Study population and sample collection
In the present study, a total of 216 women comprising 111 PCOS patients and 105 controls were recruited from the Endocrinology Clinic of Valiasr Hospital and Gynecology Clinic of Mousavi Hospital, Zanjan, Iran. According to the Rotterdam consensus, PCOS is characterized by two out of three of the following: clinical and/or biochemical signs of hyperandrogenism, polycystic ovaries on sonography, and oligo−/anovulation [21]. Patients with inflammatory diseases, acute or chronic infections, Cushing’s syndrome, and androgen-secreting tumors were excluded. The selected participants had not taken any hormonal or anti-inflammatory medicine for 3–6 months before entering the study. All subjects were new cases for PCOS.
This study was approved by ethics committee (No. ZUMS.REC.1394.90) of Zanjan University of Medical Sciences of Iran. Written informed consent was received from all the subjects before blood sampling.
For sample size calculation, we conducted a pilot study including 20 PCOS patients and 20 healthy individuals. After completion of the experiment, 25% of PCOS cases and 10% of healthy control were carrier. Sample size was calculated 100 per group based on P1 = 0.10, P2 = 0.25, α = 0.05, β = 0.20 using the formula of comparing two proportions.
Blood specimens were collected from subjects in two separate tubes on days 3–5 of their menstrual period, following the World Health Organization (WHO) guidelines. Anticoagulated whole blood samples were taken for DNA extraction and serum for biochemical parameters. Serum samples were kept at − 20 °C until determination of the biochemical parameters. Genomic DNA was extracted from white blood cells using the Bioneer genomic DNA extraction kit (Bioneer, Korea, Cat. No. K-3032). The DNA quality was determined with 260/280 optical density (OD) ratios in all samples, which were stored at − 20 °C until use.
Genotyping
Polymerase chain reaction (PCR)
In this study, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used for genotyping of G238A (rs361525) in the promoter region of the TNF-α gene. Semi-nested PCR was performed using two pairs of primers with the following sequences: forward, 5′-AGGAAACAGACCACAGACC-3′; reverse, 5′-ATCTGGAGGAAGCGGTAGTGG-3′. These were used in the first PCR reaction. The PCR product size was 264 bp. The primers for the second PCR reaction were 5′-GAAGACCCCCCTCGGAACC-3′ (forward) and 5′ATCTGGAGGAAGCGGTAGTGG-3′ (reverse), with a product size of 151 bp. The restriction site was designed to be situated on the forward primer in the second PCR reaction (Fig. 1).
PCR was performed according to the manufacturer’s protocol (Ampliqon, Denmark; PCR Master Mix 2× containing Taq DNA polymerase, buffer, MgCl2, and dNTP). DNA template (200 ng) and 10 μmol/L of each primer were added to the PCR reaction mix (25 μL). Amplification was carried out with a thermal cycler (Flex Cycler2, Germany) under the following conditions: for the first PCR, an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 92 °C for 30 s, annealing at 62 °C for 30 s, and an extension at 72 °C for 45 s. For the second PCR, the initial denaturation took place at 95 °C for 5 min, followed by 40 cycles of denaturation at 92 °C for 30 s, annealing for 63 °C for 30 s, and an extension at 72 °C for 45 s. Finally, both PCRs were followed by a final extension at 72 °C for 5 min. The PCR products were separated using 2.5% agarose (Invitrogen, USA) gel electrophoresis and visualized with an ultraviolet transilluminator after staining with DNA safe stain (EURx, Poland).
Restriction fragment length polymorphism (RFLP)
Products of the first PCR were used as a template for the second PCR, so digestion was performed on the products of the second PCR (151 bp) using 0.5 units of HpaII restriction endonuclease (CinnaGen, RD1171), following the manufacturer’s recommendations. Briefly, the reaction was incubated at 37 °C for 6 h. Digested fragments were separated on 3% agarose gel by electrophoresis. Fragments of 133 bp and 18 bp were considered to represent the homozygous GG genotype, while segments of 151 bp, 133 bp, and 18 bp represented the heterozygous GA genotype. A single band of a 151-bp fragment was considered a homozygous AA genotype.
Clinical and biochemical parameter measurement
Waist and hip circumferences, body weight, and height were measured in all subjects as anthropometric variables. The body mass index (BMI) and waist–hip ratio (WHR) were calculated as follows:
BMI = body mass/ (height)2[kg/m2],
WHR = waist circumference (cm)/hip circumference (cm).
For measurement of the serum levels of follicle-stimulating hormone (FSH; Monobind kit, USA), luteinizing hormone (LH; Monobind kit, USA), testosterone (Monobind kit, USA), estrogen (Monobind kit, USA), insulin (Monobind kit, USA), and TNF-α (eBioscience, Austria), the enzyme-linked immunosorbent assay (ELISA) method was used according to the manufacturer’s recommendations. Color intensities at the final step were recorded using an ELISA reader (Stat Fax-2100 microplate reader, Awareness Technology, USA). The biochemical parameters including fasting blood glucose (Pars azmoon, Iran), triglyceride (Pars azmoon, Iran), total cholesterol (Pars azmoon, Iran), low-density lipoprotein (LDL; Pars azmoon, Iran), and high-density lipoprotein (HDL; Pars azmoon, Iran) were measured using a BT3000 autoanalyzer (Biotechnica Instruments, USA). Homeostasis model assessment (HOMA) as an insulin resistance index was computed using the following formula: HOMA = fasting glucose (mg/dl) × fasting insulin (mU/ml)/405.
Statistical analysis
All statistical analyses were performed using SPSS 22.0 (Chicago, IL, USA). Data were tested for normal distribution using the Kolmogorov–Smirnov test. Differences between two variables were measured with an independent sample t-test for normal distributions, while the Mann–Whitney test was used for non-normally distributed data. The qualitative or quantitative results were expressed as the frequency or mean ± SD, respectively.
The association between groups and biochemical factors were evaluated using regression logistic binary test by calculating the odds ratios (OR) at a 95% CI. Differences in serum levels of FSH, LH, testosterone, estrogen, insulin, TNF-α, fasting blood glucose, triglyceride, total cholesterol, LDL, and HDL between the groups were tested using the independent student t-test or Mann–Whitney test, as appropriate. The correlation between continuous variables was assessed using Pearson’s correlation coefficient. A p-value less than 0.05 was considered significant.
Differences in the frequency of the alleles and genotypes between the PCOS patients and age-matched healthy subjects were tested using Chi-square tests. The Hardy–Weinberg equilibrium (HWE) was estimated using the Chi-square test.