Transcriptome and DNA methylome profiling reveal molecular responses of bovine blastocysts to trophectoderm biopsy and post-biopsy vitrification

Trophectoderm biopsy and post-biopsy vitrification are used in bovine embryo selection and cryopreservation, but their molecular effects remain incompletely understood. We investigated associated transcriptomic and DNA methylation changes in individual bovine blastocysts. Fifteen morphologically intact in vitro-produced blastocysts were allocated to non-biopsied fresh (NB), biopsied (B), and biopsied and vitrified (BV) groups (5 each). Cytoplasmic RNA and nuclear DNA from each blastocyst were profiled by Smart-seq2 and whole-genome bisulfite sequencing, respectively. Differentially expressed genes (DEGs), differentially methylated regions (DMRs), and coordinated promoter methylation-expression changes were analyzed through pairwise comparisons and Gene Ontology and KEGG enrichment. B vs NB yielded 822 DEGs (390 up/432 down), BV vs B 1295 (777/518), and BV vs NB 1494 (887/607). Biopsy-associated DEGs were enriched in glycolysis/gluconeogenesis, pyruvate metabolism, mitochondrial biogenesis, steroid biosynthesis, and peroxisome pathways. Vitrification was additionally associated with endocytosis, cell adhesion, and MAPK, TGF-β, FoxO, and estrogen signaling pathways; apoptosis and carbon metabolism featured in BV vs NB. B vs NB had the fewest DMRs, BV vs NB the most hypermethylated, and BV vs B the most hypomethylated. Genes with concurrent hypermethylation and downregulation were enriched in the tricarboxylic acid cycle and fatty-acid, amino-acid, sulfur, and glycerophospholipid metabolism, whereas those with hypomethylation and upregulation were associated with cytoskeleton remodeling, focal adhesion, and PI3K-Akt, Ras, MAPK, and calcium signaling. Biopsy was associated with transcriptional and epigenetic remodeling, and vitrification with broader metabolic, intercellular-signaling, and cellular-stress alterations. These candidate responses need functional validation and may help refine embryo biopsy and cryopreservation protocols.