Successful production of blastocysts following ultrarapid vitrification with step-wise equilibriation of germinal VesicleStage mouse oocytes

The purpose of this study was to evaluate the viability and subsequent developmental ability of murine germinal vesicle (GV) oocytes ultrarapidly vitrified after step-wise exposure to cryoprotectants (CPAs). Oocytes were transferred to a vitrification solution composed of 15% ethylene glycol, 15% dimethyl sulfoxide and 0.5 M sucrose in a direct manner (non-preequilibrium) or in a step-wise manner (single-, two-, or ten-step preequilibrium). After ultrarapid vitrification and storage in liquid nitrogen, the oocytes were thawed, washed by diluting the CPAs in five steps, and then subjected to in vitro maturation, fertilization and culture. In the non-preequilibrium group, the rates of post-thawed oocytes surviving, maturing to metaphase-II, developing to blastocysts and to hatching/hatched blastocysts were 91.8, 87.1, 15.9 and 2.3%, respectively. In the single- and two-step groups, the corresponding rates were 97.0-98.2%, 92.2-95.0%, 22.0-29.4% and 8.8-15.6%, whereas in the ten-step group they were 98.2, 91.8, 38.6 and 22.8%, respectively. In the non-vitrified control group, the rates of oocytes maturing to metaphase-II, developing to blastocysts and to hatching/hatched blastocysts were 90.2, 75.2 and 51.5%, respectively. The present study shows that the ultrarapid vitrification of murine GV oocytes by a step-wise manner involving 10 steps preequilibrium may have an advantage in maintaining the viability and subsequent production of blastocysts.