Superovulation Influences Methylation Reprogramming and Delays Onset of DNA Replication in Both Pronuclei of Mouse Zygotes

Abstract

Although an essential component of assisted reproductive technologies, ovarian stimulation, or superovulation, may interfere with the epigenetic reprogramming machinery during early embryogenesis and gametogenesis. To investigate the possible impact of superovulation particularly on the methylation reprogramming process directly after fertilization, we performed immunofluorescence staining of pronuclear (PN) stage embryos with antibodies against 5mC and 5hmC. PN stage embryos obtained by superovulation displayed an increased incidence of abnormal methylation and hydroxymethylation patterns in both maternal and paternal pronuclear DNA. Subsequent single-cell RT-qPCR analyses of the Tet1, Tet2, and Tet3 genes revealed no significant expression differences between PN stage embryos from spontaneously and superovulated matings that could be causative for the abnormal methylation and hydroxymethylation patterns. To analyze the possible contribution of TET-independent replication-associated demethylation mechanisms, we then determined the 5mC and 5hmC levels of PN stage mouse embryos using immunofluorescence analyses after inhibition of DNA replication with aphidicolin. Inhibition of DNA replication had no effect on abnormal methylation and hydroxymethylation patterns that still persisted in the superovulated group. Interestingly, the onset of DNA replication, which was also analyzed in these experiments, was remarkably delayed in the superovulated group. Our findings imply an impact of superovulation on both replication-dependent and -independent or yet unknown demethylation mechanisms in PN stage mouse embryos. In addition, they reveal for the first time a negative effect of superovulation on the initiation of DNA replication in PN stage mouse embryos.

Embryo responses to stress induced by assisted reproductive technologies

“…An increased incidence of abnormal methylation and hydroxymethylation patterns has been observed in both maternal and paternal pronuclear DNA in zygotes obtained by superovulation. Although no differences in the expression of Tet1 , Tet2 , and Tet3 genes were observed, a delay in the onset of DNA replication was induced by superovulation (Diken et al, ). Gonadotropin stimulation has also been associated with epimutations in somatic tissues and delayed epigenetic reprogramming in spermatogenic cells in the offspring (de Waal et al, ).…”

The proteome, not the transcriptome, predicts that oocyte superovulation affects embryonic phenotypes in mice

“…Some studies reported an increase of the rate of embryo aneuploidy and fetal malformation by use of gonadotropins 13 – 17 , while others contradicted these findings 4 , 18 , 19 . Some studies linked the superovulation to a direct effect on the acquisition of DNA methylation in oocytes 20 – 24 , including epimutations 25 , while other studies found no such effect 26 , 27 . In two studies that correlated oocyte diameters to oocyte maturity and DNA methylation 28 , one study suggested that superovulation results in smaller immature oocytes that have not completed the acquisition of DNA methylation 29 , whereas the other study found that oocyte diameters do not differ between superovulation and natural ovulation 27 .…”

“…Some studies reported an increase of the rate of embryo aneuploidy and fetal malformation by use of gonadotropins [13][14][15][16][17] , while others contradicted these findings 4,18,19 . Some studies linked the superovulation to a direct effect on the acquisition of DNA methylation in oocytes [20][21][22][23][24] , including epimutations 25 , while…”

Does the elevated estradiol level on the day of human chorionic gonadotropin administration affect embryo quality among women undergoing in vitro fertilization?

“…In addition, exogenous hormone stimulation contributes to epigenetic changes in oocytes and developing embryos. One study has shown that murine embryos conceived after superovulation may have superovulation‐induced DNA lesions and reduced viability, 22 whereas another found that hydroxymethylation and methylation in both the maternal pronucleus and paternal pronucleus of the zygote and the onset of zygotic DNA replication are delayed in IVF cycles 23 . Fauque et al 24 showed that superovulation disrupted H19 gene expression in individual embryos and that developmental kinetics and methylation of the H19 imprinting control region were affected by ART.…”