The Effect of Equilibration Temperature and Time Prior to Cryopreservation on the Outcome of Cryopreservation of 2-Cell Mouse Embryos

Ko, Hee Eun; Chung, Kyung Nam; Kim, Yong Bong

Abstract

OBJECTIVE
One-cell human zygotes have been successfully frozen and thawed using 1,2-propanediol (PROH) during freezing and thawing. This study was performed to assess the effect of equilibration temperature and time on cryopreservation of 2-cell mouse embryos by investigating the equilibration temperature and time during initial PROH exposure prior to cryopreservation.
MATERIALS AND METHODS
The late 2-cell mouse embryos were obtained from 5-6 week old ICR mice and were exposed to 1.5 M PROH in phosphate-buffered saline (PROH-PBS) at 37degrees C, room temperature (22- 24degrees C), and 4degrees C for 30 min. The PROH was washed off the 2-cell mouse embyos by incubating them for 5 min each in 1, 0.5, and then 0 M PROH-PBS in the order named. The 2-cell mouse embryos were subsequently cultured in Ham's F-10 medium and embryo development was assessed at 24, 48, and 96 hours.
RESULTS
Incubation of 2-cell mouse embryos at 37degrees C for 30 min significantly impaired embryo development to blastocysts. Embryo development after exposure to PROH at 37degrees C for 10 min was 8.3% (P=0.047) and embryo development for 30 min was 5.4% (P=0.038). Incubation of 2-cell mouse embryos at room temperature or 4degrees C for up to 30 min did not significantly reduce embryo development. Cryosurvival of 2-cell mouse embryos exposed to PROH at room temperature or 4degrees C was similar.
CONCLUSION
These findings suggested that pronanediol is toxic to 2-cell mouse embryos in a temperature- and time-dependent fashion. Cryopreservation of 2-cell mouse embryos after exposure at 4degrees C appears to be no better than after exposure at room temperature.