Understanding the effect of in vitro procedures on day15 elongated bovine embryo through transcriptomic data analysis and performing ML validation of key genes

Abstract

The objectives were to identify how the in vitro (IVP) and in vivo (IVV) procedures impact the trophectoderm (TE) and embryonic disk (ED) of the same length, day 15 bovine embryo by identifying differentially expressed genes (DEG) between IVV and IVP and quantifying the effect on the regions by transcriptomic analyses. DESeq2 package in R was used to determine DEG (FDR < 0.05). Enriched biological processes (FDR < 0.05) associated with DEG in the TE or ED between IVP and IVV embryos were determined using the DAVID database. There were 801 and 1530 DEG in the TE and ED, respectively, between IVP and IVV embryos. Cohen’s d score, which was used to calculate biological significance, showed a score of 0.55, indicating that genes of TE region are more impacted than ED due to IVP processes. The functional analysis showed that upregulated DEG in the TE of IVP embryos (429) enriched cell migration, while down-regulated genes (372) were associated with transcription and ontological terms involved in chromatin structure, such as core histones, DNA binding, cellular biosynthesis, RNA metabolism and nucleosome. Upregulated DEG in the ED of IVP embryos (626) were involved in glycerophospholipid and fatty acid metabolism. Notably, downregulated (or inhibited) DEG (963) enriched anatomical morphogenesis processes and pathways playing critical roles in embryonic development, such as canonical Wnt, bone morphogenetic protein and transforming growth factor beta signaling pathways. Top 100 significant features were selected based on high expression values, log fold change (LFC)> 2 and FDR <0.0001 of genes in top GO terms. These features were used to develop machine learning models for predicting procedures based on the region. svmLinear and Random Forest models achieved the accuracy of 0.9091 and 0.7273 respectively. In conclusion, these results demonstrated that the in vitro procedure deregulated the molecular signatures of TE and ED regions during early gastrulation and negatively impacted the developmental process of IVP embryos, even when they were of similar length to the IVV counterparts, which can explain why most of the IVP conceptuses are lost after this period.