Prenatal antiepileptic exposure associates with neonatal DNA methylation differences

Antiepileptic drugs (AEDs) are used to treat a variety of neuropsychiatric illnesses commonly encountered in women during their reproductive years, including epilepsy and bipolar disorder. Despite their widespread use, the impact of prenatal exposure on fetal development remains obscure. To evaluate whether AEDs taken by pregnant mothers influence DNA methylation patterns in their neonates, DNA was extracted from the umbilical cord blood of 201 neonates whose mothers were treated for neuropsychiatric illness during pregnancy and interrogated across 27,578 CpG sites using the Illumina HumanMethylation27 BeadChip. The association of each methylation value with the cumulative duration of prenatal AED exposure was examined using a linear mixed model. The average methylation level across all CpG sites was calculated for each subject, and this global methylation measure was evaluated similarly. Neonates with a longer duration of AED exposure in pregnancy showed a decrease in average global methylation (p = 0.0045). Further, DNA methylation of CpG sites in 14 genes significantly decreased with the duration of prenatal AED exposure even after adjusting for multiple comparisons (FDR < 0.05). For a small subset (n = 19) of these neonates, a second tissue, placenta, was available in addition to cord blood. Methylation of 3 of these 14 CpG sites was also significantly decreased in placental tissue. These novel data suggest decreased DNA methylation in neonates of mothers who took AEDs during pregnancy. The long-term stability and potential impact of these changes warrant further attention, and caution may be warranted before prescribing AEDs to pregnant women.     

DNA methylation in neonates born to women receiving psychiatric care

Prenatal exposure both to maternal psychiatric illness and psychiatric medication has been linked with adverse child outcomes that affect physiological, emotional and psychiatric development. Studies suggest that epigenetic mechanisms, such as DNA methylation, may facilitate these effects. In this report, we explore the association between maternal psychiatric illness and treatment during pregnancy and neonatal DNA methylation patterns in a prospectively-characterized clinical cohort of 201 dyads. Associations between the percent of umbilical cord blood DNA methylated at 27,578 CpG sites and maternal psychiatric diagnosis, symptoms and antidepressant use were evaluated by fitting a separate linear mixed effects model for each CpG site. There were no significant changes in neonatal DNA methylation attributable to maternal psychiatric diagnosis or depressive symptoms during pregnancy. Exposure to an antidepressant medication was associated with differential methylation of CpG sites in TNFRSF21 and CHRNA2 (false discovery rate < 0.05), but the average difference in methylation for both CpG sites was less than 3% between each group. The results were not specific to type of antidepressant or duration of the exposure. This study suggests that there are no large effects of maternal psychiatric illness, depressive symptoms or prenatal exposure to antidepressants on neonatal DNA methylation. Delineation of the influence of maternal psychiatric illness and pharmacological exposures on the developing fetuses has critical implications for clinical care during pregnancy.     

DNA methylation in neonates born to women receiving psychiatric care

Prenatal exposure both to maternal psychiatric illness and psychiatric medication has been linked with adverse child outcomes that affect physiological, emotional and psychiatric development. Studies suggest that epigenetic mechanisms, such as DNA methylation, may facilitate these effects. In this report, we explore the association between maternal psychiatric illness and treatment during pregnancy and neonatal DNA methylation patterns in a prospectively-characterized clinical cohort of 201 dyads. Associations between the percent of umbilical cord blood DNA methylated at 27,578 CpG sites and maternal psychiatric diagnosis, symptoms and antidepressant use were evaluated by fitting a separate linear mixed effects model for each CpG site. There were no significant changes in neonatal DNA methylation attributable to maternal psychiatric diagnosis or depressive symptoms during pregnancy. Exposure to an antidepressant medication was associated with differential methylation of CpG sites in TNFRSF21 and CHRNA2 (false discovery rate < 0.05), but the average difference in methylation for both CpG sites was less than 3% between each group. The results were not specific to type of antidepressant or duration of the exposure. This study suggests that there are no large effects of maternal psychiatric illness, depressive symptoms or prenatal exposure to antidepressants on neonatal DNA methylation. Delineation of the influence of maternal psychiatric illness and pharmacological exposures on the developing fetuses has critical implications for clinical care during pregnancy.     

