DNA Methylation In Placenta Research Articles

Prenatal social support in low-risk pregnancy shapes placental epigenome

BackgroundPoor social support during pregnancy has been linked to inflammation and adverse pregnancy and childhood health outcomes. Placental epigenetic alterations may underlie these links but are still unknown in humans.MethodsIn a cohort of low-risk pregnant women (n = 301) from diverse ethnic backgrounds, social support was measured using the ENRICHD Social Support Inventory (ESSI) during the first trimester. Placental samples collected at delivery were analyzed for DNA methylation and gene expression using Illumina 450K Beadchip Array and RNA-seq, respectively. We examined association between maternal prenatal social support and DNA methylation in placenta. Associated cytosine-(phosphate)-guanine sites (CpGs) were further assessed for correlation with nearby gene expression in placenta.ResultsThe mean age (SD) of the women was 27.7 (5.3) years. The median (interquartile range) of ESSI scores was 24 (22–25). Prenatal social support was significantly associated with methylation level at seven CpGs (PFDR < 0.05). The methylation levels at two of the seven CpGs correlated with placental expression of VGF and ILVBL (PFDR < 0.05), genes known to be involved in neurodevelopment and energy metabolism. The genes annotated with the top 100 CpGs were enriched for pathways related to fetal growth, coagulation system, energy metabolism, and neurodevelopment. Sex-stratified analysis identified additional significant associations at nine CpGs in male-bearing pregnancies and 35 CpGs in female-bearing pregnancies.ConclusionsThe findings suggest that prenatal social support is linked to placental DNA methylation changes in a low-stress setting, including fetal sex-dependent epigenetic changes. Given the relevance of some of these changes in fetal neurodevelopmental outcomes, the findings signal important methylation targets for future research on molecular mechanisms of effect of the broader social environment on pregnancy and fetal outcomes.Trial registrationNCT00912132 (ClinicalTrials.gov).

Profiling placental DNA methylation associated with maternal SSRI treatment during pregnancy

Selective serotonin reuptake inhibitors (SSRIs) for treatment of prenatal maternal depression have been associated with neonatal neurobehavioral disturbances, though the molecular mechanisms remain poorly understood. In utero exposure to SSRIs may affect DNA methylation (DNAme) in the human placenta, an epigenetic mark that is established during development and is associated with gene expression. Chorionic villus samples from 64 human placentas were profiled with the Illumina MethylationEPIC BeadChip; clinical assessments of maternal mood and SSRI treatment records were collected at multiple time points during pregnancy. Case distribution was 20 SSRI-exposed cases and 44 SSRI non-exposed cases. Maternal depression was defined using a mean maternal Hamilton Depression score > 8 to indicate symptomatic depressed mood (“maternally-depressed”), and we further classified cases into SSRI-exposed, maternally-depressed (n = 14); SSRI-exposed, not maternally-depressed (n = 6); SSRI non-exposed, maternally-depressed (n = 20); and SSRI non-exposed, not maternally-depressed (n = 24). For replication, Illumina 450K DNAme profiles were obtained from 34 additional cases from an independent cohort (n = 17 SSRI-exposed, n = 17 SSRI non-exposed). No CpGs were differentially methylated at FDR < 0.05 comparing SSRI-exposed to non-exposed placentas, in a model adjusted for mean maternal Hamilton Depression score, or in a model restricted to maternally-depressed cases with and without SSRI exposure. However, at a relaxed threshold of FDR < 0.25, five CpGs were differentially methylated (|Δβ| > 0.03) by SSRI exposure status. Four were covered by the replication cohort measured by the 450K array, but none replicated. No CpGs were differentially methylated (FDR < 0.25) comparing maternally depressed to not depressed cases. In sex-stratified analyses for SSRI-exposed versus non-exposed cases (females n = 31; males n = 33), three additional CpGs in females, but none in males, were differentially methylated at the relaxed FDR < 0.25 cut-off. We did not observe large-scale alterations of DNAme in placentas exposed to maternal SSRI treatment, as compared to placentas with no SSRI exposure. We also found no evidence for altered DNAme in maternal depression-exposed versus depression non-exposed placentas. This novel work in a prospectively-recruited cohort with clinician-ascertained SSRI exposure and mood assessments would benefit from future replication.

Placental Abnormalities are Associated With Specific Windows of Embryo Culture in a Mouse Model.

