The journey through infertility carries a high emotional toll, but treatment with assisted reproductive technology (ART) has provided successful options for family building. This success is often measured by live birth by both the patient and provider. Success, however, has its trade-offs. With an increasing number of children born by ART, we are gaining more insight regarding the possible adverse effects of ART. For example, pregnant women who conceive via ART have a higher likelihood of preeclampsia, gestational diabetes, and placental abnormalities as well as delivering a baby with low birth weight. Despite much observational data, it is still unclear by what mechanism ART causes these differences.

Epigenetics has emerged as a potential etiology of some of these observed results, challenging the idea that in vitro fertilization outcomes should be measured by live birth rates alone. The data to date exploring the effect of ART on deoxyribonucleic acid (DNA) methylation disruptions in newborns has been far from consistent. In multiple studies, DNA methylation changes have been observed in children conceived by ART, particularly when global DNA methylation has been assessed utilizing genome-wide assays (1Mani S. Ghosh J. Coutifaris C. Sapienza C. Mainigi M. Epigenetic changes and assisted reproductive technologies.). Although confounding factors such as age and infertility are likely contributory, the differences in methylation appear to be a consequence of ART itself and not simply because of underlying infertility (2Song S. Ghosh J. Mainigi M. Turan N. Weinerman R. Truongcao M. et al.DNA methylation differences between in vitro- and in vivo-conceived children are associated with ART procedures rather than infertility.). These differences, in general, have been restricted to a small number of genes and have been difficult to identify in heterogeneous populations. In both humans and mouse models, a hallmark of DNA methylation changes after ART is that it occurs to a greater extent in some infants than others. Some “outliers” are uniquely susceptible to the epigenetic disruptions of ART, and methylation differences are more pronounced when these groups are studied (1Mani S. Ghosh J. Coutifaris C. Sapienza C. Mainigi M. Epigenetic changes and assisted reproductive technologies., 3Choufani S. Turinsky A.L. Melamed N. Greenblatt E. Brudno M. Berard A. et al.Impact of assisted reproduction, infertility, sex and paternal factors on the placental DNA methylome.). Despite the current data, we still do not have a complete grasp on how newborns conceived by ART are affected and whether these changes persist into childhood or adulthood.In this interesting article, Yeung et al. (4Yeung E.H. Mendola P. Sundaram R. Zeng X. Guan W. Tsai M. et al.Conception by fertility treatment and offspring deoxyribonucleic acid methylation.) attempt to answer this question. They studied the effects of ART and ovulation induction with intrauterine insemination (OI/IUI) on DNA methylation using the Illumina EPIC platform in peripheral blood of newborns and young children aged 8–10 years (4Yeung E.H. Mendola P. Sundaram R. Zeng X. Guan W. Tsai M. et al.Conception by fertility treatment and offspring deoxyribonucleic acid methylation.). Methylation differences were found in 12 CpG sites in newborns conceived by ART, specifically hypomethylation of 9 maternally imprinted genes, including IGF1 and GNAS. GNAS has been consistently identified as a gene whose methylation is perturbed by ART in prior studies (1Mani S. Ghosh J. Coutifaris C. Sapienza C. Mainigi M. Epigenetic changes and assisted reproductive technologies.). Similar methylation differences were observed in newborns conceived with intracytoplasmic sperm injection as well. However, these differences were not noted to be significant after samples associated with the diagnosis of male infertility were excluded, suggesting that these differences are related to the male infertility. Despite citing 12-CpG-site methylation changes, these changes were not significant in children conceived with ART at the ages of 8–10 years, although there was a trend toward persistent hypomethylation of 3 of the previously identified genes. Additionally, no significant variations in methylation profile were observed in the dried blood spots obtained from newborn screening tests of newborns conceived with OI/IUI.Many of the DNA methylation differences in this study were found on imprinted genes, which has been a consistent finding in studies of epigenetic disruptions after ART. Imprinted genes have been considered to be more susceptible to changes in methylation because of the fact that these genes maintain their methylation signatures during the crucial preimplantation embryonic development period; however, disruptions to imprinted genes may additionally signal wider epigenetic disruption that may differentially affect a small subset of newborns (1Mani S. Ghosh J. Coutifaris C. Sapienza C. Mainigi M. Epigenetic changes and assisted reproductive technologies., 3Choufani S. Turinsky A.L. Melamed N. Greenblatt E. Brudno M. Berard A. et al.Impact of assisted reproduction, infertility, sex and paternal factors on the placental DNA methylome.).Longitudinal studies similar to Yeung et al. (4Yeung E.H. Mendola P. Sundaram R. Zeng X. Guan W. Tsai M. et al.Conception by fertility treatment and offspring deoxyribonucleic acid methylation.) are quite limited; therefore, this study adds significant value to the literature with its relatively large sample size and follow-up data. In the analyses, the investigators compared fertile controls (newborns conceived without fertility treatment) with newborns conceived with any treatment and by either ART or OI/IUI. This differentiation is significant to understand that ART has its separate, not yet understood risks from OI/IUI and that the diagnosis of infertility cannot be used to explain away these differences. The investigators acknowledge that the severity of infertility may play a role because of the findings, as previously discussed, when the diagnosis of male infertility was excluded. Additionally, the long-term data with 8–10-year-old children emphasizes the possible impact of ART over time beyond the pregnancy and newborn period. Despite the fact that there were no methylation differences in children at age of 8–10 years compared with fertile controls, the trend toward persistent hypomethylation of 3 genes suggests that these methylation changes are cumulative. Although phenotypic differences are not yet observed at this time, differential outcomes may be demonstrated in future generations to come.In addition, this study has several limitations. Dried blood spots from newborn screening tests were a source of samples for this study. The investigators reference this technique as a validated test for determining DNA methylation. Prior studies of newborn cord blood have shown methylation differences in ART newborns; however, other studies have found more robust differences assessing global methylation in placental tissue, where the effects of ART and other variables (including sex and infertility) may be more pronounced (1Mani S. Ghosh J. Coutifaris C. Sapienza C. Mainigi M. Epigenetic changes and assisted reproductive technologies., 3Choufani S. Turinsky A.L. Melamed N. Greenblatt E. Brudno M. Berard A. et al.Impact of assisted reproduction, infertility, sex and paternal factors on the placental DNA methylome.). In addition, the study was limited by the heterogeneity of the ART sample, which did not distinguish between fresh and frozen embryo transfer among other differences in ART treatments, such as treatment protocol, embryo culture environment, and use of embryo vitrification and biopsy. The study, therefore, cannot answer the question as to which aspect of the ART is responsible for disruptions in DNA methylation. Although no DNA methylation changes persisted in older children conceived by ART, there are differences between neonatal outcomes in children conceived after fresh vs. frozen cycles. In addition, there are changes in gene expression after manipulations, such as vitrification and embryo transfer. It has been studied that increasing manipulation with vitrification (e.g., single vitrification vs. double vitrification with biopsy) affects the gene expression and cytoskeletal structure of the embryo (5Van Heertum K. Lam L. Richardson B. Cartwright M. Mesiano S. Cameron M. et al.Blastocyst vitrification and trophectoderm biopsy cumulatively alter embryonic gene expression in a mouse model.). Therefore, further investigation is needed to understand whether the endometrial environment after a fresh or frozen embryo transfer plays a role in epigenetic programming as well as whether certain manipulations (i.e., treatment protocol, trigger medication, vitrification, and embryo biopsy) contribute to DNA methylation.

