International Journal of

biologically active receptor and GHS-R type 1b encodes a C terminally truncated protein (9). The human GHS-R type 1a is a polypeptide of 366 amino acids with seven transmembrane domains and is the functional form of the receptor (10). Ghrelin, apart from its GH-releasing activity, also significantly stimulates prolactin, ACTH and cortisol secretion. Recently, a number of different central and peripheral effects have been described: on appetite and weight regulation, carbohydrate metabolism, heart function, the gonadal axis, exocrine function, and cell proliferation (11-17). mRNA expression of ghrelin and GHS-R1b were reported in many different human tissues including ovary (18). Using immunohistichemistry method Gaytan et al (2003) showed the presence of ghrelin and its cognate functional receptor in cyclic human ovary with distinct but partially overlapping patterns of cellular distribution (19). Plasma ghrelin concentrations have been reported to be correlated to body mass index (BMI) with reduced levels in obesity that increases in response to weight loss (20). Schofl et al (2002) showed that in PCOS women, serum ghrelin were significantly lower than in healthy controls and there was a close correlation of ghrelin to insulin sensitivity (21).  No  difference  in  plasma  ghrelin  concentrations between the PCOS and the control groups were reported (22). It is unknown wether abnormal expression of ghrelin and GHS-Rs in ovary might play a role in the pathogenesis of women with PCOS. This study was performed in order to determine the role of ghrelin and GHS-Rs in pathogenesis of PCOS.  
 
Materials and Methods

     Patient recruitment and follicular fluid collection

     This study was carried out as case-control study on two different groups. Eleven patients were diagnosed as having PCOS according to criteria derived from the 1990 National Institutes of Health (NIH)/National Institute of Child Health and Human Development (NICHHD) conference. Briefly, the criteria include: 1) ovulatory dysfunction, 2) clinical hyperandrogen (hirsutism, acne, androgenic alopecia) 3) exclusion of other related disorders, such as hyperprolactinemia, nonclassic adrenal hyperplasia, or thyroid disease (23). Twenty-two non PCOS women matched for age and weight, undergoing IVF-ET for other causes of infertility, including 6 for tubal disease, 9 for male factor, 4 for endometriosis and 3 for unexplained infertility, were selected as the control group. The Research Ethics Committee of The Queen Elizabeth Hospital and The University of Adelaide approved the study protocols and consent forms. Written informed consent was obtained from each patient before follicular fluid collection. Prior to oocyte collection, a general clinical examination and hormonal evaluations were performed including baseline levels of testosterone (T), luteinizing hormone (LH), sex hormone binding globulin (SHBG), follicle stimulating hormone (FSH), and mid-luteal phase estradiol and progesterone. The average age, weight, body mass index  (BMI),  testosterone  and  resulting  pregnancies for both groups are shown in Table 1. Samples underwent a standard IVF protocol; with recombinant FSH (N.V. Organon, The Netherlands) -stimulated follicles (15-25mm in diameter) aspirated 32-34 h after hCG (Profasi, Serono, Rockland, MA) administration. After removal of the cumulus-oocyte complexes, follicular fluid was pooled for each sample and placed on ice.
 
     Cell isolation from follicular fluid
     Samples were centrifuged at 200g for 10 mins at 4°C and interface cells were aspirated and resuspended in Hank’s balanced salt solution (HBSS, GibcoBRL) containing 0.06 % gentamycin (DBL, Mulgrave, Victoria, Australia), 0.01% sodium azide (Sigma Aldrich) and 2mM ethylenediamine tetraacetic acid (EDTA, Sigma Aldrich) (HBSS/gent/EDTA/Az). The cells were then centrifuged over 1:1 v:v PBS:Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) at 400 g for 20 mins at 4°C to remove contaminating red blood cells. Interface cells were removed and resuspended in HBSS/gent/EDTA/ Az, centrifuged at 200g for 10 min. Finally, the cell pellet was resuspended in HBSS/gent/EDTA/Az, filtered through a 70μm diameter cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA) and washed with HBSS/gent/EDTA/Az containing 0.5% BSA. Total cells were counted and viability assessed by trypan blue dye exclusion (Sigma Aldrich).
 
     RNA extraction and quantitation
     Total RNA was extracted from isolated follicular fluid cells by Tri-Reagent (Sigma Aldrich), followed by a series of phenol-chloroform extraction and ethanol precipitations as stipulated by Tri-Reagent manufacturers. Contaminating residual DNA was eliminated by treatment with deoxyribonuclease I (DNase I, Promega, Madison, WI). Resulting RNA was dissolved in 20μl ribonuclease-free water and concentration was determined using a Ribogreen® RNA quantitation kit (Molecular Probes, Eugene, OR) according to manufacturer’s instructions.
 
     Quantitative real time RT-PCR
     For each sample, 2 mg RNA was diluted in PCR-grade water and was reverse transcribed using random primers (Roche Diagnostics GmbH, Mannheim, Germany) and a Superscript II Reverse Transcription kit (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Negative controls omitting RNA or Superscript II enzyme were included. Specific primers for the ghrelin, GHS-R1a and GHS-R1b were designed on using primer express software (Applied Biosystems, Foster City, CA) and manufactured by Geneworks (Adelaide, Australia) (Table I). Samples were run in triplicate on an ABI Geneamp 5700 sequence detection system (Applied Biosystems). They were subjected to 40 cycles of amplification under condition 92°C-20S and 62°C-1min using 3 µl diluted cDNA (1:7), 10 µl 2× SYBR green buffer (Applied Biosystems) and 5 pmol of each primer. Negative controls omitting template cDNA were included in each run. Following RT-PCR amplification, a dissociation analysis was run on the products to ensure that a single product was amplified during the PCR process. To ensure that β-actin was an acceptable housekeeping or control gene for this study, it was necessary to determine if its mRNA levels were constant for the two treatment groups. β-actin mRNA was assayed and then normalised to total RNA measurements for each sample. For β-actin to be an acceptable control gene, the cycle threshold (C_T) value per µg RNA should not vary statistically across treatment groups. It was also necessary to check that the primers for the housekeeping gene, β-actin and the other genes of interest had similar amplification efficiencies. Therefore, each primer set was run with serially diluted cDNA and the resultant C_T values plotted against log dilutions. The slopes of each graph were determined and statistically compared. To determine if primer amplification efficiencies were comparable to that of the housekeeping gene, t-tests were performed on the slopes produced by each primer set for log dilutions plotted against C_T value, along with the number of data points and the standard error generated for each slope. For all genes of interest, mRNA content was calculated for each sample relative to the housekeeping gene, β-actin. This was performed using the equation 2^-ΔΔCt (as described in PE-ABI, Sequence Detector User Bulletin 2), where ΔC_T is the difference between the gene of target and the housekeeping gene, and ΔΔC_T is the change between the ΔC_T for each sample and the control group (e.g. non-PCOS patients). Each target gene is then described as the fold change from the control group. Statistical significance was analyzed by a Students t-test. In all cases, p<0.05 was considered statistically significant.