Between December 2015 and June 2016, infertile women who were eligible for IVF/intracytoplasmic sperm injection (ICSI) treatment at the Reproductive Medicine Centre of Tianjin Central Hospital of Obstetrics and Gynecology were examined for eligibility and those that consented to participate in the present study were recruited. The study was approved by the hospital's Institutional Review Board (approval no. TJCOB2016002) and was performed in accordance with the Declaration of Helsinki. No additional interventions were included in the routine clinical and laboratory standards for IVF/ICSI preparation and treatment. Informed written consent was obtained from all participants. The exclusion criteria were as follows: Diabetes type 1 or 2, impaired thyroid, renal or hepatic function, congenital adrenal hyperplasia, endometriosis and hypothalamic amenorrhea.

Table I summarizes data from a cohort of 45 patients who underwent oocyte retrieval. A consensus was reached on the minimal criteria needed to define poor ovarian response (POR) and polycystic ovary syndrome (PCOS). POR was defined in accordance with the Bologna criteria (13). At least two of the following three features had to be present to be categorized as POR: i) Advanced maternal age (≥40 years) or any other risk factor for POR; ii) previous POR (≤3 oocytes with a conventional stimulation protocol); iii) an abnormal ovarian reserve test [for example, antral follicle count, 5-7 follicles or anti-Müllerian hormone (AMH), 0.5-1.1 ng/ml].

Only subjects with a diagnosis of PCOS according to the Rotterdam criteria (14) were included and one of the features had to be polycystic ovaries on ultrasound examination. At least two of the following three features had to be present to be categorized as PCOS: i) Oligo- or anovulation; ii) clinical and/or biochemical signs of hyperandrogenism; iii) polycystic ovaries and exclusion of other etiologies (congenital adrenal hyperplasia, androgen-secreting tumors, Cushing's syndrome).

Follicular granulosa-luteal cells were obtained after oocyte retrieval from 38 patients undergoing IVF treatment at the Reproductive Medicine Centre of Tianjin Central Hospital of Obstetrics and Gynecology. All patients participating in the present study underwent controlled ovarian stimulation according to routine long or short gonadotropin-releasing hormone agonist protocols. The follicular aspirates from each patient were pooled in conical bottomed 15-ml polypropylene centrifuge tubes. These were then centrifuged at 800 x g for 10 min at room temperature after which the supernatant was discarded. After adding a small quantity of saline to resuspend the cells, the cell suspension was transferred to an equal volume of 50% (v/v) Percoll (Sigma-Aldrich; Merck KGaA) by centrifugation at 500 x g for 20 min at room temperature. After resuspending the cells using saline, they were separated with an equal volume of 0.25% trypsin digestion solution(Sigma-Aldrich; Merck KGaA) for 10 min at room temperature. Then, after the addition of the same volume of Gibco DMEM/F-12 media (Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (ES-Cult^™ FBS; Stemcell Technologies, Inc.) to neutralize trypsin, a centrifugation was performed at 800 x g for 5 min at room temperature and density gradient centrifugation was repeated. Then, to perform a second density gradient centrifugation, the supernatant was discarded, and the cells were mixed well with normal saline and then centrifuged at 500 x g for 20 min at room temperature in an equal volume of 50% Percoll. GCs were carefully aspirated with pipettes, mixed well with saline and centrifuged at 800 x g for 5 min at room temperature, after which the supernatant was discarded. The collected human GCs were cultured in DMEM/F12 containing 10% FBS and 100 U/ml penicillin/streptomycin. Samples were taken for cell counting, viability testing via Trypan blue exclusion, and immunocytochemistry and western blot analysis. The remainder was cultured in an incubator under a constant temperature of 37˚C and 5% CO₂. Nine samples were processed for RNA extraction to analyze the integrity.

The follicular aspirates from seven POR patients were pooled. These were then centrifuged at 800 x g for 10 min at room temperature after which the supernatant was discarded. After resuspending cells using saline, they were separated using an equal volume of 0.25% trypsin for digestion at room temperature for 10 min. Subsequent processing was consistent with the abovementioned protocol. One sample was processed for RNA extraction to analyze the integrity.