As successful as ART has been for several families, this study emphasizes how much we still do not yet understand. Assisted reproductive technology practices are continually evolving to provide the best live birth rates for our patients seeking fertility treatment. However, in addition, we need to recognize how these treatments can affect the health of ART children in the long term and who is at risk. Therefore, with research similar to this current study, we can continue to foster an interest in ART outcomes beyond live birth rates and provide therapies that optimize not only the likelihood of pregnancy and live birth but also healthy longevity for the generations to come of our patients with ART.

ReferencesMani S. Ghosh J. Coutifaris C. Sapienza C. Mainigi M.

Epigenetic changes and assisted reproductive technologies.

Epigenetics. 15: 12-25Song S. Ghosh J. Mainigi M. Turan N. Weinerman R. Truongcao M. et al.

DNA methylation differences between in vitro- and in vivo-conceived children are associated with ART procedures rather than infertility.

Clin Epigenetics. 7: 41Choufani S. Turinsky A.L. Melamed N. Greenblatt E. Brudno M. Berard A. et al.

Impact of assisted reproduction, infertility, sex and paternal factors on the placental DNA methylome.

Hum Mol Genet. 28: 372-385Yeung E.H. Mendola P. Sundaram R. Zeng X. Guan W. Tsai M. et al.

Conception by fertility treatment and offspring deoxyribonucleic acid methylation.

Fertil Steril. ()Van Heertum K. Lam L. Richardson B. Cartwright M. Mesiano S. Cameron M. et al.

Blastocyst vitrification and trophectoderm biopsy cumulatively alter embryonic gene expression in a mouse model.

Reprod Sci. https://doi.org/10.1007/s43032-021-00560-zArticle InfoPublication History

Published online: July 10, 2021

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DOI: https://doi.org/10.1016/j.fertnstert.2021.06.032

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©2021 American Society for Reproductive Medicine, Published by Elsevier Inc.

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