Patients and endometrial sample collection
Tissues were obtained from the endometria of patients with PCOS (n=18) and healthy women (n=18) who underwent hysteroscopy at the Division of Reproductive Medicine Center, Peking University Third Hospital. The diagnosis of PCOS was based on the 2003 Rotterdam ESHRE/ASRM criteria, and was finalized if any two of the following three criteria were met and other causes were ruled out: (1) Oligo-and/or anovulation (2) clinical hyperandrogenism, and (3) polycystic ovaries. Women who received any hormonal treatment within 3 months prior to the start of the study, as well as patients with pelvic inflammatory disease, genital tract infection, chromosome abnormality, hysteromyoma, or endometriosis, were excluded from the study. Written informed consent was obtained from each patient before study participation and ethics approval was obtained from the Research Ethics Committees of the Reproductive Center, Peking University Third Hospital.
The endometrial tissues were divided into three equal pieces. Two pieces of each sample were frozen in liquid nitrogen and maintained at – 80°C for quantitative real-time polymerase chain reaction and western blot analyses. One-piece was used for histological and immunohistochemical examinations. Endometria were obtained during the proliferative phase of the menstrual cycle (cycle days 5–11).
Histology and immunohistochemistry
Five-micrometer-thick sections were cut from formalin-fixed, paraffin-embedded tissue blocks, placed on coated slides, dewaxed in xylene, and then rehydrated in descending grades of ethanol (100–70%). Half of the sections were stained with hematoxylin and eosin. Antigen retrieval was performed using citric acid buffer (0.1 M, pH 6.0) and microwaving for 10 min on high power. After cooling to room temperature and washing three times in phosphate-buffered saline (PBS), endogenous peroxidase was quenched using 3% hydrogen peroxide for 10 min. After washing with PBS three times, the sections were incubated with anti-C/EBP-β antibody (diluted 1:50, ab32358, Abcam) and anti-SIRT1 antibody (diluted 1:50, ab32441, Abcam) diluted in PBS for 2h at 37 °C in a humidified chamber. The negative controls were incubated with a solution devoid of any primary antibody. The secondary antibody was goat anti-rabbit IgG conjugated with horseradish peroxidase (1:250; Beijing Zhongshan Biotechnology Co., Beijing, China). After incubation with the secondary antibody for 1h at 37 °C in a humidified chamber, the signals were viewed under an Axiokop2 microscope (Carl Zeiss, Thornwood, New York, NY, USA).
HEK293T cells (Beijing qualityard and biotechnology Co., Ltd, Peking, China) in logarithmic phase were cultured in cell suspension, counted, and inoculated in 24-well plates (the number of cells was about 105, depending on the size of cells) in an incubator at 37°C and 5% CO2 until the degree of cell fusion reached about 60%. ROCHE: X-tremegene HP transfection reagent was used for plasmid transfection. The expression of fluorescently-labeled genes was observed 24-48 hours after transfection to determine transfection efficiency. As a control, equal amounts of GFP plasmid were separately transfected from the target plasmid. Luciferase was detected 48 hours after transfection. The culture medium in 24-well plates was sucked out and 300 ml Passive Lysis Buffer was added to the plate. Reactions were continued at 4°C for 20 minutes before cell lysis.
The cells were added into Lockwell maxisorp detection board, and Luciferase Assay Reagent was applied. Immediately after shaking and mixing, firefly luminescence was detected by enzyme-labeled instrument. After detecting firefly luminescence, 20 μl Stop & Glo Reagent was added to each well. Renilla luminescence was detected by enzyme-labeled instrument after shaking and mixing for 3 minutes. Relative fluorescence values of fluorescence intensity/renilla luminescence intensity were used as indicators to determine the difference among groups.
Quantitative RT-PCR was performed following a previously reported method (14). Dissociation curves for both target and housekeeping genes were utilized to ensure the absence of primer-dimers and other non-specific amplification. PCR and real-time measurements of fluorescence were performed in the 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) in at least triplicates, using SYBR Select Master Mix (Applied Biosystems). The primers were as follows: 5′-CCAAGAAGAC CGTGGACAAG-3′(forward) and 5′-TTGCGCATCTTGGCCTT-3′(reverse) for C/EBP-β; 5′-AGAACCCATGGAGGATGAAAG -3′ (forward) and 5′-TCATCTCCATCAGTCCCAAATC-3′(reverse) for SIRT1. The comparative ΔΔCt method was performed to measure relative gene expression (ABI User Bulletin 2).
Western blotting was performed to detect C/EBP-β (14) according to the previously described method. Briefly, ten endometrium samples from PCOS group and ten endometrium samples from normal control groups, each containing 60 μg of protein were electrophoresed on 10% polyacrylamide gels, after which they were denatured for five minutes in 100°C and transferred to PVDF (polyvinylidene fluoride) membranes. The membranes were blocked in Tris-buffered saline solution with 0.1% Tween 20 and 5% nonfat milk for 1 h at room temperature. The primary antibodies were anti-C/EBP-β (diluted 1:500, ab32358, Abcam) and anti-SIRT1 (diluted 1:50, ab32441, Abcam). Blots were incubated with primary antibodies overnight at 4 °C. After washing three times in Tris-buffered saline, the membranes were incubated for 1 h at room temperature with 1:500 horseradish peroxidase-conjugated secondary antibodies. Finally, the membranes were processed and visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotechnology, Piscataway, NJ, USA). The relative band density normalized to that of β-actin was determined from light scans of the resulting films.
Statistical analyses were performed using SPSS 18.0 (Chicago, IL, USA). The Shapiro–Wilk test was performed to determine whether continuous variables were normally distributed. All error bars in figures indicate standard errors (SE). The data were analyzed by t-tests and Mann–Whitney U tests. P-value of < 0.05 was considered statistically significant.