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. 2019 Aug;14(8):780-790.
doi: 10.1080/15592294.2019.1615353. Epub 2019 May 13.

Superovulation alters global DNA methylation in early mouse embryo development

Bo Yu  1   2 , Thomas H Smith  3 , Stephanie L Battle  1   3 , Shannon Ferrell  1 , R David Hawkins  2   3
Affiliations

Superovulation alters global DNA methylation in early mouse embryo development

Bo Yu et al. Epigenetics. 2019 Aug.

Abstract

Assisted reproductive technologies are known to alter the developmental environment of gametes and early embryos during the most dynamic period of establishing the epigenome. This may result in the introduction of errors during active DNA methylation reprogramming. Controlled ovarian hyperstimulation, or superovulation, is a ubiquitously used intervention which has been demonstrated to alter the methylation of certain imprinted genes. The objective of this study was to investigate whether ovarian hyperstimulation results in genome-wide DNA methylation changes in mouse early embryos. Ovarian hyperstimulation was induced by treating mice with either low doses (5 IU) or high doses (10 IU) of PMSG and hCG. Natural mating (NM) control mice received no treatment. Zygotes and 8-cell embryos were collected from each group and DNA methylomes were generated by whole-genome bisulfite sequencing. In the NM group, mean CpG methylation levels slightly decreased from zygote to 8-cell stage, whereas a large decrease in mean CpG methylation level was observed in both superovulated groups. A separate analysis of the mean CpG methylation levels within each developmental stage confirmed that significant genome-wide erasure of CpG methylation from the zygote to 8-cell stage only occurred in the superovulation groups. Our results suggest that superovulation alters the genome-wide DNA methylation erasure process in mouse early pre-implantation embryos. It is not clear whether these changes are transient or persistent. Further studies are ongoing to investigate the impact of ovarian hyperstimulation on DNA methylation re-establishment in later stages of embryo development.

Keywords: DNA methylation; assisted reproductive technologies; embryo development; epigenetics; ovarian hyperstimulation.

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Figures

Figure 1.
Figure 1.
Distribution of CpG methylation at shared sites. Violin plots displaying the distribution of methylation levels at shared CpG sites within each stage or treatment group. The x-axis represents the CpG methylation level, 0–100%. The number of shared CpG sites, ‘n’, and the percentage of total CpG sites (%) are provided above each graph and the mean methylation level (xˉ1) is provided beneath its respective violin plot. White dots: median methylation levels. (a) Within each treatment group, the distribution of methylated CpGs in zygote and 8-cell stage is plotted. (b) Within each developmental stage, NM was compared to 5 IU or 10 IU treated groups.
Figure 2.
Figure 2.
Differentially methylated CpGs genome-wide. (a) The number of significant (p < 0.01) differentially methylated CpGs (DMCs) is shown in the table for each comparison. DMCs can only be calculated for shared CpG sites in each pairwise comparison. The percentage after each number represents the percentage of shared CpG sites. (b) Circos plot showing the methylation levels of DMCs across the genome. Outer circle: DMCs for the 8-cell NM versus 5IU comparison; Inner circle: DMCs for 8-cell NM versus 10IU. Red dots: DMC methylation levels in the NM group; Blue dots: DMC methylation levels in superovulation (SO) groups.
Figure 3.
Figure 3.
Global and local CpG methylation changes. (a) Top: mean CpG methylation levels at each stage and treatment group. Bottom: Percent of DMCs in each genome annotation. Only the DMCs with higher methylation levels in the NM group in the NM versus SO comparisons were shown here. (b) Two examples of genes with DMCs (in red bars, NM versus SO) in the ATAC-seq peaks. Top: Igf2bp1; Bottom: Bach1.
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