ABSTRACT: This study compared the gene expression of extraembryonic membranes (EEM) from in vitro produced (IVP) and in vivo (AI) derived embryos. Samples of EEM from AI and IVP pregnancies were collected on days 18 and 32. A piece of the conceptus (day 18) or chorioallantois (day 32) were used for DNA and RNA isolation and subsequent determination of the sex by amplification of a Y-linked region of the DNA. The male and female ratios were analyzed by Chi-square of SAS. A subset of three female and three male EEM samples from each group (AI and IVP, days 18 and 32) were used for transcriptome analysis. Differentially expressed genes (DEGs) between groups, sex, and days were determined using edgeR-robust. A false discovery rate (FDR) <0.05 was used for statistical significance, and Toppgene platform was used for biological processes analysis. Sex ratio was similar on day 18 for AI and IVP groups. On day 32, the IVP group had a greater number of females than males (75 vs 25%, P=0.004). On day 18 female embryos, out of 71 DEGs identified, 50 were upregulated in IVP, and 21 in AI. The trophectoderm marker, TKDP2, was upregulated in the AI group, whereas IGF2, which is involved in placenta development, and APOA2, APOB, and APOE, which are important for lipid metabolism, were upregulated in the IVP group. Male embryos on day 18 had 22 DEGs, being 15 upregulated in AI and 7 in IVP. Female embryos on day 32 had 74 DEGs, with 21 upregulated in AI and 53 in IVP embryos. Some of the genes with increased expression in the IVP group were PTGIS, APOB, and APOH, which are related to lipid synthesis and metabolism. Male embryos on day 32 presented 899 DEGs, 564 upregulated in AI and 335 in IVP. Embryos from IVP had increased expression of genes related to embryo development, morphogenesis, neurogenesis, and developmental growth. In addition, male IVP embryos had decreased expression of genes related to lipid and carbohydrate metabolism. Interestingly, PLAC8B, BRB, pregnancy-associated glycoproteins (PAG) 7, 9, 10, and 19, were also downregulated in IVP male EEM. When comparing IVP males and females, there were 6753 DEGs, with 3091 being upregulated in females, and 3662 in male conceptuses on day 18. Some of the genes upregulated in females were related to placental function such as TKDP4, PLAC8A and PAGs 6, 8, 10, 12, 17, 18 and 19. On males, IFNT-related genes were upregulated (IFNT, IFNT2 IFNT3 and IFN-T). On day d32, there were 6772 DEGs, with 3252 genes with increased expression in female, and 3520 in male placentas. Among the genes increased in female placentas are genes involved in placentation: PAGs 17, 19, 9 and PLAC8B. On the other hand, PAG1 was detected as highly expressed in male placentas. In conclusion, IVP-derived male embryos were more susceptible to alterations in gene expression and these effects extend to the peri-implantation period including genes associated with placental development and markers of placental function.
