Study on the Development of Efficient Vitrification of Human Blastocysts

Lee, Sang Min; Lee, Ju Hee; Lee, Sang Won; Lee, Seoung Min; Yoon, San Hyun; Lim, Jin Ho; Park, Huem Dai; Lee, Seong Goo

Abstract

OBJECTIVE
The purpose of this study was to evaluate the survival rate of vitrified blastocyst according to the freezing vessels, equilibration time in cryoprotectant and artificial dehydration method.
METHODS
Human blastocysts were vitrified after loading onto the plastic straw, open-pulled straw (OPS), electron microscopy grid (EM grid) for 1.5 min or 3 min. They also were directly plunged into LN2 within 30sec. For artificial shrinkage of blastocysts, 36 gauge fine needle was pushed at the cellular junction of the trophectoderm into the blstocoele cavity until it shrank without damage of inner cell mass.
RESULTS
The survival rate of vitrified blastocysts on plastic straw, OPS, EM grid as freezing vessels were 26.7, 13.0 and 60.5%, respectively. The survival rate of EM grid was significantly higher than that of plastic straw and OPS (p<0.05). For 1.5 min equilibrium, the survival rates of early blastocyst (EB), middle blastocyst (MB) and late blastocyst (LB) were 64.4, 81.0, and 20.0% respectively. For 3 min equilibrium, the survival rates of EB, MB, and LB were 69.9, 50.0 and 57.5% respectively. The survival rates of EB and MB were significantly higher than that of LB in 1.5 min equilibrium group (p<0.05), however, the significance was not observed in 3 min equilibrium groups. In cytoplasmic shrinkage before vitrification, the survival rates of EB, MB and LB were 92.9, 100 and 75.9% respectively. The survival rate of MB was significantly higher than that of LB (p<0.05). The survival rates of vitrified blastocysts by artificial dehydration and slow-frozen blastocysts were not significantly different as 88.9 and 66.7%, respectively.
CONCLUSION
This study showed that the vitrification of human blastocysts using EM grid and artificial dehydration is an effective method. Therefore, these methods would be an useful techniques for blastocyst cryopreservation.