Hormones and reagents
Equine chorionic gonadotropin (eCG/PMSG), human chorionic gonadotropin (hCG), and chemical inhibitors including indomethacin, nordihydroguaiaretic acid, GW9662 were purchased from Sigma (St. Louis, MO, USA). RU486 was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA).
Animals for superovulation induction and administration of ovulation-inhibiting agents
Immature female Sprague-Dawley rats were purchased from Korea Basic Science Institute (Gwangju, Korea) and Samtako BioKorea (Seoul, Korea). They were housed in groups in a room with controlled temperature and photoperiod (10-h dark/14-h light; lights on from 0600 to 2000 h). The animals had ad libitum access to food and water. Immature rats (26 days old; body weight, 60–65 g) were s.c. injected with 10 IU of eCG to induce multiple follicle growth. Two days later, some eCG-primed rats were i.p. injected with 10 IU hCG to induce superovulation. All animals were maintained and treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, as approved by the Institutional Animal Care and Use Committee at Chonnam National University.
Five eCG-primed rats for each treatment group were i.p. injected 30 min before hCG administration with ovulation-inhibiting agents including progesterone receptor antagonist (RU486, 10 mg/kg body weight), cyclooxygenase inhibitor (indomethacin, 10 mg/kg body weight), lipoxygenase inhibitor (nordihydroguaiaretic acid, 3 mg/kg body weight), or proliferator-activated receptor γ (PPARγ) antagonist (GW9662, 2 mg/kg body weight) . Six hours after hCG injection, the rats were euthanized using CO2 administration method and ovaries, upon removal of oviduct and fat pad, were collected for RNA isolation.
Preparation of the rat model of ovarian hyperstimulation syndrome (OHSS)
To prepare the OHSS rat model, immature rats (22 days old) were s.c. injected with 10 IU eCG at 0900 for four consecutive days to promote follicular development; this was followed by an i.p. injection of 30 IU hCG on the 5th day (on the 26th day of life) to induce OHSS (Figure 1). As the control, rats were injected with 0.9% saline instead of hCG on the 5th day. Manifestation of OHSS includes the increased ovarian weight, VEGF expression and vascular permeability 48 h after hCG administration . Subsequently, the rats were euthanized 48 h after hCG administration (on the 28th day of life); then, the ovaries were collected for RNA isolation. Ovaries were also collected from rats that were stimulated for superovulation in a routine manner 0 h and 48 h after hCG administration.
Collection of ovaries and isolation of granulosa and theca cells of preovulatory follicles
Ovaries were collected from immature rats at different time points (0, 3, 6, 9 and 12 h) after eCG/hCG administration for RNA and protein detection of tissue factor, TFPI-1 and TFPI-2. For the isolation of the granulosa and theca cells of preovulatory follicles, the ovaries were incubated in DMEM/Ham’s F-12 medium (Gibco, Grand Island, NY, USA) containing 0.5 M sucrose and 10 mM EGTA at 37°С for 30 min. The ovaries were then washed thrice with phosphate buffered saline (PBS), and flattened to a single layer to easily identify the preovulatory follicles using fine forceps under a dissection microscope. The granulosa and theca cells were isolated from the preovulatory follicles using a 21-gauge needle for the measurement of mRNA levels.
RNA isolation and real-time PCR analysis
To detect mRNA levels of tissue factor, TFPI-1 and TFPI-2 in ovaries and preovulatory follicles after hCG treatment (0, 3, 6, 9 and 12 h), total RNA was extracted using TRIzol reagent (Molecular Research Center, Inc., Cincinnati, OH, USA), according to the manufacturer’s instructions. Ten or twenty micrograms of total RNA was reverse-transcribed using the RevertAid M-MuLV reverse transcriptase kit (Fermentas, St. Leon-Rot, Germany) to evaluate gene expression. Real-time PCR was then performed on a Rotor-Gene Q 5plex (QIAGEN, Hilden, Germany), located at Korea Basic Science Institute (Gwangju, Korea), using the QuantiTect SYBR Green PCR Kit (QIAGEN) at 95°С for 20 seconds, 60°С for 20 seconds, and 72°С for 30 seconds. Specific primers were designed using the PRIMER3 software (Table 1). The average Ct value in triplicate for each gene was divided by the linear Ct value of β-actin to obtain relative abundance of the transcripts. β-Actin was used as an internal control for all measurements.
