Germinal vesicle (GV)-stage cumulus-oocyte complexes (COC) were obtained from women undergoing controlled ovarian hyperstimulation for in vitro fertilization. After suppression of endogenous gonadotropin with leuprolide acetate (Lupron; TAP Pharmaceuticals, Chicago, IL; 0.5 mg/ day, s.c.), patients were treated with human menopausal gonadotropin (Pergonal; Serono, Randolph, MA) and/or human FSH (Metrodin; Serono) to stimulate follicular development. When the lead follicle was equal to or greater than 16 mm in diameter, hCG (Profasi; Serono, Randolph, MA) was administered, and follicle aspiration was performed 36 h later. GV-stage oocytes isolated from the follicle aspirates that were not required for patient treatment were transported to the laboratory and processed as described below. This protocol was approved by the Massachusetts General Hospital Subcommittee on Human Studies, and informed consent was obtained from all patients. flovent inhaler
COC were either processed within 2-3 h of aspiration or cultured for up to 24 h in IVF50 medium (Scandinavian IVF Science, Vero Beach, Florida), under 5% CO2 at 37°C, until spontaneous maturation to metaphase 1 (M1) or metaphase 2 (M2). Before RNA extraction, oocytes were mechanically detached from the bulk of the cumulus mass and treated with hyaluronidase (200 IU/ml; Sigma), and all remaining granulosa cells were removed by repeat pipetting.