Epigenetically regulated imprinted gene expression associated with IVF and infertility: possible influence of prenatal stress and depression.

PURPOSE: Despite the growing body of research implying an impact of in vitro fertilization (IVF) on imprinted genes and epigenetics, few studies have examined the effects of underlying subfertility or prenatal stress on epigenetics, particularly in terms of their role in determining infant birthweights. Both subfertility and prenatal stressors have been found to impact epigenetics and may be confounding the effect of IVF on epigenetics and imprinted genes. Like IVF, both of these exposures-infertility and prenatal stressors-have been associated with lower infant birthweights. The placenta, and specifically epigenetically regulated placental imprinted genes, provides an ideal but understudied mechanism for evaluating the relationship between underlying genetics, environmental exposures, and birthweight. METHODS AND RESULTS: In this review, we discuss the impacts of IVF and infertility on birthweight, epigenetic mechanisms and genomic imprinting, and the role of these mechanisms in the IVF population and discuss the role and importance of the placenta in infant development. We then highlight recent work on the relationships between infertility, IVF, and prenatal stressors in terms of placental imprinting. CONCLUSIONS: In combination, the studies discussed, as well as two recent projects of our own on placental imprinted gene expression, suggest that lower birthweights in IVF infants are secondary to a combination of exposures including the infertility and prenatal stress that couples undergoing IVF are experiencing. The work highlighted herein emphasizes the need for appropriate control populations that take infertility into account and also for consideration of prenatal psychosocial stressors as confounders and causes of variation in IVF infant outcomes.

Birthweight in infants conceived through in vitro fertilization following blastocyst or cleavage-stage embryo transfer: a national registry study.

PURPOSE: In vitro fertilization (IVF) infants have lower birthweights than their peers, predisposing them to long-term health consequences. Blastocyst transfer (BT), at day 5-6 post-fertilization, is increasing in usage, partially due to improved pregnancy outcomes over cleavage-stage transfer (CT, day 2-3). Data to date, however, have been inconclusive regarding BT's effects on birthweight. METHODS: Participants included all US autologous, single-gestation, fresh embryo transfer cycles initiated from 2007 to 2014 that resulted in a term infant (N = 124,154) from the National Assisted Reproductive Technology Surveillance System. Generalized linear models including obstetric history, maternal demographics, and infant sex and gestational age were used to compare birthweight outcomes for infants born following BT (N = 67,169) with infants born following CT (N = 56,985) and to test for an interaction between transfer stage and single embryo transfer (SET). RESULTS: Infants born following BT were 6 g larger than those born following CT (p = 0.04), but rates of macrosomia (RR 1.00, 95% CI 0.96-1.04) and low birthweight (LBW, RR 1.00, 95% CI 0.93-1.06) were not different between the groups. The interaction between SET and transfer stage was significant (p = 0.02). Among SET infants, BT was associated with 19.26 g increased birthweight compared to CT (p = 0.008). CONCLUSIONS: The increase in birthweights identified following BT is unlikely to be clinically relevant, as there were no differences in rates of macrosomia or LBW. These findings are clinically reassuring and indicate that the increasing use of BT is unlikely to further decrease the on average lower birthweights seen in IVF infants compared to their naturally conceived peers.

Prenatal exposure to maternal depression and anxiety on imprinted gene expression in placenta and infant neurodevelopment and growth.

BackgroundDepression and/or anxiety during pregnancy have been associated with impaired fetal growth and neurodevelopment. Because placental imprinted genes play a central role in fetal development and respond to environmental stressors, we hypothesized that imprinted gene expression would be affected by prenatal depression and anxiety.MethodsPlacental gene expression was compared between mothers with prenatal depression and/or anxiety/obsessive compulsive disorder/panic and control mothers without psychiatric history (n=458) in the Rhode Island Child Health Study.ResultsTwenty-nine genes were identified as being significantly differentially expressed between placentae from infants of mothers with both depression and anxiety (n=54), with depression (n=89), or who took perinatal psychiatric medications (n=29) and control mother/infant pairs, with most genes having decreased expression in the stressed group. Among placentae from infants of mothers with depression, we found no differences in expression by medication use, indicating that our results are related to the stressor rather than the treatments. We did not find any relationship between the stress-associated gene expression and neonatal neurodevelopment, as measured using the Neonatal Intensive Care Unit Network Neurobehavioral Scale.ConclusionsThis variation in expression may be part of an adaptive mechanism by which the placenta buffers the infant from the effects of maternal stress.

Effect of frozen/thawed embryo transfer on birthweight, macrosomia, and low birthweight rates in US singleton infants.