Prenatal exposure to polycyclic aromatic hydrocarbons could increase the risk of low birth weight by affecting the DNA methylation states in a Chinese cohort

Polycyclic aromatic hydrocarbons (PAHs), as a kind of endocrine disruptors, can enter the fetus body cross the placental barrier from prenatal PAHs exposure to cause adverse birth outcomes. However, it is controversial association between prenatal PAHs exposure and low birth weight (LBW) of their infants. So the present study aimed to estimate the effects of prenatal PAHs exposure during the pregnancy on the risk of LBW in a Chinese cohort through modifying the DNA methylation states. A longitudinal prospective study with 407 pregnant women was established from May to October 2019. The prenatal PAHs exposure during the pregnancy was assessed using the internal dose such as the PAHs metabolites and PAH-DNA adducts in the umbilical cord blood. The methylation levels of genomic DNA and growth-related genes (IGF1 and IGF2) were assessed, while the expressions of these genes were both determined by RT-PCR and Elisa methods. The growth outcomes and relevant Z-scores were recorded at birth. The correlations between the DNA methylation status and concentrations of PAHs, expression levels of growth-related genes and body weight/WAZ were investigated as the measures. According to the PAH-DNA adducts, the subjects were divided into two groups: PAHs-exposed group (PAH-DNA adducts>0, n = 55) and non-exposed group (PAH-DNA adducts = 0, n = 352). Compared with the non-exposed group, it displayed marked decreased birth weight, and increased concentrations of PAHs and DNA methylation levels of the global genomic, IGF1 and IGF2 with their lower expressions in the PAHs-exposed group. These hypermethylation (global genomic, CpG14 and CpG15 of IGF1, and CpG14 of IGF2) were positively correlated with the contents of PAHs in the umbilical cord blood, and negatively correlated with the growth outcomes and their expressions. Totally, prenatal PAHs exposures may contribute to an increased risk of LBW of their infants by modulating the DNA methylation states of genomic DNA and growth-related genes (IGF1 and IGF2) in the umbilical cord blood, which could provide the prenatal prevention of PAHs exposure from possible environmental media except from the occupation and tobacco usage to ensure the health of their infants.     

In Utero Exposure to Diethylstilbestrol and Blood DNA Methylation in Women Ages 40–59 Years from the Sister Study

In utero exposure to diethylstilbestrol (DES) has been associated with increased risk of adverse health outcomes such as fertility problems and vaginal as well as breast cancer. Animal studies have linked prenatal DES exposure to lasting DNA methylation changes. We investigated genome-wide DNA methylation and in utero DES exposure in a sample of non-Hispanic white women aged 40–59 years from the Sister Study, a large United States cohort study of women with a family history of breast cancer. Using questionnaire information from women and their mothers, we selected 100 women whose mothers reported taking DES while pregnant and 100 control women whose mothers had not taken DES. DNA methylation in blood was measured at 485,577 CpG sites using the Illumina HumanMethylation450 BeadChip. Associations between CpG methylation and DES exposure status were analyzed using robust linear regression with adjustment for blood cell composition and multiple comparisons. Although four CpGs had p<10⁵, after accounting for multiple comparisons using the false discovery rate (FDR), none reached genome-wide significance. In conclusion, adult women exposed to DES in utero had no evidence of large persistent changes in blood DNA methylation.     

