In vitro development and gene expression of frozen-thawed 8-cell stage mouse embryos following slow freezing or vitrification

Abstract

Objective: This study was performed to compare the efficiency of slow freezing and vitrification based on survival, development to blastocysts, and cell numbers of blastocysts. Changes in embryonic gene expression in fresh and frozen-thawed embryos were also examined.

Methods: Eight-cell stage embryos were collected from superovulated female BDF1 mice. The collected embryos were randomly divided into three groups. One group was maintained as fresh controls (n=42), one was frozen by slow freezing (n=43), and one was cooled by vitrification (n=43). After thawing or cooling, survival rates, development to blastocyst, and cell numbers and inner cell mass (ICM) cell numbers of blastocysts were compared with those of the control group. The expressions of eight genes (Rbm3, Birc5, Sod1, Sod2, Cirbp, Caspase3, Trp53, Hsp70.1) were examined by real time-quantitative polymerase chain reaction in the fresh and frozen-thawed embryos.

Results: There were no significant differences in the slow freezing and vitrification groups' survival rate after thawing (88.4% vs. 88.4%), development to blastocyst (100% vs. 97.4%), cell numbers (107.0±21.0 vs. 115.0±19.7), or ICM cell numbers of blastocysts (11.3±5.2 vs. 11.1±3.7). Cell numbers of blastocysts were significantly (p<0.05) lower in the frozen-thawed embryos than the fresh embryos. There were no significant differences in the slow freezing and the vitrification groups' expressions of the eight genes. The expressions of CirbP and Hsp70.1 were higher in the frozen-thawed embryos than in the fresh embryos but there were no significant differences.

Conclusion: These results suggest that there were no significant differences between embryos that underwent slow freezing and vitrification.

Keywords: Cryopreservation; Gene expression; Human; RT-qPCR; Slow freezing; Vitrification.

Figure 1

Relative expressions of the eight genes in thawed 8-cell mouse embryos frozen by slow freezing or vitrification. There was no significant difference in gene expressions among the groups. Relative expression levels are expressed as mean±SE.