Which Stage of Mouse Embryos Is More Appropriate for Vitrification?

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Abstract

BACKGROUND:

Vitrification has been shown as one of the most effective methods of cryopreservation for mammalian embryos. However, there is no consensus which stage of embryonic development is the most appropriate for vitrification with subsequent maximal development after thawing. This study was carried out to explore and compare the effect(s) of vitrification on mouse 2-cell, 4-cell, 8-cell, morula and blastocyst stage embryos and subsequent blast formation and hatching after thawing.

MATERIALS AND METHODS:

In this experimental study, 2-cell embryos were obtained from the oviducts of super ovulated female NMRI mice. Some embryos were randomly selected and vitrified through a two-step media protocol and cryotop. Other embryos were cultured to assess their development. During the ensuing days, some of these cultured embryos were vitrified at 4-cell, 8-cell, morula and blastocyst stages. After 10 to 14 days, the embryos were thawed to assess their survival and also cultured to determine the rate of blastocyst formation and hatching. The results were analyzed using one-way ANOVA and Tukey's post-hoc tests.

RESULTS:

There was no significant difference in the survival rates of vitrified embryos at 2-cell, 4-cell, 8-cell, morula and blastocyst stages after thawing (P>0.05). The blastocyst formation rate of vitrified 8-cell embryos was significantly higher than that of 2-cell embryos (P<0.05). The hatching rate of vitrified 4-cell, 8-cell and blastocysts were significantly higher than that of 2-cell embryos (P<0.05).

CONCLUSION:

Vitrification is suitable for cryopreservation of all stages of mouse embryonic development. However, the best tolerance for vitrification was observed at 4and 8-cell stages of development. Accordingly, the development of vitrified embryos to blastocysts, following thawing, was most efficacious for 4 and 8-cell embryos. Compared to mouse 2-cell embryos, embryos vitrified as blastocysts had the highest rate of hatching.

Table 1

Mouse embryonic development after early cleavage-stage embryo vitrification-warming


Stage of vitrification	Embryos (n)	Survival rate (%) ± SD	Blastocysts formation (%) ± SD	Hatched(%) ± SD

Vitrified 2-cell	157	82.1%±16.8	56.5%±23.4	21.2%±14.7
Vitrified 4-cell	145	83.2%±11	79.8%±13.6	49.1%±25.5*
Vitrified 8-cell	166	85.8%±22.4	84%±21.2*	50.6%±25.7*
Vitrified morula	180	77%±17.8	75.8%±21	30%±16.3
Vitrified blastocyst	140	60.5%±22.3	-	59.8%±25.7*

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2017年2月21日