Application of vitrification to human embryo freezing.

To solve the problem of multiple pregnancies during the in vitro fertilization (IVF) and embryo transfer procedure, excess embryos must be cryopreserved for embryo transfer in future. We applied the vitrification method to cryopreservation of human embryos. A total of 31 frozen-thawed embryo transfer cycles were analyzed at the Yamagata University Hospital, Yamagata, Japan. The patients were introduced to IVF treatment and had an excess of valuable embryos to be frozen after the transfer of three fresh embryos that did not result in establishing a pregnancy. Excess human 8- to 16-cell stage embryos were exposed to vitrification solution and then frozen in liquid nitrogen. The cryoprotectant was removed by washing the embryos in media containing different concentrations of cryoprotectant. Three days after LH surge and/or 2 days after ultrasonographic ovulation the embryos were transferred. The rate of poor quality embryos significantly increased and the rate of good quality embryos decreased after thawing the embryos frozen by the vitrification method. In menstrual cycles with good quality embryo transfer, a higher rate of pregnancies was established than in the cycles in which fair or poor quality embryos were the highest grade of embryos transferred into the uterus. In total, 5 pregnancies were established from 31 embryo tansfers; 4 pregnancies were in cycles associated with the transfer of good quality embryos, and 1 pregnancy was in a cycle in which the highest grade of embryo was fair. When compared with slow embryo freezing methods, vitrification has marked advantages for clinical application in terms of cost and time. Vitrification will be an alternative method for embryo freezing.

The optimal equilibration time for mouse embryos frozen by vitrification with trehalose.

To determine the best equilibration time in the cryoprotectant before rapid cooling, 8-cell mouse embryos were exposed to a vitrification solution containing ethylene glycol, Ficoll and trehalose in modified phosphate-buffered saline at 5 degrees C for varying periods of time. They were frozen using an ultra rapid freezing method, thawed in a 20 degrees C water bath and cultured for 24 h with 5-bromo-2-deoxyuridine. Embryo development and the number of sister chromatid exchanges, a sensitive indicator of genetic damage, were observed. The results demonstrated that embryo development after freezing and thawing was similar among the groups exposed for periods of 5-40 min. However, the number of sister chromatid exchanges was significantly smaller in the group exposed for 5 min, indicating that this was the safest equilibration time in the vitrification solution.