Marked blastomere herniation is the only post-thaw morphological characteristic associated with a reduced pregnancy...

It is not known whether the likelihood of pregnancy after transfer of cryopreserved equine in vitro produced (IVP) blastocysts is related to post-thaw morphological characteristics, or could be impacted by other factors such as embryo biopsy prior to cryopreservation. This study examined associations between post-thaw morphological appearance of equine IVP embryos, and biopsy prior to slow-freezing, on the likelihood of pregnancy. Biopsy of IVP blastocysts was performed by harvesting cells herniated through the intra-cytoplasmic sperm injection (ICSI)-derived hole in the zona pellucida (ZP). Morphological characteristics (shape, extent of presumptive trophectoderm layer, % extruded blastomeres, expansion and % herniated cells) of frozen-thawed, biopsied (n = 36) and non-biopsied (n = 444) IVP blastocysts were recorded prior to transfer, and pregnancy outcome was recorded. A binomial multivariate generalized linear model was used to identify factors influencing pregnancy outcome. The overall pregnancy rate was 73.3 %, but was significantly affected by the interval between ICSI and confirmation of blastocyst formation, with lower pregnancy rates for later developing embryos (p < 0.01). Furthermore likelihood of pregnancy was significantly influenced by the stallion used for ICSI (p < 0.01) and the biological mother (p = 0.02), although no single biological sire or dam could be identified as contributing significantly. The biopsy procedure per se did not compromise pregnancy outcome (p = 0.59). On the other hand, pregnancy was less likely (p = 0.02) for blastocysts with more than 10 % herniated cells post-thaw (39 %; n = 13) than for those with fewer than 10 % (69 %; n = 166) or no (77 %; n = 301) herniated cells. No other post-thaw morphological characteristic affected pregnancy outcome. In conclusion, the sporadic herniation of a large part of the embryo through the ICSI-derived hole in the ZP during thawing negatively influences pregnancy outcome.