Neonatal DNA methylation patterns associate with gestational age

Risk for adverse neonatal outcome increases with declining gestational age (GA), and changes in DNA methylation may contribute to the relationship between GA and adverse health outcomes in offspring. To test this hypothesis, we evaluated the association between GA and more than 27,000 CpG sites in neonatal DNA extracted from umbilical cord blood from two prospectively-characterized cohorts: (1) a discovery cohort consisting of 259 neonates from women with a history of neuropsychiatric disorders and (2) a replication cohort consisting of 194 neonates of uncomplicated mothers. GA was determined by obstetrician report and maternal last menstrual period. The associations between proportion of DNA methylated and GA were evaluated by fitting a separate linear mixed effects model for each CpG site, adjusting for relevant covariates including neonatal sex, race, parity, birth weight percentile and chip effects. CpG sites in 39 genes were associated with GA (false discovery rate < 0.05) in the discovery cohort. The same CpG sites in 25 of these genes replicated in the replication cohort, with each association replicating in the same direction. Notably, these CpG sites were located in genes previously implicated in labor and delivery (e.g., AVP, OXT, CRHBP and ESR1) or that may influence the risk for adverse health outcomes later in life (e.g., DUOX2, TMEM176A and CASP8). All associations were independent of method of delivery or induction of labor. These results suggest neonatal DNA methylation varies with GA even in term deliveries. The potential contribution of these changes to clinically significant postnatal outcomes warrants further investigation.     

Epigenetic effects of prenatal stress on 11β-hydroxysteroid dehydrogenase-2 in the placenta and fetal brain

Maternal exposure to stress during pregnancy is associated with significant alterations in offspring neurodevelopment and elevated maternal glucocorticoids likely play a central role in mediating these effects. Placental 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) buffers the impact of maternal glucocorticoid exposure by converting cortisol/corticosterone into inactive metabolites. However, previous studies indicate that maternal adversity during the prenatal period can lead to a down-regulation of this enzyme. In the current study, we examined the impact of prenatal stress (chronic restraint stress during gestational days 14-20) in Long Evans rats on HSD11B2 mRNA in the placenta and fetal brain (E20) and assessed the role of epigenetic mechanisms in these stress-induced effects. In the placenta, prenatal stress was associated with a significant decrease in HSD11B2 mRNA, increased mRNA levels of the DNA methyltransferase DNMT3a, and increased DNA methylation at specific CpG sites within the HSD11B2 gene promoter. Within the fetal hypothalamus, though we find no stress-induced effects on HSD11B2 mRNA levels, prenatal stress induced decreased CpG methylation within the HSD11B2 promoter and increased methylation at sites within exon 1. Within the fetal cortex, HSD11B2 mRNA and DNA methylation levels were not altered by prenatal stress, though we did find stress-induced elevations in DNMT1 mRNA in this brain region. Within individuals, we identified CpG sites within the HSD11B2 gene promoter and exon 1 at which DNA methylation levels were highly correlated between the placenta and fetal cortex. Overall, our findings implicate DNA methylation as a mechanism by which prenatal stress alters HSD11B2 gene expression. These findings highlight the tissue specificity of epigenetic effects, but also raise the intriguing possibility of using the epigenetic status of placenta to predict corresponding changes in the brain.     

Prenatal antidepressant exposure associated with CYP2E1 DNA methylation change in neonates

Some but not all neonates are affected by prenatal exposure to serotonin reuptake inhibitor antidepressants (SRI) and maternal mood disturbances. Distinguishing the impact of these 2 exposures is challenging and raises critical questions about whether pharmacological, genetic, or epigenetic factors can explain the spectrum of reported outcomes. Using unbiased DNA methylation array measurements followed by a detailed candidate gene approach, we examined whether prenatal SRI exposure was associated with neonatal DNA methylation changes and whether such changes were associated with differences in birth outcomes. Prenatal SRI exposure was first associated with increased DNA methylation status primarily at CYP2E1(β_Non-exposed = 0.06, β_SRI-exposed = 0.30, FDR = 0); however, this finding could not be distinguished from the potential impact of prenatal maternal depressed mood. Then, using pyrosequencing of CYP2E1 regulatory regions in an expanded cohort, higher DNA methylation status—both the mean across 16 CpG sites (P < 0.01) and at each specific CpG site (P < 0.05)—was associated with exposure to lower 3rd trimester maternal depressed mood symptoms only in the SRI-exposed neonates, indicating a maternal mood x SRI exposure interaction. In addition, higher DNA methylation levels at CpG2 (P = 0.04), CpG9 (P = 0.04) and CpG10 (P = 0.02), in the interrogated CYP2E1 region, were associated with increased birth weight independently of prenatal maternal mood, SRI drug exposure, or gestational age at birth. Prenatal SRI antidepressant exposure and maternal depressed mood were associated with altered neonatal CYP2E1 DNA methylation status, which, in turn, appeared to be associated with birth weight.     

