98 Capturing the miracle: time-lapse imaging of equine embryos reveals cleavage patterns impact pregnancy success

In vitro production (IVP) of embryos has become a routine procedure in the equine industry. Despite an increase in the quality and quantity of embryos produced per intracytoplasmic sperm injection session, a low pregnancy rate and a high pregnancy loss are observed with the IVP embryos. Of special importance is the rate of early embryonic loss (EEL), which doubles in IVP embryos compared to those produced in vivo. Thus, a better understanding of embryonic developmental events might provide an insight for the cause of this higher embryonic loss, as well as aid in the classification of embryos based on their pregnancy potential. Embryo developmental events were analysed retrospectively in IVP embryos with known pregnancy rate at 14 days (n = 36) and 42 days (n = 21). Time-lapse imaging was used to evaluate morphokinetics and temporal checkpoints of embryonic development. Type of cleavage (normal (NC) or abnormal (AC)), percentage of extruded blastomeres (at morula compaction and initiation of pulsing), and developmental time points were associated with pregnancy and EEL likelihood. Statistical analysis was performed using the comparisons of proportions (‘N-1’ Chi-squared test) using MedCalc Software Ltd and JMP (SAS Institute, version 14 Pro). Pregnancy rate at 14 days was 72%. From the pregnant mares only 27% of the embryos presented AC (direct or indirect, 7/26) whereas 73% had NC (P = 0.001). From the mares that maintained pregnancy over 42 days, 87% had NC patterns (13/15) compared to AC 13%, (P = 0.0001). Both nonpregnant mares or those that presented EEL had a 50% incidence of AC. Of the embryos presenting AC and pregnant at 14 days, 60% were lost before 42 days (3 out of 5 pregnancies with available data). Further evaluation of the embryos with abnormal cleavage pattern revealed a difference in the percentage of extruded blastomeres at morula compaction between the embryos that yielded a pregnancy at 14 days (n = 7) in comparison to the embryos that did not yield a pregnancy (n = 5). In the AC embryos that lead to pregnancy, around 29% (20–50%; minimum–maximum) of extruded blastomeres were observed, while in the AC embryos that did not lead to pregnancy the extruded material accounted for 43% (10–70%) of the area. A minority of embryos with cleavage aberrations might be capable of compensating these early errors by the time of morula compaction. Cleavage pattern evaluation and blastomere exclusion percentage at morula stage presents an opportunity for embryo classification based on pregnancy risk. These results suggest an association between abnormal cleavage patterns and the likelihood of pregnancy maintenance in horses, similar to those observed in other species. Further investigation of the events leading to the first mitotic division could shed more light on the differences of IVP embryos.