Antioxidants in Mouse Embryo Culture and Vitrification Media Maintain an In Vivo Like Gene Expression Profile

Abstract

Background: Embryo culture and cryopreservation causes detrimental effects on blastocyst gene expression. While combined antioxidants (acetyl-L-carnitine/N-acetyl-L-cysteine/[Formula: see text]-lipoic acid (A3)) in culture and vitrification media have beneficial effects on embryo and fetal development, their impact on fetal gene expression has not been assessed. Therefore, the effects of antioxidants on global gene expression were examined from fetal and placental tissue of transferred blastocysts and compared to in vivo derived embryos. Aim: To determine the effect of A3 in culture and vitrification media on mouse blastocyst, fetal and placental gene expression. Method: Embryo transfers were conducted on in vivo flushed blastocysts or blastocysts cultured or vitrified with and without A3. Transcriptional profiles of E14 fetal liver and placental tissue in all groups were quantified using RNAseq and functional analyses (KEGG). Results: Compared to in vivo derived embryos, in vitro culture altered the expression of 3601 fetal liver and 408 placental genes. Functional analysis showed upregulation/enrichment of oxidative phosphorylation and mitochondrial function and activity. Similarly, vitrification led to 2018 liver and 216 placental differentially expressed genes (DEGs). Upregulated KEGG pathways were enriched for cell and tissue development and cell cycle regulation. Down regulated pathways were associated with metabolism and immune response. Interestingly, exposure to A3 during development significantly reduced the number of DEGs with 1855 and 4 identified in liver and placentae respectively with no KEGG pathways identified. Correspondingly, exposure to A3 during vitrification reduced the number of DEGs to 1017 in liver and 206 in placentae. Functional pathway enrichment was similar to embryos vitrified without A3, although with decreased expression. Conclusion: Supplementation with antioxidants reduce the number of DEGs and biological processes altered by in vitro culture and vitrification, in fetal liver and placentae, establishing a more in vivo like gene expression profile which may help maintain the viability of vitrified human embryos.