BACKGROUND: Singleton infants conceived using assisted reproductive technology have lower average birthweights than naturally conceived infants and are more likely to be born low birthweight (<2500 gr). Lower birthweights are associated with increased infant and child mortality and poor adult health outcomes, including cardiovascular disease, hypertension, and diabetes. Data from registry and single-center studies suggest that frozen/thawed embryo transfer may be associated with larger birthweights. To date, however, a nationwide, full-population study on United States infants born using frozen/thawed embryo transfer has not been reported. OBJECTIVES: The objective of this study was to compare the effect of frozen/thawed vs fresh embryo transfer on birthweight outcomes for singleton, term infants conceived using in vitro fertilization in the United States between 2007 and 2014, including average birthweight and the risks of both macrosomia (>4000 g) and low birthweight (<2500 g). STUDY DESIGN: We used data from the Centers for Disease Control and Prevention's National Assisted Reproductive Technology Surveillance System to compare birthweight outcomes of live-born singleton, autologous oocyte, term (37-43 weeks) infants. Generalized linear models for all infants and stratified by infant sex were used to assess the relationship between frozen/thawed embryo transfer and birthweight, in grams. Infertility diagnosis, year of treatment, maternal age, maternal obstetric history, maternal and paternal race, and infant gestational age and sex were included in the models. Missing race data were imputed. The adjusted relative risks for macrosomia and low birthweight were evaluated using multivariable predicted marginal proportions from logistic regression models. RESULTS: In total, 180,184 singleton, term infants were included, with 55,898 (31.02%) having been conceived from frozen/thawed embryos. Frozen/thawed embryo transfer was associated with, on average, a 142 g increase in birthweight compared with infants born after fresh embryo transfer (P < .001). An interaction between infant sex and embryo transfer type was significant (P < .0001), with frozen/thawed embryo transfer having a larger effect on male infants by 16 g. The adjusted risk of a macrosomic infant was 1.70 times higher (95% confidence interval, 1.64-1.76) following frozen/thawed embryo transfer than fresh embryo transfer. However, adjusted risk of low birthweight following frozen/thawed embryo transfer was 0.52 (95% confidence interval, 0.48-0.56) compared with fresh embryo transfer. CONCLUSION: Frozen/thawed embryo transfer, in comparison with fresh embryo transfer, was associated with increased average birthweight in singleton, autologous oocytes, term infants born in the United States, with a significant interaction between frozen/thawed embryo transfer and infant sex. The risk of macrosomia following frozen/thawed embryo transfer was greater than that following fresh embryo transfer, but the risk of low birthweight among frozen/thawed embryo transfer infants was significantly decreased in comparison with fresh embryo transfer infants.

Placental imprinting variation associated with assisted reproductive technologies and subfertility.

Infertility affects one in 6 couples in developed nations, resulting in an increasing use of assisted reproductive technologies (ART). Both ART and subfertility appear to be linked to lower birth weight outcomes, setting infants up for poor long-term health. Prenatal growth is, in part, regulated via epigenetically-controlled imprinted genes in the placenta. Although differences in DNA methylation between ART and control infants have been found, it remains unclear whether these differences are due to the ART procedures or to the underlying parental subfertility and how these methylation differences affect imprinted gene expression. In this study, we examined the expression of 108 imprinted genes in placental tissues from infants born to subfertile parents (n = 79), matched naturally-conceived controls (n = 158), and infants conceived using in vitro fertilization (IVF, n = 18). Forty-five genes were identified as having significantly different expression between the subfertile infants and controls, whereas no significant differences were identified between the IVF and control groups. The expression of 4 genes-IGF2, NAPIL5, PAX8-AS1, and TUBGCP5-was significantly downregulated in the IVF compared with the subfertile group. Three of the 45 genes significantly dysregulated between subfertile and control placentae-GRB10, NDN, and CD44 -were found to have a significant positive correlation between expression and birth weight. Methylation levels for these 3 genes and 4 others-MKRN3, WRB, DHCR24, and CYR61-were significantly correlated with expression. Our findings indicate that epigenetic differences in placentas resulting from IVF pregnancies may be related to the underlying subfertility in parents using IVF rather than the IVF procedure itself.

MiR-338-3p regulates neuronal maturation and suppresses glioblastoma proliferation.

Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral "sponge" to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.

Maternal smoking during pregnancy is associated with mitochondrial DNA methylation.

Maternal smoking during pregnancy (MSDP) has detrimental effects on fetal development and on the health of the offspring into adulthood. Energy homeostasis through ATP production via the mitochondria (mt) plays a key role during pregnancy. This study aimed to determine if MSDP resulted in differences in DNA methylation to the placental mitochondrial chromosome at the transcription and replication control region, the D-Loop, and if these differences were also present in an alternate neonatal tissue (foreskin) in an independent birth cohort. We investigated mtDNA methylation by bisulfite-pyrosequencing in two sections of the D-Loop control region and in long interspersed nuclear element-1 (LINE-1) genomic sequences in placenta from 96 mother-newborn pairs that were enrolled in a Rhode Island birth cohort along with foreskin samples from 62 infants from a Kentucky birth cohort. In both placenta and foreskin, mtDNA methylation in the light chain D-Loop region 1 was positively associated with MSDP in placenta (difference+2.73%) (P=0.001) and foreskin (difference+1.22%) (P=0.08). Additionally, in foreskin, a second segment of the D-Loop-heavy chain region 1 showed a small but significant change in methylation with MSDP (+0.4%, P=0.04). No methylation changes were noted in either tissue at the LINE-1 repetitive element. We identified a similar pattern of epigenetic effect to mitochondria arising in cells from different primordial lineages and in different populations, associated with MSDP. These robust and consistent results build evidence that MSDP may impact mt D-Loop methylation, as one mechanism through which this exposure affects newborn health.