insulin concentrations were determined by Immunolite 2000 using two-site chemiluminescent immunometric assay.

All reagents were purchased from Sigma and Merck. MDA was determined by a modified spectrophometric method of Yagi K [¹⁵] using tetrametoxypropan as Standard and BioTek MicroQuant microplate reader. NO was determined by measuring stable NO end-products-nitrite and nitrate levels using Miranda's spectrophometric method [¹⁶], while total sulfhydryl groups was measured using Ellman's reagent by Sedlak and Lindsay's method [¹⁷].

Venous blood samples were centrifuged at 1000× g for 10 min and the serums were stored at -80°C until the analysis -not exceeding three months. In this study, serum total homocysteine levels were measured by Fluorescence Polarization Immunoassay Method (IMX Homocysteine Assay, Abbott Diagnostics No: 7D29-20). Dilution method was used for those whose serum homocysteine levels were higher than 50 μmol/L. All samples were prepared as 200 μl. Three controls were used in each sample for calibration of the device. For each of the controls, the results including ranges were accepted as low control (5, 25-8, 75), medium control (10.0-15.0), and high control (20.0-30.0) μmol/ml.

DNA isolations were carried out by using High Pure PCR Template Preparation Kit (Roche Applied Science, Germany) from peripheral blood samples of control and study group cases taken to tubes with EDTA.

For ABCA 1 G1051A polymorphism analysis, by using Forward Primer: 5'-CTC CAA AAGACT TCA AGG ACC C-3', Reverse Primer: 5'-GGC CCA AAA GTC TGA AAG AAC AC-3' primer pair, a DNA fragment of 433 base pairs is amplified by PCR method. For each sample, 25 μl of prepared PCR reaction mixture contains 16, 75 μl of sterile distilled water, 0,5 μl of Forward primer (100 p μl), 0,5 μl of Reverse primer (100 p μl), 2,5 μl Mg⁺² (25 mM), 2 μl of dNTP mixture (2 mM), 0,25 μl of Taq DNA polymerase (5 U/μl), and 2,5 μl of genomic DNA.

Denaturation is carried out at 95°C for 2 minutes, amplification at 94°C for 30 seconds as 35 cycles, 1 minute at 72°C, prolongation at 72°C for 7 minutes by applying PCR protocol.

For ABCA1 G2706A polymorphism analysis, DNA fragment of 350 base pairs is amplified by using Forward Primer: 5'-CAA GTG AGT GCT TGG GAT TG-3', Reverse Primer: 5'-TGC TTT TAT TCA GGG ACT CCA-3' primer pairs by PCR method. For each sample, 25 μl of PCR reaction mixture contains 17, 25 μl of sterile distilled water, 0,5 μl of Forward primer (100 p μl), 0,5 μl of Reverse primer (100 p μl), 2,5 μl Mg⁺² (25 mM), 1,5 μl of dNTP mixture (2 mM), 0,25 μl of Taq DNA polymerase (5 U/μl), and 2,5 μl of genomic DNA.

Denaturation is carried out for ten minutes at 94°, amplification for 30 seconds at 94°C as 40 cycles, for 45 seconds at 52°C, 1 minutes at 72°C, prolongation for 7 minutes at 72°C by applying PCR protocol. The size of the products obtained after PCR is controlled at gel electrophoresis. PCR products made for the analysis of ABCA 1 G1051A and G2706A gene polymorphisms are evaluated in agarose gel of 2%.

G1051A polymorphism analysis of -ABCA 1 gene is carried out at 37°C for 6 hours by incubating RFLP reaction mixture of 30 μl containing 17 μl of distilled water, 10 μl of PCR product, 2 μl of Buffer O, and 1 μl of EcoO109I enzyme.

G2706A polymorphism analysis of -ABCA 1 gene is carried out by incubating at 30°C for 3 hours the mixture of RFLP reaction of 30 μl containing 17 μl of distilled water, 10 μl of PCR product, 2 μl of Buffer O, and 1 μl of BsaAI enzyme.

Genotyping is carried out after having conducted and displayed the products obtained as a result of enzyme cut for G1051A polymorphism analysis of ABCA 1 gene in agarose gel of 2% and in agarose gel of 3% for G2706A polymorphism analysis.

As a result of G1051A polymorphism analysis, DNA fragments of sizes of 189 base pairs, 131 base pairs, and 113 base pairs in cases of wild genotype, 320 base pairs, 189 base pairs, 131 and 113 base pairs in heterozygote cases, and 320 base pairs and 113 base pairs in mutant cases are obtained (Figure 1). As a result of G2706A polymorphism analysis, DNA fragments of sizes of 252 base pairs and 98 base pairs in cases of wild genotype, 350 base pairs, 252 base pairs and 98 base pairs in heterozygote cases, and 350 base pairs in mutant cases are obtained (Figure 2).

The demographic, hormonal and metabolic parameters of the PCOS and control groups are shown in Table 1. In the PCOS group fasting glucose, DHEAS, 17-OHP, free testosterone, total-cholesterol, triglyceride, LDL-cholesterol and fibrinogen were significantly (P < 0.05) different in comparison with healthy women (Table 1). The fasting glucose levels were significantly (P < 0.05) higher in PCOS than in control women, whereas no difference was observed in fasting insulin concentrations between the groups (Table 1). No significant differences were detected in age, weight, BMI, homocystein, MDA, NO, SH, estradiol, DHEAS, prolactin, HDL-cholesterol levels between two groups (Table 1).

The frequency of ABCA G1051A and G2706A polymorphism are shown in Table 2. The allelic distribution of ABCA genotypes was in Hardy-Weinberg equilibrium for both groups of women.

The genotype ABCA G1051A distribution didn't differ between the control group (GG 31.2%, AG 53.8%, AA 15.1%) and the PCOS patients (GG 36.6%, AG 52.7%, AA 10.8%) (P > 0.05) The genotype ABCA G2706A distribution differed between the control group (GG 60.7%, GA 32.1%, AA 7.1%) and the PCOS patients (GG 8.7%, GA 8.7%, AA 76.8%) (P < 0.05). The frequency of the polymorphic A allele (ABCA G2706A) was higher in PCOS patients than control group with 13,0% and 23,2%, respectively (p = 0.027, OR:2.016; Table 2).

There was no statistically significant difference in PCOS patients between ABCA G1051A genotypes (AA, GA and GG) and BMI, fasting insulin, fasting glucose, triglyceride levels, HDL levels, LDL levels, fasting blood glucose levels, f-testosterone, fibrinogen and 17-OHP levels (p > 0.05; Table 3). The fasting insulin level was significantly higher in PCOS patients with ABCA G1051A mutant genotype than those with heterozygote and wild genotypes (Table 3). No statistically meaningful difference was determined between polymorphism and lipid and other parameter levels in patients carrying ABCA1 G2706A polymorphism (Table 4).

In our study, the homocystein levels were significantly higher in PCOS patients with ABCA G1051A mutant genotype than those with heterozygote and wild genotypes (Table 3). The ABCA genotypes do not appear to have significant correlation with the plasma Hcy levels, MDA, NO and SH in PCOS patients.