Is embryo grade classification associated with pregnancy outcome for in vivo and ICSI-derived vitrified embryos?

The ability to predict the potential of an embryo to develop into pregnancy is valuable in any embryo transfer program. Equine embryos are typically graded using a system designed for in vivo embryos. Embryos are now routinely produced in vivo or in vitro by intracytoplasmic sperm injection (ICSI) and often transferred after vitrification and thawing. The purpose of this study was to investigate the relationship between embryo grade and pregnancy outcome for vitrified embryos. Blastocysts were graded during vitrification at 8 days of development for in vivo (N = 91) or ICSI (N = 73) derived embryos. Blastocoel aspiration was performed on in vivo derived embryos >300µm prior to vitrification. The membrane integrity of the embryos was confirmed during equilibration in the hypertonic vitrification solution. The blastocyst grading system was defined as Grade 1: Intact plasma membrane, differentiation of cells into trophectoderm and inner cell mass, shrinkage during vitrification. Grade 2: Intact plasma membrane, extrusions of cells, debris or lipid between zona pellucida and trophectoderm, shrinkage during vitrification, poorly defined differentiation. Grade 3: Intact plasma membrane, extrusions of cells between zona pellucida and trophectoderm, shrinkage during vitrification, poorly defined differentiation, low cell numbers and other irregularities. This grading system was then applied to commercially produced blastocysts which were subsequently vitrified, thawed, andtransferred to recipient mares. Overall, 56% of the in vivo (N=91) and 65% of in vitro ICSI (N=73) derived vitrified embryos resulted in pregnancy at 15 days of gestation. Generalized linear model analysis of factors influencing blastocyst survival after vitrification and transfer revealed a significant effect of origin (in vivo vs ICSI, p=0.034), embryo grade (1,2,3, p = 0.00), and recipient source (client vs on farm, p=0.02) on blastocyst survival. Logistic regression analysis revealed that the probability of pregnancy declined by 87% with each increase in embryo grade. However, there was no significant difference in the mean (±SEM) percentage of mares pregnant at 15 days of gestation after vitrification and transfer of in vivo (64.5±0.05%) or ICSI-derived (78.2±0.05%) Grade 1 blastocysts. Further analysis revealed that ICSI-derived commercial embryos after vitrification and transfer experience 17% pregnancy loss between days 15 and 60 of gestation, resulting in 54.2% of vitrified, transferred ICSI embryos producing a pregnancy at 60days. This compares with 4% pregnancy loss experienced between day 15 and 60 by vitrified in vivo derived embryos, resulting in 53.8% of the embryos transferred producing a pregnancy >60days of gestation. In conclusion, there was a significant effect of grade on embryo survival observed at day 15 of gestation but there was no significant difference in the percentage of mares pregnant at day 60 after transfer of vitrified in vivo or ICSI embryos.