Critical temperature point for storage of vitrified embryos

Abstract

OBJECTIVE: Objective of this study was to determine the critical temperature for storage of vitrified embryos with minimal cryo-injury and adverse effects on the embryonic development following thawing. DESIGN: Mouse embryos were vitrified and stored under liquid nitrogen (control group) or at −90 °C, −100°C, −110 °C, −122.5°C (experimental groups) for 5 hours. Embryos were then thawed and their developmental potential was evaluated. MATERIALS AND METHODS: 8-cell mouse embryos, previously frozen at 2-cell stage and cultured to the 8-cell stage after thawing were vitrified by treatment with 7.5% Ethylene Glycol + 7.5% DMSO, and 15% Ethylene Glycol + 15% DMASO + 0.5M sucrose and loading into V-Kim vitrification straws, a recently developed closed vitrifcation straw system (Isatis Scientific, California) before plunging into liquid nitrogen. Some of the straws were kept in liquid nitrogen (control group) and the rest were placed in a controlled freezing chamber at temperatures of −90 °C, −100°C, −110 °C, −122.5°C (experimental groups) for a period of 5 hours. Embryos were then removed from their respective temperature of storage and thawed by transferring to 1 and 0.5 M sucrose, and wash medium supplemented with 10% serum. Embryo survival and blastocyst formation rates were evaluated. Results were analyzed by one-way ANOVA. RESULTS: A total of 163 embryos were vitrified and divided into ‘Control’, ‘-90 °C’, ‘-100°C’, ‘-110 °C’, ‘-122.5°C’ groups. After thawing, 92 % of embryos survived in the control group and 82% developed to the blastocyst stage. Survival rates after thawing were 56%, 84%, 87%, and 90%; and blastocyst formation rates were 39%, 78%; 78%, and 79% for the −90 °C, −100°C, −110 °C, −122.5°C groups respectively. There were no significant differences in survival and blastocyst formation rates in all groups (P>0.05) except in the −90 °C group which had lower survival and blastocyst formation rates when compared with all other groups (P<0.05). CONCLUSIONS: The critical temperature for storage of vitrified mouse embryos is around −100 °C’. Storing below this temperature even for a short period of time has adverse effects on thaw rates and blastocyst formation. Experiments are underway to determine if this finding applies to other stages of mouse embryonic development and to human embryos.