Assisted Reproductive Technologies (ART) employ gamete/embryo handling and culture in vitro to produce offspring. ART pregnancies have an increased risk of low birth weight, abnormal placentation, pregnancy complications, and imprinting disorders. Embryo culture induces low birth weight, abnormal placental morphology, and lower levels of DNA methylation in placentas in a mouse model of ART. Whether preimplantation embryos at specific stages of development are more susceptible to these perturbations remains unresolved. Accordingly, we performed embryo culture for several discrete periods of preimplantation development and following embryo transfer, assessed fetal and placental outcomes at term. We observed a reduction in fetal:placental ratio associated with two distinct windows of preimplantation embryo development, one prior to the morula stage and the other from the morula to blastocyst stage, whereas placental morphological abnormalities and reduced imprinting control region methylation were only associated with culture prior to the morula stage. Extended culture to the blastocyst stage also induces additional placental DNA methylation changes compared to embryos transferred at the morula stage, and female concepti exhibited a higher loss of DNA methylation than males. By identifying specific developmental windows of susceptibility, this study provides a framework to optimize further culture conditions to minimize risks associated with ART pregnancies.

Evaluating the Effects of BPA and TBBPA Exposure on Pregnancy Loss and Maternal-Fetal Immune Cells in Mice.

Background:Bisphenol A (BPA) exposure has been linked to miscarriages and pregnancy complications in humans. In contrast, the potential reproductive toxicity of BPA analogs, including tetrabromobisphenol A (TBBPA), is understudied. Furthermore, although environmental exposure has been linked to altered immune mediators, the effects of BPA and TBBPA on maternal–fetal immune tolerance during pregnancy have not been studied. The present study investigated whether exposure resulted in higher rates of pregnancy loss in mice, lower number of regulatory T cells (Tregs), and lower indoleamine 2,3 deoxygenase 1 (Ido1) expression, which provided evidence for mechanisms related to immune tolerance in pregnancy.Objectives:The purpose of this investigation was to characterize the effects of BPA and TBBPA exposure on pregnancy loss in mice and to study the percentage and number of Tregs and Ido1 expression and DNA methylation.Methods:Analysis of fetal resorption and quantification of maternal and fetal immune cells by flow cytometry were performed in allogeneic and syngeneic pregnancies. Ido1 mRNA and protein expression, and DNA methylation in placentas from control and BPA- and TBBPA-exposed mice were analyzed using real-time quantitative polymerase chain reaction, immunofluorescence, and bisulfite sequencing analyses.Results:BPA and TBBPA exposure resulted in higher rates of hemorrhaging in early allogeneic, but not syngeneic, conceptuses. In allogeneic pregnancies, BPA and TBBPA exposure was associated with higher fetal resorption rates and lower maternal Treg number. Importantly, these differences were associated with lower IDO1 protein expression in trophoblast giant cells and higher mean percentage Ido1 DNA methylation in embryonic day 9.5 placentas from BPA- and TBBPA-exposed mice.Discussion:BPA- and TBBPA-induced pregnancy loss in mice was associated with perturbed IDO1-dependent maternal immune tolerance. https://doi.org/10.1289/EHP10640

Pregnancy exposure to phthalates and DNA methylation in male placenta - An epigenome-wide association study.

Exposure to phthalates during pregnancy may alter DNA methylation in the placenta, a crucial organ for the growth and development of the fetus. We studied associations between urinary concentrations of phthalate biomarkers during pregnancy and placental DNA methylation. We measured concentrations of 11 phthalate metabolites in maternal spot urine samples collected between 22 and 29 gestational weeks in 202 pregnant women. We analyzed DNA methylation levels in placental tissue (fetal side) collected at delivery. We first investigated changes in global DNA methylation of repetitive elements Alu and LINE-1. We then performed an adjusted epigenome-wide association study using IlluminaHM450 BeadChips and identified differentially methylated regions (DMRs) associated with phthalate exposure. Monobenzyl phthalate concentration was inversely associated with placental methylation of Alu repeats. Moreover, all phthalate biomarkers except for monocarboxy-iso-octyl phthalate and mono(2-ethyl-5-hydroxyhexyl) phthalate were associated with at least one DMR. All but three DMRs showed increased DNA methylation with increased phthalate exposure. The largest identified DMR (22 CpGs) was positively associated with monocarboxy-iso-nonyl phthalate and encompassed heat shock proteins (HSPA1A, HSPA1L). The remaining DMRs encompassed transcription factors and nucleotide exchange factors, among other genes. This is the first description of genome-wide modifications of placental DNA methylation in association with pregnancy exposure to phthalates. Our results suggest epigenetic mechanisms by which exposure to these compounds could affect fetal development. Of interest, four identified DMRs had been previously associated with maternal smoking, which may suggest particular sensitivity of these genomic regions to the effect of environmental contaminants.

