In Vitro Culture Increases the Frequency of Stochastic Epigenetic Errors at Imprinted Genes in Placental Tissues from Mouse Concepti Produced Through Assisted Reproductive Technologies1

Abstract

Assisted reproductive technologies (ART) have enabled millions of couples with compromised fertility to conceive children. Nevertheless, there is a growing concern regarding the safety of these procedures due to an increased incidence of imprinting disorders, premature birth, and low birth weight in ART-conceived offspring. An integral aspect of ART is the oxygen concentration used during in vitro development of mammalian embryos, which is typically either atmospheric (~20%) or reduced (5%). Both oxygen tension levels have been widely used, but 5% oxygen improves preimplantation development in several mammalian species, including that of humans. To determine whether a high oxygen tension increases the frequency of epigenetic abnormalities in mouse embryos subjected to ART, we measured DNA methylation and expression of several imprinted genes in both embryonic and placental tissues from concepti generated by in vitro fertilization (IVF) and exposed to 5% or 20% oxygen during culture. We found that placentae from IVF embryos exhibit an increased frequency of abnormal methylation and expression profiles of several imprinted genes, compared to embryonic tissues. Moreover, IVF-derived placentae exhibit a variety of epigenetic profiles at the assayed imprinted genes, suggesting that these epigenetic defects arise by a stochastic process. Although culturing embryos in both of the oxygen concentrations resulted in a significant increase of epigenetic defects in placental tissues compared to naturally conceived controls, we did not detect significant differences between embryos cultured in 5% and those cultured in 20% oxygen. Thus, further optimization of ART should be considered to minimize the occurrence of epigenetic errors in the placenta.

Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant

“…Allele-specific bisulfite sequencing was performed as previously described using nested PCR for the H19 ICR (de Waal et al 2014). Briefly, bisulfite mutagenesis was performed with 1 µg of isolated genomic DNA from MEFs using the Epitect bisulfite kit (Qiagen).…”

Epigenetics and imprinting in human disease

“…Bovine models demonstrate that ART leads to increased large offspring syndrome with macrosomia, macroglossia, and abdominal wall defects and biallelically expressed imprinted genes seen in BWS . Interestingly, the ART conceptuses show a much greater imprinting perturbation in the placentas than in embryonic tissues (de Waal et al, 2014). While there are a number of possible explanations for this result, one of the most compelling explanations is that imprinted genes have redundant mechanisms to maintain parental-specific imprinting, including DNA methylation and post-translational histone modifications, in the embryonic lineages whereas extraembryonic tissues are less likely to employ both sets of epigenetic machinery in the maintenance of imprinted gene expression.…”

Humanized H19/Igf2 locus reveals diverged imprinting mechanism between mouse and human and reflects Silver–Russell syndrome phenotypes

“…RNA isolation and cDNA synthesis was performed as described previously (42). For qRT-PCR, total expression levels of H19 and Igf2 were measured relative to the geometric mean of expression levels of Arbp (acidic ribosomal phosphoprotein P0), Nono (non-POU domain-containing, octamer-binding protein), and Rpl13a (ribosomal protein L13A).…”