Embryo vitrification affects the methylation of the H19/Igf2 differentially methylated domain and the expression of H19 and Igf2

Objective

To investigate the effects of embryo vitrification on the methylation status of the H19/Igf2 differentially methylated domain (DMD) and the expression of those genes.

Design

Comparative and controlled study.

Setting

A university-affiliated hospital.

Animals

ICR female and male mice.

Intervention(s)

Day 14 fetuses that arose from copulation of male and female mice were taken as the control group. Embryos resulting from superovulation, IVF, in vitro culture, and embryo transfer were taken as the IVF group. Embryos that were vitrified were taken as the vitrified group.

Main Outcome Measure(s)

The methylation status of H19/Igf2 DMD was analyzed by cloning and sequencing following DNA bisulfite treatment. The expression of H19 and Igf2 was quantified by real-time reverse-transcription polymerase chain reaction.

Result(s)

Loss of methylation was found in the H19/Igf2 DMD of the fetuses from the IVF group, but it was more severe in the vitrified group. H19 expression was significantly increased in the IVF and vitrified groups, as compared with the control group; however, the quantity in the vitrified group was less than that detected in the IVF group. Igf2 expression in the IVF and vitrified groups was found to have decreased significantly, whereas the Igf2 expression in the vitrified group was greater than in the IVF group.

Conclusion

Embryo vitrification aggravated the loss of methylation in the H19/Igf2 DMD, and compensated for the perturbed expression of H19 by IVF procedures.

Key Words: Embryo, vitrification, mouse, imprinting