Vitrification of mouse embryos at 2-cell, 4-cell and 8-cell stages by cryotop method

Abstract

Purpose The objective of this study was to investigate the effects of vitrification on the preimplantation developmental competence of mouse 2-cell, 4-cell and 8-cell stage embryos. Methods Mouse 2-cell, 4-cell and 8-cell stage embryos were cryopreserved using the cryotop vitrification method and subsequently warmed on a later date. The embryos were then assessed by their morphology, blastocyst formation and hatching rates. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in hatched blastocysts from the control and experimental groups. Results Vitrified embryos at the 2-cell, 4-cell and 8-cell stages appeared morphologically normal after warming. The overall survival rate of vitrified embryos at various stages after warming was 96.7% and there were no significant differences among 2-cell stage (96.0%), 4-cell stage (96.8%) and 8-cell stage (97.1%) embryos (P>0.05). The blastocyst formation rate (69.4%) and hatching rate (52.6%) of vitrified 2-cell embryos were significantly lower than that from the control group and vitrified 8-cell embryos (P<0.05). In the vitrified 4-cell embryo group, the blastocyst formation rate (90.3%) was similar to the 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of the non-vitrified control ( 84.1%) and vitrified 8-cell embryo (78.4%) groups (P<0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell and 8-cell embryos had significantly lower cell counts both in the ICM and TE, as compared to fresh blastocysts (P<0.05). Among the vitrified 2-cell, 4-cell and 8-cell embryo groups, there were no significant differences in the cell counts of ICM and TE (P>0.05). Conclusions Although cryotop vitrification was suitable for the cryopreservation of mouse embryos from the 2-cell stage, 4-cell stage and 8-cell stage without significant loss of survival, vitrification had an adverse effect on the development of 2-cell embryos. Mouse embryos at the 8-cell stage had the best tolerance for vitrification and would yield the highest level of post-vitrification developmental competence among early cleavage stage embryos. Nevertheless, it is unclear how these findings can be extrapolated to human embryos.

Cryopreservation of mammalian embryos: Advancement of putting life on hold

“…Hatched blastocysts developed from vitrified zygotes by cryotube, and in-straw methods (Bagis et al, 2004(Bagis et al, , 2005 and from vitrified two-cell, four-cell, and eightcell embryos by cryotop method (Zhang et al, 2009b) did not show significant alteration in cell numbers in developed blastocysts.…”

“…Reciprocal studies on the effect of vitrification on the number of cells in blastocysts were conducted by Bagis et al (2004) and Zhang et al (2009b), independently. Hatched blastocysts developed from vitrified zygotes by cryotube, and in-straw methods (Bagis et al, 2004(Bagis et al, , 2005 and from vitrified two-cell, four-cell, and eightcell embryos by cryotop method (Zhang et al, 2009b) did not show significant alteration in cell numbers in developed blastocysts.…”

Melatonin modulates the expression of BCL-xl and improve the development of vitrified embryos obtained by IVF in mice

“…Several studies on mammalian embryos have proven that the best tool for vitrification is cryotop [28,[34][35][36]. In the Zhang study [36], the vitrified mouse embryos derived in vivo with cryotop showed survival rates of 96 % for two-cell vitrified embryos. Yan and coworkers [37] vitrified two-cell mouse embryos derived from IVF with OPS, and reported the survival rate of 51 %.…”

Human cleavage-stage embryo vitrification is comparable to slow-rate cryopreservation in cycles of assisted reproduction

“…The vitrification of cleavage-stage embryos has also been tested [9][10][11]. At the present, a wide variety of protocols and procedures are available [12][13][14].…”