Differential methylation of genes in the human placenta associated with bisphenol A exposure

Prenatal exposure to bisphenol A (BPA) is associated with numerous adverse health outcomes among offspring. Although DNA methylation is considered one of the underlying causes of these associations, few studies have focused on the association between prenatal BPA exposure and DNA methylation in the human placenta. In this study, we examined the association between prenatal BPA exposure and DNA methylation in the placenta of 146 mother-infant pairs from the Shanghai-Minhang Birth Cohort Study. BPA concentrations in maternal urine samples were measured using high-performance liquid chromatography. Six placenta samples were selected for whole-genome methylation analysis using Infinium Human Methylation 450K Beadchip, followed by pyrosequencing-based methylation analysis of three selected genes in 146 placentas. Among 282 differentially methylated CpGs, representing 208 genes, 127 were hypermethylated, and 155 were hypomethylated in the BPA exposure group. Prenatal BPA exposure was associated with a higher methylation level of HLA-DRB6 in individuals as determined using pyrosequencing, which was consistent with the whole-genome methylation analysis results. Compared with that subjects with low BPA exposure, the methylation level (ln-transformed) of HLA-DRB6 in placentas from those with high BPA exposure increased by 0.29% (95% confidence interval[CI]: 0.02%, 0.56%) at the CpG2 site, and the average methylation level (ln-transformed) of the three CpG sites increased by 0.30% (95%CI: −0.03%, 0.63%). Our findings provide evidence that prenatal BPA exposure might alter DNA methylation levels in the placenta.

Sex-specific effects of in vitro fertilization on adult metabolic outcomes and hepatic transcriptome and proteome in mouse.

Although in vitro fertilization (IVF) is associated with adverse perinatal outcomes, there is increasing concern about the long-term and sex-specific health implications. Augmenting our IVF mouse model to longitudinally investigate metabolic outcomes in offspring from optimal neonatal litter sizes, we found sex-specific metabolic outcomes in IVF offspring. IVF-conceived females had higher body weight and cholesterol levels compared to naturally conceived females, whereas IVF-conceived males had higher levels of triglycerides and insulin, and increased body fat composition. Through adult liver transcriptomics and proteomics, we identified sexually dimorphic dysregulation of the sterol regulatory element-binding protein (SREBP) pathways that are associated with the sex-specific phenotypes. We also found that global loss of DNA methylation in placenta was linked to higher cholesterol levels in IVF-conceived females. Our findings indicate that IVF procedures have long-lasting sex-specific effects on metabolic health of offspring and lay the foundation to utilize the placenta as a predictor of long-term outcomes.

Exposure to Gestational Diabetes Mellitus (GDM) alters DNA methylation in placenta and fetal cord blood

BackgroundFetal exposure to maternal GDM increases offspring risk for adult-onset metabolic syndromes. Epigenetic modifications such as DNA methylation are modulators for fetal metabolic programming and susceptibility to adult-onset disease. This study investigates genome-wide DNA methylation in GDM exposed cord blood and placenta. MethodsOral glucose tolerance testing between 24 and 28 weeks of pregnancy was used to determine severity of glucose intolerance. We measured DNA methylation (DNAm) using the Illumina Infinium 450 K array in 42 fetal cord blood and 36 placenta samples. ResultsWe identified 662 and 99 CpG sites in GDM placenta and cord blood, respectively at p-value <0.01 and a methylation difference >5% after adjustment for confounders. Annotated sites for AHRR and PTPRN2 were common to cord blood and placenta. Adding published GDM cord blood DNAm data we segregated patients based on treatment (Diet Only vs. +Insulin) and identified altered CpG sites to be unique to each GDM treatment group. ConclusionConsistency in findings with other studies provides evidence for the role of DNAm in placental and fetal responses to hyperglycemia. However, segregating DNAm analysis of GDM samples based on treatment may help delineate whether observed DNAm alterations are reflective of adaptive responses or treatment effects in utero.

