Analysis of androgen receptor and anti-Müllerian hormone pathways in human granulosa cells under luteinizing hormone treatment

539 Patients undergoing their first ART treatment with a long COH protocol were enrolled in the study. This study was approved by the Ethics Committee of Chang Gung Memorial Hospital. Approval from the institutional review board was obtained for the analysis of this series (CGMH94-360). The study included patients who underwent IVF/intracytoplasmic sperm injection in our institution between March 1, 2004 and September 30, 2006. Within this study period, 35 patients received rLH supplementation cycles (12 patients with antral follicle count (AFC) <6; 2 patients with previous intrauterine insemination (IUI) poor response history; 7 patients with age > 35 years old; 10 patients with mid-follicular serum estradiol <100 pg/mL or LH < 0.5mIU/mL; 2 patients with poor follicular growth rate in the mid-follicular phase; 1 patient with deep infiltrating endometriosis history and 1 patient due to obesity). To match appropriate candidates to compare outcome efficiency, a 2:1 individual matching case-control design [28] was used to recruit 70 patients who did not receive rLH supplementation cycles but matched the demographic characteristics of the 35 patients receiving rLH therapy (Figure1). At that time, it has been suggested that recombinant LH can be used in a group of unselected IVF patients [29]. All patients provided informed consent for their participation in the molecular investigation. The COH procedure was followed by the standard down-regulation protocol, as published previously [30]. Briefly, GnRHa (Lupron; Abbott Australasia PTY, Ltd., Kurnell, New South Wales, Australia) was administered from the mid-luteal phase, and rFSH (Gonal-F; Serono Laboratories, Aubonne, Switzerland) was administered after pituitary desensitization was achieved. Patients received one of the following fixed starting doses (age < 35: 225 IU/day; age≥35: 300 IU/day) of rFSH (Gonal-F, 75 IU/ampoule, Serono Laboratories, Aubonne, Switzerland) after pituitary suppression with GnRHa. Gonadotropin was administered daily for 5 days, after which the dose was individualized according to ovarian follicular growth, as in our previous reports [30]. Monitoring of transvaginal ultrasound and serum estradiol (E2) was performed every 2 to 3 days starting on the 5^th day of stimulation. After 6 days of rFSH stimulation, patients who had ever received co-treatment with 75 IU of rLH (Luveris; Merck Serono SA, Aubonne Branch, Switzerland) were submitted to the rLH supplementation group. rFSH, with or without rLH, was continuously administered until 2 of the dominant follicles reached 18 mm, and then the patients received 250 μg of hCG (Ovidrel; Merck Serono, Inc., West Orange, NJ, USA).

Thirty-four to 36 hours after hCG administration, follicular fluid (FF) and luteinized granulosa cells were collected by means of transvaginal ultrasound-guided oocyte retrieval. A double-lumen needle was used for aspiration because the puncture needle used to obtain a sample of uncontaminated luteinized granulosa cells was not withdrawn before entering the next follicle. Only the first aspirate of pre-ovulatory follicles without blood contamination from one ovary was used for this study. After follicular aspiration, the cumulus-oocyte complexes were separated for IVF, and the luteinized granulosa cells were flushed with IVF medium to disperse the cells. The cells were then immediately transported to the laboratory. The follicular fluid was stored under the same conditions as serum. The FF samples were analyzed for testosterone, estradiol, progesterone, and androstenedione. The dispersed cells were transferred to a 15-mL centrifuge tube containing 4 mL of Histopaque 1077 (Sigma Chemical Co., St. Louis, MO, USA). Human luteinized granulosa cells were separated from red blood cells by centrifugation at 600 × g for 10 minutes. Granulosa cells formed a thin layer between the Histopaque and the medium [31] and were collected as previously reported [17].

