The study protocol was approved by the medical ethics committee of the Research Institute for Endocrine Sciences (RIES) of I.R.Iran. Written informed consent was obtained from all participants.
A total of 25 cases of PCOS, based on the Rotterdam criteria (23, 24) and aged 18-45 years, who referred to Reproductive Endocrinology Research Center diagnosis, were recruited for the purpose of the present study. These women had at least two of the following criteria: i: / oligo/anovulation (defined as either regular or irregular menstruation ≥ 34 days or history of eight or fewer menstrual cycles in a year); ii: / clinical symptoms of hyperandrogenism (including hirsutism diagnosed based on a standardized scoring system of modified Ferriman-Gallwey ≥ 8, acne and androgenic alopecia) or biochemical hyperandrogenism ,defined as the increase of one or more serum level of androgens ( testosterone or androstenedione ) to> 95th percentile, presented in the selected healthy non-hirsute eumenorrheic women of the study population) (25); iii:/ polycystic ovaries (polycystic ovaries with >12 follicles /ovary, 2–9 mm in diameter and/or increased ovarian volume (10 cm3)) and exclusion of other etiologies. A total of 25 non hirsute eumenorrheic healthy age and BMI-matched women, aged 18-45 years, were selected as controls.
All women with histories of cancers, primary or acquired immunodeficiency, suffering from infectious diseases and systemic disorders were exclude. We also excluded women with a family history of cancer, those with early menopause (occurring at or before age 45 years) (26), and substance dependents (addiction). Characteristics of the study groups are presented in table 1.There were no statistically significant differences in baseline characteristics of the participants.
In this study we evaluated the proliferative response of lymphocytes as the mean proliferation score (presented through absorbance), measured of TNF-α concentration using a standard graph and determined percentage of the CD3+ CD8+ lymphocytes as the main effectors cell population.
Tumor cell line culture
Two human breast cancer cell lines (MCF-7, MDA-MB-468) were purchased from the Cell Bank of Institute Pasteur Karaj, Iran. Cell lines were cultured in RPMI-1640 (Gibco, Invitrogen, USA) and DMEM (Gibco, Invitrogen, USA) media supplemented with 10% fetal bovine serum (FBS), 100 u/ml penicillin/streptomycin and (Atocel, Austria) in humidified 5% CO2 incubator at 37°C. After reaching the log phase of growth and 80% confluency in the T25 tissue culture flask, growing cells were detached from the culture bed using trypsin-EDTA solution and divided into 24 well culture plates or maintained at -80°C for future use or as backup data.
Isolation of Peripheral Blood Mononuclear Cells
Heparinized blood samples were taken and diluted with RPMI medium at a ratio of 1:1. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation (Ficoll hypauque 1077 Sigma, USA). The inter phase layer including mononuclear cells was collected and washed 3 times with RPMI medium PBMCs were then suspended in Complete Tissue Medium (CTM) and cell suspension was stained with trypan blue and viability was determined.
Tumor cell lines (5×105cell/well) were pre-seeded in the lower chamber at Transwell 24-well plates (SPL, Korea) in RPMI-1640/DMEM medium supplemented with 10% FBS and incubated overnight in humidified CO2 incubator at 37°C. Subsequently, PBMCs (2×106 cells) were added into the upper insert chamber with a pore size of 0.4 µm (SPL, Korea) at a ratio of E:T (1:4). For an incubation period of 72 h. As a negative control, PBMCs were cultured in the upper chamber of the plates in the absence of tumor cell lines. The samples for cytokines assay were separately created from the supernatants of cancer cell lines and PBMCs cultures in Transwell chambers at two time intervals of 48 and 72 h incubation following co-culture.
Evaluation of proliferative response of Peripheral Blood Mononuclear Cells
The non-radioactive Cell Proliferation assay (colorimetric) ELISA kit, BrdU (Roche Diagnostic, Germany; Ref:11647229001) was used to evaluate PBMCs proliferation rate during co-culture with tumor cell lines as well as in the absence of tumor cell lines as controls at two time intervals. The BrdU cell proliferation assay was performed according kit instructions. Absorbance was measured at 450 nm with a reference wave length at 690 nm.
Immunophenotyping of Peripheral Blood Mononuclear Cells
To determine the percentages of TCD3+ CD8+ lymphocytes in cultured cells, we used a combination of phycoerythrin-conjugated anti-CD8 (PE, clone SK1), and fluorescein isothiocyanate-conjugated anti-CD3 (FITC; Leu 4 FITC, clone SK7) mAbs (BD Biosciences, Cat No: 340044); Percentages of total mononuclear cells, as well as TCD3+ CD8+ lymphocytes, were evaluated by flow cytometry (FACSCalibur) at two different time intervals.
Measurement of Tumor Necrosis Factor-alpha concentration
TNF-α concentration in the supernatants collected from PBMCs co-cultured with and without tumor cell lines was determined and reported in pg/dL using the sandwich ELISA kit (Quantikine, R&D Systems, USA), for human TNF-α, according to the manufacturer’s instruction, Results were calculated using a standard curve.
All continuous variables were checked for normality using the one-sample Kolmogorov–Smirnoff test and expressed as mean (standard deviation) if variables had a normal distribution, or median with inter-quartile range (IQ25-75) for variables with skewed distribution. Characteristics of study participants were compared between the PCOS and control groups using two independent-sample t-test or the equivalent nonparametric Mann-Whitney U test.; categorical variables, expressed as percentages, were compared using Pearson’s test. Statistical analysis was performed using software package SPSS (version 15) and GraphPad Prism. Significance level was set at P<0.05, and confidence interval (CI) as 95%.