Effects of maternal dietary omega-3 polyunsaturated fatty acids and methionine during late gestation on fetal growth, DNA methylation, and mRNA relative expression of genes associated with the inflammatory response, lipid metabolism and DNA methylation in placenta and offspring\u2019s liver in sheep

BackgroundOmega-3 PUFA or methionine (Met) supply during gestation alters offspring physiology. However, the effect of both nutrients on fetal development has not been explored. Our objective was to determine the effects of supplementation of these two nutrients during late gestation on fetal growth, DNA methylation, and mRNA expression of genes associated with the inflammatory response, and DNA methylation. Ewes (n = 5/treatment) were fed from day 100 to 145 of gestation one of the following treatments: 1) basal diet (NS) without fatty acids (FS) or methionine (MS) supplementation; 2) FS (10 g/kg Ca salts, source omega-3 PUFA); 3) MS (1 g/kg rumen protected methionine); and 4) FS and MS (FS-MS). On day 145, ewes were euthanized, and data from dams and fetus was recorded. Placenta (cotyledon), fetal liver, and blood samples were collected.ResultsA treatments interaction on fetal liver weight, ewe body weight and body condition score (BCS) was observed; FS-MS were heavier (P < 0.01) than FS and MS, and FS-MS ewes had a better (P = 0.02) BCS than NS. Methionine increased (P = 0.03) ewe plasma glucose concentration. Fetal liver global DNA methylation increased (P < 0.01) in FS and MS. Dietary treatments modify the mRNA relative expression on some of the genes evaluated. In the fetal liver, FS increased (P = 0.04) the mRNA relative expression of arachidonate-5-lipoxygenase-activating-protein and tended to decrease (P = 0.06) methionine-adenosyltransferase-1A. Moreover, MS decreased (P = 0.04) DNA-methyltransferase-1 and tended to decrease (P = 0.08) free-fatty-acid-receptor-1 mRNA relative expression. Furthermore, FS-MS decreased mRNA relative expression of tumor-necrosis-factor-alpha (P = 0.05), peroxisome-proliferator-activated-receptor-delta (P = 0.03) and gamma (P = 0.04), tended to decrease (P ≤ 0.09) interleukin-6, fatty-acid-transport-protein-1, and delta-5-desaturase, and increased adenosylhomocysteinase (P = 0.04) mRNA relative expression. In cotyledon, FS tended to decrease fatty acid binding protein 4 (P = 0.09) mRNA relative expression.ConclusionOmega-3 PUFA and Met supplementation improves dam’s performance in late gestation, which was positively correlated with an increase in offspring’s liver development. Moreover, FS-MS decreased mRNA relative expression of proinflammatory cytokines, and lipogenic genes, and increased the expression on an enzyme that has an important role in methylation.

DNA methylation loci in placenta associated with birthweight and expression of genes relevant for early development and adult diseases

BackgroundBirthweight marks an important milestone of health across the lifespan, including cardiometabolic disease risk in later life. The placenta, a transient organ at the maternal-fetal interface, regulates fetal growth. Identifying genetic loci where DNA methylation in placenta is associated with birthweight can unravel genomic pathways that are dysregulated in aberrant fetal growth and cardiometabolic diseases in later life.ResultsWe performed placental epigenome-wide association study (EWAS) of birthweight in an ethnic diverse cohort of pregnant women (n = 301). Methylation at 15 cytosine-(phosphate)-guanine sites (CpGs) was associated with birthweight (false discovery rate (FDR) < 0.05). Methylation at four (26.7%) CpG sites was associated with placental transcript levels of 15 genes (FDR < 0.05), including genes known to be associated with adult lipid traits, inflammation and oxidative stress. Increased methylation at cg06155341 was associated with higher birthweight and lower FOSL1 expression, and lower FOSL1 expression was correlated with higher birthweight. Given the role of the FOSL1 transcription factor in regulating developmental processes at the maternal-fetal interface, epigenetic mechanisms at this locus may regulate fetal development. We demonstrated trans-tissue portability of methylation at four genes (MLLT1, PDE9A, ASAP2, and SLC20A2) implicated in birthweight by a previous study in cord blood. We also found that methylation changes known to be related to maternal underweight, preeclampsia and adult type 2 diabetes were associated with lower birthweight in placenta.ConclusionWe identified novel placental DNA methylation changes associated with birthweight. Placental epigenetic mechanisms may underlie dysregulated fetal development and early origins of adult cardiometabolic diseases.Clinical trial registrationClinicalTrials.gov, NCT00912132

98-OR: Placental DNA Methylation Changes Associated with Birthweight and Overlaps with Adult Type 2 Diabetes