The HO23 human immortalized luteinized granulosa cell line, provided by Dr. Abraham Amsterdam, Weizmann Institute of Science, Rehovot, Israel [32], was used for in vitro analyses. HO23 cells were established by triple transfection of SV40 DNA, the Ha-ras oncogene, and a temperature-sensitive (ts) mutant of the p53 tumor suppressor gene (p53val135) in primary granulosa cells obtained from IVF patients. The cells were maintained at 37°C and 5% CO₂ in Dulbecco’s minimal essential medium (DMEM)/Ham’s F12 (1:1), supplemented with 5% fetal calf serum and antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin). To test the effect of rLH or androgen on AR and AMH expression, cells (5 × 10⁵) were seeded on 100-mm culture dishes and incubated for 24 hours. The media were then removed, and the cells were reincubated in medium supplemented with charcoal-dextran-treated serum and then treated with different concentrations of DHT or rLH for 24 hours.

Total RNA was extracted from granulosa cells (5 × 10⁵) using TRIzol reagent (Gibco-BRL, Grand Island, NY, USA). Briefly, 1 mL of TRIzol was added to the granulosa cells. The mixture was pipetted and allowed to sit for 5 minutes at room temperature. Chloroform (0.2 mL) was added, mixed, and allowed to incubate at room temperature for 3 minutes. The mixture was then processed by centrifugation at 12,000 × g for 15 minutes, and the supernatant was transferred to a fresh tube. Isopropanol (0.5 mL) was added, mixed, and incubated for 10 minutes at room temperature. The solution was then processed by centrifugation at 12,000 × g for 10 minutes, and the RNA was purified.. The pellet was washed once with 70% ethanol, resuspended in H₂O, and stored at -80°C. The concentrations of the RNA samples were determined by measuring the absorbance at 260 nm in a spectrophotometer (DU® 640B, Beckman Coulter, USA).

Two micrograms of total RNA from the sample preparation were reverse-transcribed in 25 μL as follows: 0.1 μg of random hexamer primers (Amersham Pharmacia Biotech, Inc., Buckinghamshire, UK) were denatured for 10 minutes at 70°C in a Gradient Cycler (DNA Engine, Watertown, MA, USA), and then the reverse transcription reaction was performed at 42°C for 1 hour by adding 5× reverse transcriptase buffer (500 mM of each dNTP, 3 mM MgCl₂, 75 mM KCl, and 50 mM Tris-HCl [pH 8.3]), 1 mM dNTP (Promega, Madison, WI, USA), 10 IU RNase inhibitor (Promega), and 100 U MMLV reverse transcriptase (Promega). The reverse transcriptase was inactivated by heating at 95°C for 5 minutes and cooling at 4°C for 5 minutes.

Specific PCR amplification products were detected using an ABI PRISM 7700 sequence detector system (Perkin-Elmer Applied Biosystems, Los Angeles, CA, USA) and the SYBR green PCR master mix kit (Applied Biosystems, Foster City, USA), according to the manufacturer’s protocol. The forward and reverse primer sequences for AR, AR co-factors, SOX9, AMH, LHR and ß-actin and 18S are listed in Table 1. Duplicate experiments were performed for each set of experimental conditions, and we retested any sample with a > 1% coefficient of variation for the Ct value. Quantitative values are obtained from the threshold cycle (Ct) number at which the increase in signal associated with an exponential growth of PCR product starts to be detected (using 7500 Fast System SDS software (Applied Biosystems)), according to the manufacturer’s manual. The precise amount of total RNA added to each reaction (based on absorbance) and its quality (i.e., lack of extensive degradation) are both difficult to assess. We quantified ß-actin or 18S gene transcripts as an endogenous RNA control, and normalized each sample with respect to its ß-actin or 18S content. Final results, express as N-fold differences in target gene expression relative to the β-actin gene termed “N target,” are determined as follows: N target=2^{△Ct sample}where the △Ct values of the sample were determined by subtracting the average Ct value of the target gene from the average Ct value of the ß-actin or 18S gene in each sample.

Data analyses were performed with SPSS 10.0 software (Statistical Package for Social Sciences, Inc., Chicago, IL, USA). Continuous data were summarized as the mean ± standard deviation (SD). For the purpose of this analysis, correlations in gene expression were examined. A multiple regression analysis with the stepwise forward procedure (multivariate analysis) was used to identify independent factors and to test for interactions between the covariates. The clinical outcome comparison used the Mann-Whitney rank-sum test for the comparison of means and the Fisher’s exact test for proportions. All P values were two-sided, and P < 0.05 was considered statistically significant.