GCs were seeded at 70% confluence onto small glass coverslips placed into 24-well plates. After 24 h of culture the coverslips were removed, washed with PBS three times, fixed with 100% methanol (-20˚C) for 15 min at room temperature, washed with PBST three times, and blocked for 1 h with 5% (v/v) goat serum (Gibco; Thermo Fisher Scientific, Inc.) in PBS. Next, the cells were incubated overnight at 4˚C with the following primary antibodies: Polyclonal goat anti-human IgG follicle stimulating hormone receptor (FSHR; 1:100; cat. no. sc-7798) and polyclonal rabbit anti-human IgG to Müllerian inhibiting substance type II receptor (MISIIR; 1:200; cat. no. sc-67287) from Santa Cruz Biotechnology, Inc., and then incubated with secondary antibody rabbit anti-goat IgG and goat anti-rabbit IgG (1:200; cat. no. sc2768 and sc2004) at room temperature for 1 h and rinsed with PBS. Chromogen 3,3'-diaminobenzidine tetrachloride (SERVA Electrophoresis GmbH) was used as a substrate. The cell nucleus was stained with Harris hematoxylin solution (cat. no. HY-N0116; MedChemExpress) for 15 min at room temperature. The expression levels of FSHR and MISIIR were scored according to the extent and intensity of staining. The extent of staining was scored by the percentage of the positively stained area in each region of interest using the following scale: 0 for <5%; 1 for 5-25%; 2 for 25-50%; 3 for 50-75%; and 4 for ≥75% (15). The staining intensity was scored as 0, 1, 2 and 3 for the representation of negative (no staining), mild (weak), intermediate (distinct) and intense (strong) staining, respectively (15). The staining intensity and stained area percentage were multiplied to establish a weighted score. Scoring was determined by three independent evaluators without any knowledge of the pathological and clinical characteristics of the patients.

To confirm their phenotype, the GCs were characterized for the expression of specific surface antigens defining human GCs. Cells incubated for 30 min at 4˚C with 1:200 diluted human FSHR-phycoerythrin (PE) from R&D Systems, Inc., (cat. no. FAB65591P), CD9-PE (cat. no. 312105; BioLegend, Inc.) and CD24-PE (cat. no. 12-0247-42; eBioscience; Thermo Fisher Scientific, Inc.). After staining, cells were mixed well with PBS and cell fluorescence was evaluated in 10,000 viable cells using a BD FACSCanto^™ II flow cytometer (BD Biosciences).

Cells were seeded in multi-well plates (96-well) in DMEM/F12 medium containing 10% FBS, at a density of 1x10⁴ cells/well, and allowed to attach to the DMEM/F12 medium containing 10% FBS for 8 h following analysis. Cells were counted using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocol, and the absorbance was measured at 450 nm. The cell viability was analyzed using enzyme-linked immunosorbent assay and optical density values were read at 450 nm. The survival rate was calculated with the following formula: [(As-Ab)/(Ac-Ab)] x100% (16), where As is the absorbance of PCOS and POR groups; Ab is the absorbance of the blank group, which contains complete medium without cells; and Ac is the healthy group.

Cell lines were seeded into 12-well plates and cultured at 37˚C for 72 h. The cells were collected into centrifuge tubes, washed three times with PBS and stained with propidium iodide (PI) and FITC-Annexin V (AnV) from the FITC Annexin V Apoptosis Detection kit I (BD Biosciences), followed by flow cytometry using the BD FACSCanto^™ II. The percentage of the cell population in the process of apoptosis was calculated using FlowJo software (v10.4; FlowJo LLC).

