2013 ASRM 69th Annual Meeting
S.Y. Yoon, S.K. Cha, N.J. Yang, J.H. Eum, W.S. Lee, D.R. Lee
Vitrification induces damage to the antioxidant systems of oocytes, with consequent increases in the levels of reactive oxygen species (ROS) in vitrified oocytes. ROS are known to have detrimental effects on the embryonic development through such as alteration of mitochondrial membrane potential, ATP synthesis, and calcium oscillation during fertilization. We investigated the possible causes for low development to blastocyst of vitrified/warmed oocytes by extended prophase stage after vitrification to induce recovery of antioxidant system.
Materials and Methods
GV oocytes and sperm were collected from BDF1 mice. GV oocytes were vitrified in EG+ DMSO with an EM grid and slush-nitrogen. As phosphodietsterase 3 inhibitor, a milrinone, were used to block spontaneous GVBD during procedures, and to extend prophase I stage after vitrification for 1 or 3h. In vitro matured MII oocytes were fertilized in vitro and cultured in KSOM for 5 days to analyze embryonic development. As ROS indicator, carboxy-H2DCFDA were used and monitored with fluorescence microscopy.
There is no difference in in vitro maturation and fertilization rate between fresh and vitrified/warmed oocyte. The development rate to blastocyst in thawed/warmed oocyte was significantly lower than those in fresh oocyte (p< 0.05). However, the development rate to blastocyst was recovered if these oocytes were incubated in milrinone for more than 3hr for recovery of mitochondrial function before oocyte maturation. Thawed/warmed oocyte showed high level of ROS compare to control oocytes, on the other hand, for 3hr incubated oocyte in milrinone showed similar level of ROS to non-vitrified 3hr in vitro cultured oocyte (p< 0.05).
Extended culture of prophase I stage in milrinone of vitrified oocytes before in vitro maturation may help the recovery of antioxidant system.