Background: There is cumulating evidence for associations between birthweight-lowering genetic factors and increased risk of type 2 diabetes (T2D). Given the crucial role of the placenta in supporting fetal growth, epigenetic mechanisms in placenta may lend insights into prevention of T2D in early life. We investigated epigenetic changes in placenta associated with birthweight and those that overlap with T2D risk. Methods: DNA methylation was measured using Illumina’s Infinium Human Methylation450 Beadchip in placenta samples obtained from race/ethnic-diverse pregnant women who participated in the NICHD Fetal Growth Studies (n=301). Associations between DNA methylation and birthweight were tested using linear regression models. Next, we examined whether DNA methylation at loci previously implicated in adult T2D were associated with birthweight in placenta. The relationship between DNA methylation and gene expression was tested in 75 placenta samples profiled for both DNA methylation and gene expression. Statistical tests were considered significant at a false discovery rate &amp;lt; 0.05. Results: Placenta methylation at 15 cytosine-phosphate-guanine (CpG) sites was significantly associated with birthweight, of which methylation at UBXN11, LBX2, CNIH2, and PDE9A was significantly associated with expression of nearby genes. At six CpG sites (MGRN1, AMIGO2, CHD3 and three intergenic regions) known in previous studies to be associated with adult T2D, we found significant associations between DNA methylation in placenta and birthweight. For all the six CpG sites, the methylation changes related to increased risk of T2D were associated with lower birthweight. Conclusions: We found novel placental DNA methylation signatures associated with lower birthweight and higher risk of adult T2D. Our findings provide the first evidence to suggest that epigenetic changes in the placenta may be one potential mechanism that explains the link between early growth and later life T2D. Disclosure F. Tekola-Ayele: None. T. Workalemahu: None. X. Zeng: None. Funding Eunice Kennedy Shriver National Institute of Child Health and Human Development

Maternal Imbalance of Vitamin B12 and Folic Acid Alters the Expression of Fetal Growth-related Imprinted Genes in Placenta (P24-033-19)

ObjectivesThe present study aimed to elucidate the trans-generational influence of low dietary vitamin B12 with different levels of folic acid in mice model on fetal growth and expression of fetal growth-related imprinted genes in placenta. MethodsC57BL/6 mice (four-week-old) were assigned into four dietary groups: (test groups)-BDFN*, BDFO*, BDFD* & (control group)-BNFN* and were fed diet accordingly for two generations (F0 and F1). Mating was carried out and on 20th day of gestation pregnant mice were sacrificed for fetus collection in both F1 & F2 generation. In F1 generation after weaning period a sub-group of F1 offsprings born to F0 dams were rehabilitated to control diet in each test group (transient) while others were continued on same diet as their mothers (sustained) and further mating was carried out. Vitamins and homocysteine levels in serum were evaluated. Expression of genes, global DNA methylation in placenta and growth-parameters of fetuses were studied. ResultsOur data suggests that deficiency of both vitamins significantly reduced the total body weight and circulating levels of these vitamins in serum. Dietary modulation of vitamins impaired the normal fetal growth as head circumference and crown-rump length was found reduced in BDFO transient & sustained group (P < 0.01), fetal birth weight was reduced in B12 deficient conditions in both F1 & F2 generation and a decrease in placental weight was also found in BDFD & BDFO sustained group (F0 & F1, P < 0.01). Placental expression of imprinted genes were reduced in F0 while it was found to be increased in F1 in all test groups. Over-expression of PHLDA2 in F1 placenta (P < 0.001) can be corelated to the reduced placental weight (BDFD & BDFO sustained group F1, P < 0.01). GRB10 expression was increased in all vitamin B12 deficient sustained groups (F1, P < 0.001 & < 0.01). Enhanced expression of MEST was observed in FO conditions (F1 transient and sustained, P < 0.05) which can be corelated to reduced head circumference, moreover DLK1 was over-expressed in both vitamin deficiency conditions.*Abbreviations: BN: B12 Normal FN: Folate Normal FD: Folate deficient FO: Folate over supplementation BD: B12 deficient. ConclusionsDietary imbalance of vitamin B12 and folic acid levels during the pre-& post-natal development bring about growth anomalies at genetic & phenotypic level in subsequent generation. Funding SourcesThis work is funded by Department of science and technology- Science and Engineering board, New Delhi, India.

