Effect of 5-aza-2′-deoxycytidine on methylation of the putative imprinted control region of H19 during the in vitro development of vitrified bovine two-cell embryos

Objective: To determine the effect of vitrification and 5-aza-2'-deoxycytidine (5-aza-dC) on the methylation levels of the putative imprinted control region (ICR) of H19 and H19 expression in bovine two-cell embryos and their derived blastocysts. Design: Experimental animal study. Setting: Academic institution. Animal(s): Abattoir-derived bovine ovaries. Intervention(s): Vitrified two-cell embryos were cultured in vitro to blastocysts with 0.01 mu M 5-aza-dC (5-aza-dC group) or without 5-aza-dC (vitrification group). Fresh embryos and their derived blastocysts were used as control. Main Outcome Measure(s): Putative ICR methylation of H19 was measured by bisulfate mutagenesis and sequencing, blastocyst development rate; total cell number were determined; and H19 expression was measured by real-time reverse transcriptase-polymerase chain reaction (PCR). Result(s): Vitrification significantly increased putative ICR methylation of H19 in two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced putative ICR methylation of H19 in vitrified two-cell embryos and their derived blastocysts. The H19 expression level was significantly higher in blastocysts from the 5-aza-dC group than the vitrification group. The blastocyst development rate and total cell number in the 5-aza-dC and vitrification groups were similar. Conclusion(s): Putative ICR methylation levels of H19 significantly increased in vitrified two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced putative ICR methylation of H19 and increased H19 expression in blastocysts derived from vitrified two-cell embryos. (Fertil Steril (R) 2012; 98: 222-7. (C) 2012 by American Society for Reproductive Medicine.)