114 Effects of in vitro embryo production on the epigenomic profiles of day 15 bovine conceptus

In vitro-produced (IVP) embryos continue to surpass in vivo-produced (IVV) embryos worldwide. However, there is increased concern about the current drawbacks of IVP embryos, such as a higher pregnancy loss in the early stages of development often associated with impairment in conceptus elongation, which begins from approximately Day 13 of gestation. The embryonic disc (ED) and the extra-embryonic membranes (EEM), from which the fetus and the placenta respectively originate, differentiate during this period. The epigenomes of the ED and the EEM might be affected by the in vitro procedures, impairing conceptus development. This study aimed to identify changes in DNA methylation in the ED and EEM of Day 15 IVP conceptuses compared with those conceived in vivo. For IVP embryos (n = 8), oocytes aspirated from slaughterhouse ovaries were matured, fertilized, and cultivated in serum-free medium until transfer at Day 7. IVV embryos (n = 8) were generated after ovarian super-stimulation and AI. For both procedures, animals were flushed at Day 15 of gestation, and sections of the ED and EEM were snap-frozen in 0.1% PVA PBS until DNA extraction and sequencing through whole genome bisulfite sequencing. Raw fastq files were processed with the Bismark software, aligning the reads to the ARS-UCD1.3 bovine genome. IVP samples showed significantly higher methylation variation of the whole epigenome between each other than IVV samples (t-test; P < 0.001). Statistical analyses were performed with the Methylkit package for R. After subdividing the genome into tiles of 1000 bp, differentially methylated regions (DMRs) were determined separately for the ED and EEM using a logistic regression model that corrected for the effects of sex and morphology (adjusted P-value < 0.01, minimum absolute methylation difference > 20%). Only DMRs associated with genic and regulatory elements were considered for analysis. Of these DMRs, 15% (414) and 29% (400) were hypermethylated in IVP samples in the ED and EEM, respectively. The DMRs were subjected to overrepresentation analysis with the DAVID database (FDR < 0.05). DMRs (n = 2776) of the ED showed a large overrepresentation of gene ontology terms relating to development and cell differentiation, with some degree of differential methylation in regulation with GTPase activity. DMRs (n = 1361) of the EEM showed a comparatively smaller overrepresentation in developmental terms but a larger cluster of terms relating to signaling and its regulation, with a more notable overrepresentation of GTPase-mediated signal transduction. KEGG pathways related to the corpus luteum, such as Oxytocin and Relaxin pathways, and lineage segregation, such as Hippo and Wnt signaling pathways, were enriched with the hypermethylated (n = 2077) and hypomethylated (n = 4460) regions in the EEM when a broader subset of DMRs was used (adjusted P-value < 0.1, minimum absolute difference > 10). In conclusion, compared with the IVV conceptus epigenome, the Day 15 IVP conceptus epigenome was more heterogeneous, suggesting a variable effect of the in vitro procedure. However, both the ED and EEM regions were altered, which potentially affected the conceptus transcriptome and its development beyond this stage.