Bovine in vitro blastocysts with distinct morphokinetic patterns show transcriptomic differences at genome activation

Bovine embryo in vitro production (IVP) is characterised by low efficiency and variable outcomes. Monitoring early embryonic development by videomicroscopy revealed substantial morphokinetic heterogeneity in the first four embryonic cycles (EC, conventionally referred to as the 2-, 4-, 8- and 16-cell stages). Morphokinetic analysis offers a promising approach to characterize divergent developmental trajectories and provide a better understanding of underlying molecular mechanisms. We developed a Random Forest classification system (Bovine Embryo Analyser based on Morphokinetics: BEAM) to predict embryo phenotype. It is based on morphokinetic variables collected from the 1st to the 4th EC and predicts four blastocyst categories (EHB: Early Hatching Blastocyst, HB: Hatching Blastocyst, SSB: Subtle Developmental Shift Blastocyst, ADB: Arrhythmic Development Blastocyst). Classification performance on an independent dataset was good (F1 score = 0.59; Accuracy = 0.78), indicating that the BEAM can be useful for embryo development studies. The BEAM was further applied to embryos submitted to 4.3 days of culture and having completed the 4th EC (16-32 cells). RNA sequencing was performed on sixteen samples (4 x 8 pooled embryos/category). The ADB category was significantly enriched in transcripts involved in the regulation of transcriptional activity compared to the EHB category. In addition, in the ADB category, 22.7% (n = 185/816) of the upregulated genes were of maternal origin while only 6.2% were of embryonic origin (n = 54/816). In conclusion, despite being at a comparable developmental stage and transcriptionally competent, the ADB category showed delayed maternal transcript degradation suggesting delayed transition to embryonic transcriptional autonomy.