Epigenome-wide DNA methylation in placentas from preterm infants: association with maternal socioeconomic status

ABSTRACTThis study evaluated the hypothesis that prenatal maternal socioeconomic status (SES) adversity is associated with DNA methylation in the placenta. SES adversity was defined by the presence of, as well as a summative count of, four factors: less than college education, single marital status, food and nutritional service assistance, and public health insurance. Epigenome-wide DNA methylation was assessed using the Illumina EPIC array in 426 placentas from a sample of infants born < 28 weeks of gestation from the Extremely Low Gestational Age Newborn cohort. Associations between SES adversity and DNA methylation were assessed with robust linear regressions adjusted for covariates and controlled the false discovery rate at < 10%. We also examined whether such associations were sex specific. Indicators of SES adversity were associated with differential methylation at 33 CpG sites. Of the 33 identified CpG sites, 19 (57.6%) displayed increased methylation, and 14 (42.4%) displayed decreased methylation in association with at least one of the SES adversity factors. Sex differences were observed in DNA methylation associated with summative SES score; in which placentas derived from female pregnancies showed more robust differential CpG methylation than placentas from male pregnancies. Maternal SES adversity was associated with differential methylation of genes with key role in gene transcription and placental function, potentially altering immunity and stress response. Further investigation is needed to evaluate the role of epigenetic differences in mediating the association between maternal socioeconomic status during pregnancy and later life health outcomes in children.

Epigenetic marks of prenatal air pollution exposure found in multiple tissues relevant for child health

BackgroundPrenatal air pollution exposure has been linked to many adverse health conditions in the offspring. However, little is known about the mechanisms underlying these associations. Epigenetics may be one plausible biologic link. Here, we sought to identify site-specific and global DNA methylation (DNAm) changes, in developmentally relevant tissues, associated with prenatal exposure to nitrogen dioxide (NO2) and ozone (O3). Additionally, we assessed whether sex-specific changes in methylation exist and whether DNAm changes are consistently observed across tissues. MethodsGenome-scale DNAm measurements were obtained using the Infinium HumanMethylation450k platform for 133 placenta and 175 cord blood specimens from Early Autism Risk Longitudinal Investigation (EARLI) neonates. Ambient NO2 and O3 exposure levels were based on prenatal address locations of EARLI mothers and the Environmental Protection Agency's AirNOW monitoring network using inverse distance weighting. We computed sample-level aggregate methylation measures for each of 5 types of genomic regions including genome-wide, open sea, shelf, shore, and island regions. Linear regression was performed for each genomic region; per-sample aggregate methylation measures were modeled as a function of quantitative exposure level with covariate adjustment. In addition, bumphunting was performed to identify differentially methylated regions (DMRs) associated with prenatal O3 and NO2 exposures in each tissue and by sex, with adjustment for technical and biological sources of variation. ResultsWe identified global and locus-specific changes in DNA methylation related to prenatal exposure to NO2 and O3 in 2 developmentally relevant tissues. Neonates with increased prenatal O3 exposure had lower aggregate levels of DNAm at CpGs located in open sea and shelf regions of the genome. We identified 6 DMRs associated with prenatal NO2 exposure, including 3 sex-specific. An additional 3 sex-specific DMRs were associated with prenatal O3 exposure levels. DMRs initially detected in cord blood samples (n = 4) showed consistent exposure-related changes in DNAm in placenta. However, the DMRs initially detected in placenta (n = 5) did not show DNAm differences in cord blood and, thus, they appear to be tissue-specific. ConclusionsWe observed global, locus, and sex-specific methylation changes associated with prenatal NO2 and O3 exposures. Our findings support DNAm is a biologic target of prenatal air pollutant exposures and highlight epigenetic involvement in sex-specific differential susceptibility to environmental exposure effects in 2 developmentally relevant tissues.

MiR-126 regulates glycogen trophoblast proliferation and DNA methylation in the murine placenta

A functional placenta develops through a delicate interplay of its vascular and trophoblast compartments. We have identified a previously unknown expression domain for the endothelial-specific microRNA miR-126 in trophoblasts of murine and human placentas. Here, we determine the role of miR-126 in placental development using a mouse model with a targeted deletion of miR-126. In addition to vascular defects observed only in the embryo, loss of miR-126 function in the placenta leads to junctional zone hyperplasia at E15.5 at the expense of the labyrinth, reduced placental volume for nutrient exchange and intra-uterine growth restriction of the embryos. Junctional zone hyperplasia results from increased numbers of proliferating glycogen trophoblast (GlyT) progenitors at E13.5 that give rise to an expanded glycogen trophoblast population at E15.5. Transcriptomic profile of miR-126-/- placentas revealed dysregulation of a large number of GlyT (Prl6a1, Prl7c1, Pcdh12) and trophoblast-specific genes (Tpbpa, Tpbpb, Prld1) and genes with known roles in placental development. We show that miR-126-/- placentas, but not miR-126-/- embryos, display aberrant expression of imprinted genes with important roles in glycogen trophoblasts and junctional zone development, including Igf2, H19, Cdkn1c and Phlda2, during mid-gestation. We also show that miR126-/- placentas display global hypermethylation, including at several imprint control centers. Our findings uncover a novel role for miR-126 in regulating extra-embryonic energy stores, expression of imprinted genes and DNA methylation in the placenta.

