Embryonic survival, development and cryoinjury of repeatedly frozen mouse preimplantation embryos

Abstract

Objective Although guidelines about the number of embryos to be transferred differ among each country or individual IVF center, efforts to reduce the number of transferred embryo in a single cycle have been continuously made. If embryos were frozen in the past cycle and one or more embryos are transferred in the present cycle, surplus embryos should be re-frozen. It has been concerned that re-frozen embryos would have great cryoinjury, low survival, and subsequent poor pregnancy success. The aim of this study is to investigate the embryonic survival, development, and expressions of cryoinjury-related genes in once-frozen, twice-frozen, and three-time-frozen mouse preimplantation embryos using indirect vitrification methods.

Methods Experimental animal study. Six hundred 8-cell stage embryos were obtained from 60 female mice and randomly assigned to control and three experimental groups (150 embryos per each group). 8-cell stage embryos were vitrified by indirect vitrification methods and warmed once, twice, and three times, respectively. We analyzed the developmental outcome including survival rate, blastocyst formation rate, the percentage of hatching/hatched blastocyst including the cell number differences. And we used DAPI staining in hatching or hatched blastocysts for cell count. And a part of hatching or hatched blastocysts were subjected to real-time quantitative RT-PCR (qRT-PCR) for quantification of cryoinjury-related genes. We examined the expressions of cryoinjury related genes (Casp3, Cirbp, Sod 1, Gpx 3, Cat). And we also evaluated three anti-oxidant enzymes against ROS production on mouse embryo (Sod 1, Gpx 3, Cat). Data were analyzed by the comparative cycle threshold method in all experiments using Histone H2A.Z (H2afz) as endogenous reference genes. All mRNA expressions were normalized to that of H2afz mRNA. The experiments were repeated 6 times using different sets of embryos.

Results In three frozen groups, overall survival rate, blastocyst-formation rates, the proportions of hatching/hatched blastocyst, and the cell count in hatching/hatched blastocyst were all similar compared to non-frozen control group. The mRNA expression of Casp 3, Cirbp showed no significant differences between 4 groups. And the mRNA expression of Sod 1, Gpx 3, Cat was significantly elevated in the experimental group.

Conclusion This is the first study to evaluate the efficacy of three times of repetitive freezing-warming of mouse 8-cell embryos, and also the first study using indirect (closed) vitrification method. Repeatedly frozen mouse preimplantation 8-cell embryos even up to three times could preserve good survival and embryonic development. Similar expression of mRNAs for cryoinjury-related markers (Cirbp, Casp 3) or elevated anti-oxidant related enzymes (Sod 1, Gpx 3, Cat) indicates the safety of repeatedly frozen embryos at a molecular level. We demonstrated that closed (indirect) vitrification method also have efficacy and safety in frozen technologies.