Vitrification of Mature Mouse Oocytes: Improved Results Following Addition of Polyethylene Glycol to a Dimethyl Sulfoxide Solution

Abstract

Oocytes have been successfully cryopreserved using rapid and slow freezing procedures; however, variability in the success of replicates has limited its practical application. We have evaluated the potentially beneficial effects of adding 1 mg/ml of the polymer polyethylene glycol (PEG) (Mr8000) to a 6Mdimethyl sulfoxide (Me2SO) vitrification solution. Stepwise addition of cryoprotectant, either with or without PEG, was performed at room temperature (19–21°C). Oocytes were then loaded in plastic insemination straws and held in liquid nitrogen vapour at −140°C for 3 min prior to storage in liquid nitrogen. Oocytes were warmed rapidly to room temperature and removal of cryoprotective agent was effected in the presence of 1Msucrose solution. Viability was assessed byin vitrofertilization. Oocytes cryopreserved after exposure to 6MMe2SO in the absence of PEG showed 60% normality, 80% fertilization, and 55% development to blastocyst, median of 11 replicate experiments (191 oocytes). Individual replicates yielded highly variable survival which ranged from 0 to 100%. The addition of PEG significantly improved oocyte normality to 95% (range, 76–100%; median of 9 replicate experiments, 301 oocytes). Rates of fertilization (91%; 60–100%) and development to blastocyst (73%; 67–92%) were also improved. The addition of 1 mg/ml PEG to a 6MMe2SO solution resulted in greatly improved viability of oocytes following cryopreservation and vastly reduced the variability seen with the Me2SO solution alone.