The total protein from GCs was collected using RIPA buffer (Sangon Biotech Co., Ltd.). A total of 1 ml RIPA buffer was used per 100 mg. Cells were collected from a 6-well plate using 100 µl RIPA buffer per well. The lysates were homogenized and then centrifuged at 16,000 x g for 30 min at 4˚C. The membrane protein was extracted using the Membrane and Cytoplasm Protein Extraction Kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. Proteins from the supernatant were quantified using a BCA assay (Thermo Fisher Scientific, Inc.), an equal volume of 1X loading buffer was added and then the protein was denatured for 10 min at 100˚C. A total of 20 µg protein from each sample was separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Bio-Rad Laboratories, Inc.). The membranes were blocked for 1 h at room temperature in 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20. Subsequently, the membranes were incubated overnight at 4˚C with 1:1,000 diluted goat anti-FSHR (cat. no. sc-7798; Santa Cruz, Biotechnology, Inc.), 1:1,000 diluted rabbit anti-caspase-3 (cat. no. sc-65497; Santa Cruz Biotechnology, Inc.), 1:1,000 diluted rabbit anti-Bcl-2 (cat. no. sc-7382; Santa Cruz Biotechnology, Inc.) and 1:10,000 diluted mouse anti-β-actin (cat. no. C1213; Sungene Biotech, Ltd.). The next day, the membranes were incubated with the corresponding secondary antibodies rabbit anti-goat IgG, goat anti-rabbit IgG and goat anti-mouse (1:200; cat. no. sc2768, sc2004 and sc2005, respectively) at 1:1,000 for 1 h at room temperature. The immune complexes were examined by ECL detection (EMD Millipore). The western blotting bands were quantified using ImageJ Software (v1.46; National Institutes of Health) after background subtraction.

For RNA analysis, GC samples were obtained from: Patients with PCOS (n=5); healthy patients (n=2); and patients with POR (n=2). Total RNA was extracted using an RNAequeous^®-Micro kit (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The samples were analyzed for total RNA concentration using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and total RNA quality and level of degradation using an Agilent 2100 Bioanalyzer (Agilent Technologies Deutschland GmbH) according to the manufacturer's instructions. All of the RNA samples showed two distinct peaks representing 18S and 28S ribosomal RNA, which indicated good quality RNA, and presented an RIN of 8-10.

The data were analyzed using SPSS 19.0 software (IBM Corp.). Student's unpaired t-test was performed for mean comparison between two groups. The comparisons among multiple groups were analyzed using ANOVA followed by the Least significant difference post hoc test. Experimental data are presented as means ± standard deviation and P<0.05 was considered to indicate a statistically significant difference.

Following 24 h of primary culture, single adherent cells were observed in a distinct area of the culture dish. These cells were rhombic or irregular in shape, inconsistent in size and shape, and had clear boundaries. Additionally, connections between the cells were observed under a microscope (Fig. 2). As all cells plated on the cover slips exhibited MISIIR and FSHR (Fig. 2), immunocytochemistry demonstrated that the majority, if not all, of human GCs express these receptors. The mean purity of GC samples obtained using this method was >95%.

In the three groups >95% of GCs were positive for FSHR (Fig. 3). The CD9 and CD24 molecules are cell surface glycoproteins, which are also expressed on the GCs surrounding the oocytes. Flow cytometry demonstrated that CD9 is present on the cell surface of, on arithmetic average, 70% of GCs among the three groups and CD24 is present on the cell surface of 90% of GCs among the three groups. The expression of CD9 on GCs in patients with PCOS was significantly lower than that in the other groups (Fig. 3).

Cell viability was assessed by CCK-8 assay. An increase of ~50-70% in cell viability was observed in the cells over time, with no significant differences identified among the three groups. The histogram in Fig. 4 further illustrates the experimental results. Flow cytometry was then performed to evaluate GC apoptosis and the arithmetic average percentages of cells in two quadrants are presented. The early apoptotic cells (PI^-/AnV⁺) and late apoptotic cells (PI⁺/AnV⁺) are presented as percentages of events and no statistically significant differences in the levels of apoptosis were identified among the different groups (Fig. 5). Higher levels of FSHR were observed in the GCs of healthy individuals and patients with PCOS when compared with those of patients with POR (P<0.01; Fig. 6). The results showed that GCs from healthy individuals and patients with PCOS exhibited increased Bcl-2 expression levels and inhibited early cleavage of caspase-3 when compared with patients with POR.

An RIN was then determined for each sample using an established algorithm following sample assessment in an automated electrophoresis system (Agilent 2100 Bioanalyzer) (17). No statistically significant RNA degradation was observed in the nine samples of the GCs, as demonstrated by the representative electropherograms and corresponding RIN values (Fig. 7). The mean RIN for all patient samples was 9.1. Moreover, the baseline of the electrophoretic pattern was stable, therefore RNA integrity was good, the results were reliable and follow-up studies are now required.