yte quality: metaphase I oocytes, unfertilized metaphase II oocytes and MII oocytes developed to blastocyst stage embryo. We performed two analyses: firstly we assessed differences in CC gene expression according to the oocyte stage achieved, and secondly we assessed the differences in CC gene expression according to the GnRH analogue used. By comparing the CC MI and MII expression profiles we sought for gene markers linked to oocyte maturity. Materials and Methods Patients and IVF treatment In this prospective, randomized study, 21 patients undergoing classical IVF cycle at the Department of 
order BCTC Obstetrics and Gynecology, University Medical Center Ljubljana, were included. The study was approved by the national medical ethics committee and all patients have signed informed consent. Randomization was performed according to JL Fleiss; the randomization list was prepared in advance. Each patient who agreed to participate in the study was assigned to either a GnRH agonist or a GnRH antagonist group, and had an equal chance to be assigned to either group. The allocation was carried out by revealing a therapy group by a third person to medical staff and the patient at the moment of entering the study. The inclusion criteria were as follows: age less than 35 years, body mass index ranging between 17 and 26 kg/m2, indication for IVF program was tubal factor infertility, and the partner’s spermiogram had to be normal according to WHO criteria. Eleven randomly selected patients were administered GnRH agonist buserelin acetate from day 22 at a daily dose of 0.6 ml subcutaneously. When the criteria for ovarian desensitization were fulfilled, they were subcutaneously administered 225 IU of gonadotrophin folitropin a. To the remaining 10 patients 225 IU of gonadotrophin follitropin a was subcutaneously administered on day 2. When the dominant follicle measured $14 mm in diameter, GnRH antagonist cetrorelix acetate in a dose of 0.25 mg was administered subcutaneously. When at least three follicles were $17 mm and serum oestradiol was $0.40 nmol/L per follicle all patients were administered 10,000 IU of human chorionic gonadotrophin ; 3436 h later an ultrasound guided transvaginal oocyte retrieval was performed. RNA preparation RNA was extracted using TRI reagent according to slightly modified manufacturer’s instruction. Due to small sample volume, glycogen was used as a carrier to increase RNA yield. Briefly, CC from individual cumulus oocyte complexes were homogenized in 500 mL TRI reagent supplemented with 125 mg of glycogen. After 2 min incubation at room temperature, 100 mL chloroform was added and the sample was vortexed vigorously. RNA was precipitated with isopropanol from the aqueous phase and collected after 15 min centrifugation at 12,0006 g and 4uC. RNA pellet was washed 3 times by 75% ethanol, dried and dissolved in 15 mL of RNAse free water. The integrity of the RNA samples was assessed on Agilent 2100 Bioanalyzer to assure high quality of total RNA; the RIN number was more than 7 for each sample. Transcriptome analysis Transcriptome analysis was performed using the GeneChip Human Gene 1.0 ST Arrays. The GnRH Analogues on Cumulus Cells Gene Expression arrays were hybridized according to manufacturer’s recommendations. Briefly, 200 ng of RNA was amplified and converted to cDNA using the WT Expression Kit. The resulting cDNA was fragmented and labeled using the GeneChip WT Terminal Labeling and Controls Kit and hybridized to the arrays for 16
