Abstract
PURPOSE: Dimethyl sulfoxide (DMSO) alters DNA methylation in vitrified-warmed embryos and potentially affects subsequent development. This study aimed to examine possible countermeasures against DMSO-induced demethylation. METHODS: In vitro-produced bovine embryos (8-cell stage) were vitrified using a combination of DMSO and ethylene glycol (EG) or propylene glycol (PG) + EG. After warming, the lipid content and expression levels of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), DNMTs, and TETs were examined. In addition, RNA-sequencing was performed on blastocysts derived from the vitrified embryos. Furthermore, the effect of supplementation with a vitrification medium containing DMSO and N-acetyl-L-cysteine (NAC, 5 mM) on the levels of 5mC in embryos was examined. RESULTS: Vitrification decreased the levels of 5mC and increased the levels of 5hmC in 8-cell stage embryos. Low levels of 5mC persisted until the blastocyst stage in the DMSO group but increased in the PG group. The expression level of TET3A was higher in the DMSO group than in the fresh group, but not in the PG group. Both cryoprotectants reduced the lipid levels in post-warmed 8-cell stage embryos. The addition of NAC ameliorated DMSO-induced demethylation at both the 8-cell and blastocyst stages. RNA-seq analysis revealed that PG-specific pathways included ribosomes and mitochondria and that both DMSO and PG affected cGMP-PGK, MAPK, Wnt, and insulin secretion-related signaling. The K-medoids method predicted that DMSO affected cell adhesion molecules and that MAPK signaling was affected the most. CONCLUSIONS: PG and NAC may antagonize DMSO-induced demethylation; however, PG exerts adverse effects on embryos.