Fetal glucocorticoid receptor (Nr3c1) deficiency alters the landscape of DNA methylation of murine placenta in a sex-dependent manner and is associated to anxiety-like behavior in adulthood

Prenatal stress defines long-term phenotypes through epigenetic programming of the offspring. These effects are potentially mediated by glucocorticoid release and by sex. We hypothesized that the glucocorticoid receptor (Gr, Nr3c1) fashions the DNA methylation profile of offspring. Consistent with this hypothesis, fetal Nr3c1 heterozygosity leads to altered DNA methylation landscape in fetal placenta in a sex-specific manner. There was a significant overlap of differentially methylated genes in fetal placenta and adult frontal cortex in Nr3c1 heterozygotes. Phenotypically, Nr3c1 heterozygotes show significantly more anxiety-like behavior than wildtype. DNA methylation status of fetal placental tissue is significantly correlated with anxiety-like behavior of the same animals in adulthood. Thus, placental DNA methylation might predict behavioral phenotypes in adulthood. Our data supports the hypothesis that Nr3c1 influences DNA methylation at birth and that DNA methylation in placenta correlates with adult frontal cortex DNA methylation and anxiety-like phenotypes.

Methionine Supply During Late-Gestation Triggers Offspring Sex-Specific Divergent Changes in Metabolic and Epigenetic Signatures in Bovine Placenta

Nonruminant male and female offspring respond differently to gestational nutrition, with placenta contributing to the underlying mechanisms. However, similar data are lacking in large ruminants. The aim of this study was to investigate the impact of methionine supply during late-gestation on metabolism and DNA methylation in placenta from cows carrying male or female calves. During the last 28 d of pregnancy, cows were individually fed a control diet (CON) or the control diet plus rumen-protected d,l-methionine (MET; 0.9 g/kg dry matter intake). Placentomes collected at term were classified according to cow dietary treatment and offspring sex as follows: Male CON (n=7), Male MET (n=7), Female CON (n=8), and Female MET (n=8). Calf growth was measured until 9 wk of age. Targeted metabolomics, RT-PCR, global DNA methylation, and activity of selected enzymes in one-carbon metabolism and transsulfuration pathways were performed. Statistics were conducted via ANOVA using MIXED models. At birth, Male MET calves were heavier than Male CON calves (7.6%, P=0.02), but body mass was similar at 9 wk of age. In contrast, compared with Female CON, Female MET calves had greater body mass at 9 wk of age (6.3%, P=0.03). Compared with Male CON, placenta from Male MET calves had greater concentrations of tricarboxylic acid (TCA) cycle and transsulfuration intermediates (23-100%, P<0.05), along with greater 5-methyltetrahydrofolatehomocysteine methyltransferase activity (67%, P=0.03). Compared with Female CON, placenta from Female MET calves had greater concentrations of one-carbon metabolism intermediates (13-52%, P<0.05). DNA methyltransferase 3A (DNMT3A) was upregulated (43%, P<0.01) in placenta from Female MET compared with Female CON calves. Global DNA methylation was lower in placenta from Female MET compared with Female CON calves (45%, P=0.06). Methionine supply affects placental metabolism, DNA methylation, and body mass of the calf in a sex-specific manner, underscoring its importance as dietary methyl-donor for pregnant cows.

Epigenetic changes in preterm birth placenta suggest a role for ADAMTS genes in spontaneous preterm birth.

