P-523: Coculture with granulose cells of human oocytes during in vitro maturation and the influence on the maturation of ooplasm

Abstract

IVM technique plays an important role in assisted reproductive technologies, especially for PCOS patients. The current in vitro culture medium can achieve nuclear maturation of oocytes rather than cytoplasmic maturation. This study is designed:firstly, to identify whether nuclear and cytoplasmic maturation can reach simultaneously in vitro maturation. Secondly, to observe if in vitro culture time can affect nuclear and cytoplasmic maturity of oocytes . Thirdly, Many animal experiments have identified that cocultured with monolayer granulose cells(GCs) can greatly improve the ooplasmic maturation, which is manifested in high fertilization rate, high embryo quality and high pregnancy rate. Our objectives were to confirm that cocultured with GCs can surely improve ooplasmic maturation of human oocytes. In this experiment, we used the linear distribution of cortical granules(CG) as the marker of cytoplasmic maturity of oocytes, since many reports have defined that. Prospective cohort study. Immature oocytes were retrieved from PCOS patients who were undergoing treatment from March to June 2005 at the Reproductive Medical Center in the Provincial Hospital of Shandong,all of whom had signed informed consents explicitly for this study. Pick-up of immature oocytes were performed 36 hours after HCG injection in natural menstruation cycles. Granulosa cells for coculture were taken from the cumulus-oocyte complexes (COC) in classical ICSI cycles. The COCs were digested with 80IU/ml hyaluronidase no more than 30 seconds. Then GCs were suspended with M-199 medium, centrifuged twice, 1000rpm for 10 mins. Then the suspension of granulose cells were dropped into 4-well plates and half of the culture medium was changed after 24 h. At that time, monolayer of GCs was formed and prepared for coculture with immature oocytes. Randomly,some of immature oocytes were cocultured with GCs and others were not cocultured in M-199 medium as control. All of them were evaluated 24h and 48h after in vitro maturation. The oocytes reached MII stage were labeled with FITC-LCA and observed under laser-scanning confocal microscope to investigate the distribution of CG. In comparison with the oocytes cultured in M-199 medium without granulose cells, the percentage of nuclear maturity after 24h, 48h in vitro maturation of cocultured oocytes were not significantly different respectively(36/69 vs 37/71,P>0.05;43/69 vs 43/71,P>0.05).The nuclear maturation rate of oocytes cultured for 48h are significantly higher than that of 24h in both control and experimental group (37/71 vs 43/71,P<0.05;39/69 vs 43/69,P<0.05).As for CG distribution pattern, the rate of peripheral distribution pattern is significantly higher in cocultured oocytes compared with control, either after cultured for 24h (27/39 vs 10/37,P<0.01) or cultured for 48h(31/43 vs 11/43,P<0.05),but for the comparison of maturality after in vitro maturation for 24hs between for 48hs in either control group or in co-culture group,no significant difference(10/37vs11/43,P>0.05;27/39vs31/43,P>0.05). It surely occurs that nuclear maturation and cytoplasmic maturation can not reach simutaneously during in vitro maturation. Cocultured with granulose cells monolayer can prompt greatly the cytoplasmic maturation of oocytes.Prolonged culture time can increase nuclear maturate rate but seem no helpful in cytoplasmic maturation.