ABSTRACT
The collected mature oocytes (metaphase II) were subjected to insemination 2 or 3 h later using intracytoplasmic sperm injection (ICSI). The remaining immature oocytes (at germinal vesicle or metaphase I stages) were further cultured in oocyte IVM medium. We used YSmedium as oocyte IVM medium, containing 30% human follicular fluid (HFF) supplemented with 1 IU/ml FSH, 10 IU/ml hCG, and 10 ng/ml rhEGF (Daewoong Pharmaceutical Co, Korea)17,18. The HFF was prepared using the method reported by Chi et al.19. The immature oocytes were cultured in maturation medium at 37°C in 5% CO2, 5% O2, and 90% N2. After 1 day of culture, COCs were denuded of the cumulus cells using 0.03% hyaluronidase (Sigma, St Louis, MO, USA) in Hepes buffered Ham’s F-10 medium and mechanical pipetting. At 24 and 48 h of culture, the mature oocytes were inseminated by ICSI, respectively. Fertilization was assessed 17-19 h after ICSI to detect the appearance of two distinct pronuclei and two polar bodies. The zygotes were co-cultured with the cumulus cells prepared on the day of oocyte retrieval in 10 ml of YS medium supplemented with 10% HFF20.
