2. Maturity assessment and in vitro fertilization

After IVM, adherent cumulus cells were removed from oocytes mechanically by gentle micropipetting using a fine-drawn glass pipette. The in vitro matured oocytes were divided into two groups according to their maturity: oocytes that displayed a distinct first polar body (PB) were classified as the MII group, while those without a PB and a visible GV nucleus were classified as the MI-arrested group. All oocytes from the MII group and some from the MI group were inseminated using sperm collected from the cauda epididymis of B6D2-F1 males in MRC#D01 medium. The remaining MI oocytes were cultured for a further 4 hours to investigate the spontaneous maturation rate in insemination medium. Throughout the fertilization period, the maturity of MI oocytes was re-evaluated by monitoring the appearance of the first PB every hour for up to 4 hours. After insemination, oocytes were washed and placed in insemination medium. The fertilization rate was assessed by the presence of the 2-cell stage at 20 hours following insemination. The cleaved embryos were cultured sequentially in cleavage and blastocyst medium (MRC#D13 and 46, Maria Medical Foundation) for 96 hours.