Preterm birth (PTB) affects approximately 1 in 10 pregnancies and contributes to approximately 50% of neonatal mortality. However, despite decades of research, little is understood about the etiology of PTB, likely due to the multifactorial nature of the disease. In this study, we examined preterm and term placentas, from unassisted conceptions and those conceived using in vitro fertilization (IVF). IVF increases the risk of PTB and causes epigenetic change in the placenta and fetus; therefore, we utilized these patients as a unique population with a potential common etiology. We investigated genome-wide DNA methylation in placentas from term IVF, preterm IVF, term control (unassisted conception) and preterm control pregnancies and discovered epigenetic dysregulation of multiple genes involved in cell migration, including members of the ADAMTS family, ADAMTS12 and ADAMTS16. These genes function in extracellular matrix regulation and tumor cell invasion, processes replicated by invasive trophoblasts (extravillous trophoblasts (EVTs)) during early placentation. Though expression was similar between term and preterm placentas, we found that both genes demonstrate high expression in first- and second-trimester placenta, specifically in EVTs and syncytiotrophoblasts. When we knocked down ADAMTS12 or ADAMTS16in vitro, there was poor EVT invasion and reduced matrix metalloproteinase activity, reinforcing their critical role in placentation. In conclusion, utilizing a population at high risk for PTB, we have identified a role for ADAMTS gene methylation in regulating early placentation and susceptibility to PTB.

Placental DNA Methylation of miR-548 and WWTR1 Genes Influence Insulin Sensitivity during Pregnancy

Insulin sensitivity decrease in pregnancy is a physiologic adaptation to derive nutrients for the growing fetus. The placenta has a major role in this phenomenon, but exact mechanisms remain unknown. A better understanding of the placental role in maternal insulin sensitivity regulation could help us reveal pathophysiologic pathways of gestational diabetes. We tested associations between insulin sensitivity and DNA methylation (DNAm) across the genome in 439 placenta samples from our prospective cohort Gen3G using Illumina EPIC arrays. Women were recruited at 1st trimester of pregnancy and performed a 2-hour 75g oral glucose tolerance test (OGTT) at 24-28 weeks: we measured glucose and insulin levels at each OGTT point to calculate Matsuda insulin sensitivity index. We applied standard quality control procedures for DNAm arrays analyses and adjusted for technical batch effects. We tested 720,077 individual CpGs using robust linear regression on M-values (adjusted for maternal age, BMI, smoking, parity, gestational age at birth, and child sex) and used Bonferroni to account for multiple testing. We found that maternal insulin sensitivity was associated with DNAm in placenta at 318 CpGs across the genome (P&amp;lt;6.94 x10-8). Among these, we found differentially methylated CpGs at loci well known for their role in diabetes (TCF7L2, SPRY2), brown adipose tissue regulation (PRDM16), and near the Prolactin Receptor. Mendelian Randomization (MR) analyses supported that placental DNAm causally influence maternal insulin sensitivity at 28 CpGs (P&amp;lt;0.05). Among these, our two strongest MR associations (FDR adjusted P&amp;lt;0.05) pointed at CpGs near miR-548 (P=5.6x10-5) and WWTR1 (P=9.8x10-5), a gene implicated in PPARalpha regulation and previously associated with preeclampsia. Our findings highlight the role of placenta epigenetic regulation in insulin sensitivity during pregnancy and reveal novel potential biologic targets implicated in gestational metabolic disorders. Disclosure M. Hivert: None. C.M. Briggs: None. A. Cardenas: None. P. Perron: Research Support; Self; Merck &amp; Co., Inc., Pfizer Inc., Novo Nordisk Inc.. Speaker's Bureau; Self; Sanofi. Research Support; Self; AstraZeneca. L. Bouchard: None.

Offspring sex impacts DNA methylation and gene expression in placentae from women with diabetes during pregnancy.

Aims/HypothesisWe hypothesized that diabetes during pregnancy (DDP) alters genome-wide DNA methylation in placenta resulting in differentially methylated loci of metabolically relevant genes and downstream changes in RNA and protein expression.MethodsWe mapped genome-wide DNA methylation with the Infinium 450K Human Methylation Bead Chip in term fetal placentae from Native American and Hispanic women with DDP using a nested case-control design (n = 17 pairs). RNA expression and protein levels were assayed via RNA-Seq and Western Blot.ResultsGenome-wide DNA methylation analysis revealed 465 CpG sites with significant changes for male offspring, 247 for female offspring, and 277 for offspring of both sexes (p<0.001). Placentae from female offspring were 40% more likely to have significant gains in DNA methylation compared with placentae from male offspring exposed to DDP (p<0.001). Changes in DNA methylation corresponded to changes in RNA and protein levels for 6 genes: PIWIL3, CYBA, GSTM1, GSTM5, KCNE1 and NXN. Differential DNA methylation was detected at loci related to mitochondrial function, DNA repair, inflammation, oxidative stress.Conclusions/InterpretationThese findings begin to explain mechanisms responsible for the increased risk for obesity and type 2 diabetes in offspring of mothers with DDP.