Western blot analyses
The ovarian lysates (30 μg) were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Bioscience, Arlington Heights, IL, USA), as previously described . Briefly, the transferred membrane was blocked using 5% skim milk before immunoblotting using anti-tissue factor polyclonal antibodies (American Diagnostica, Inc., Stamford, CT, USA; 1:500 dilution) and horseradish peroxidase-conjugated secondary IgGs (1:1,000 final dilution). Gapdh (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the loading control. Signals were visualized via enhanced chemiluminescence (Amersham Biosciences).
The localization of the tissue factor protein was determined by immunofluorescence as previously described . Briefly, paraffin sections of ovary (5 μm thick) were incubated with 10% normal horse serum in PBS for 30 min to block non-specific binding of the antibody. The ovarian sections were probed with primary anti-tissue factor antibodies (American Diagnostica, Inc., 1:500 dilution) overnight and, then, washed thrice with PBS, followed by incubation with AlexaFluor 633 fluorescence antibodies (Invitrogen, Carlsbad, CA, USA ; 1:500 dilution) for 1 h. After washing thrice with PBS, the sections were mounted on slides and the nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) in ProLong Gold Antifade reagent (Invitrogen). Digital images were captured using a TCS SP5 AOBS laser-scanning confocal microscope (Leica Microsystems, Heidelberg, Germany), located at the Korea Basic Science Institute Gwangju center.
Collection of follicular fluid from women undergoing in vitro fertilization (IVF) and measurement of tissue factor concentrations via enzyme-linked immunosorbent assay (ELISA)
Follicular fluid was collected from 80 patients undergoing ovarian stimulation for IVF. Characteristics of patients based on the cause of infertility were presented in Supplemental Table 1. Forty-nine patients with infertility due to male (n = 22) or tubal factors (n = 27) served as controls. The male infertility patients were described as total motile count of <10 million sperms/ml or normal morphology in <4% of the sperm by strict criteria. Five women showed mild signs of OHSS after hCG administration during the IVF procedure. The causes of infertility among five OHSS patients include unknown factor (n = 3), oocyte donor and tubal factor. The inclusion criteria were age 21-42 years and normal uterine cavity on hysteroscopy. Patients who presented allergy to gonadotropins or other medications used in the treatment, or abusive use of any medications during the treatment were excluded. Our research was approved by the Institutional Review Board of Creation & Love Women’s Hospital (CLWH-IRB-2009-001).
Only clear follicular fluid, without blood or flushing medium contamination, was processed. After oocyte transfer, the follicular fluid (≈10 mL) aspirated from each patient was centrifuged for 10 min at 500 × g. Supernatants of the follicular fluid samples were stored at -80°С until the tissue factor concentrations were determined using an ELISA kit (EIAab Science Co., Wuhan, China). All the procedures were carried out according to the manufacturer's instructions. Concentrations of tissue factor were detected in follicular fluids obtained from women with different infertility factors.
Statistical analyses were performed using the statistical software GraphPad Prism 5 (GraphPad Software, Inc. La Jolla, CA, USA). Data obtained from rat ovaries were presented as the means ± SEM. One way ANOVA, followed by Dunnett’s test, was used for comparisons among multiple groups. Comparisons between any two points were evaluated using Student’s two-tailed t-test. The levels of tissue factor in human follicular fluid were presented as the mean ± SD or median (range). Correlation analysis was performed using Spearman's rho test. Pregnant and non-pregnant women were compared using the Kruskal-Wallis test or Mann–Whitney's U-test. Fisher's F-test was used to assess the relationship between two variables for parametric data. P < 0.05 was considered significant.