Sex-specific effects of early life cadmium exposure on DNA methylation and implications for birth weight

Dietary cadmium exposure was recently found to alter DNA methylation in adults, but data on effects early in life are lacking. Our objective was to evaluate associations between prenatal cadmium exposure, DNA methylation and birth weight. In total 127 mother-child pairs from rural Bangladesh were studied. For comparison, we included 56 children at 4.5 y. Cadmium concentrations in mothers’ blood (gestational week 14) and children’s urine were measured by ICPMS. Global DNA methylation was analyzed by Infinium HumanMethylation450K BeadChip in cord blood and children’s blood. Maternal cadmium exposure was associated with cord blood DNA methylation (p-value < 10^–16). The association was markedly sex-specific. In boys, 96% of the top 500 CpG sites showed positive correlations (r_S-values > 0.50), whereas most associations in girls were inverse; only 29% were positive (r_S > 0.45). In girls we found overrepresentation of methylation changes in genes associated with organ development, morphology and mineralization of bone, whereas changes in boys were found in cell death-related genes. Several individual CpG sites that were positively associated with cadmium were inversely correlated with birth weight, although none statistically significant after correction for multiple comparisons. The associations were, however, fairly robust in multivariable-adjusted linear regression models. We identified CpG sites that were significantly associated with cadmium exposure in both newborns and 4.5-y-old children. In conclusion, cadmium exposure in early life appears to alter DNA methylation differently in girls and boys. This is consistent with previous findings of sex-specific cadmium toxicity. Cadmium-related changes in methylation were also related to lower birth weight.     

The roles of DNA methylation of NR3C1 and 11β-HSD2 and exposure to maternal mood disorder in utero on newborn neurobehavior

Exposure to maternal mood disorder in utero may program infant neurobehavior via DNA methylation of the glucocorticoid receptor (NR3C1) and 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD-2), two placental genes that have been implicated in perturbations of the hypothalamic pituitary adrenocortical (HPA) axis. We tested the relations among prenatal exposure to maternal depression or anxiety, methylation of exon 1F of NR3C1 and 11β-HSD-2, and newborn neurobehavior. Controlling for relevant covariates, infants whose mothers reported depression during pregnancy and showed greater methylation of placental NR3C1 CpG2 had poorer self-regulation, more hypotonia, and more lethargy than infants whose mothers did not report depression. On the other hand, infants whose mothers reported anxiety during pregnancy and showed greater methylation of placental 11β-HSD-2 CpG4 were more hypotonic compared with infants of mothers who did not report anxiety during pregnancy. Our results support the fetal programming hypothesis and suggest that fetal adjustments to cues from the intrauterine environment, in this case an environment that could be characterized by increased exposure to maternal cortisol, may lead to poor neurodevelopmental outcomes.     

Dietary and supplemental maternal methyl-group donor intake and cord blood DNA methylation

Maternal nutrition is critically involved in the development and health of the fetus. We evaluated maternal methyl-group donor intake through diet (methionine, betaine, choline, folate) and supplementation (folic acid) before and during pregnancy in relation to global DNA methylation and hydroxymethylation and gene specific (IGF2 DMR, DNMT1, LEP, RXRA) cord blood methylation. A total of 115 mother-infant pairs were enrolled in the MAternal Nutrition and Offspring's Epigenome (MANOE) study. The intake of methyl-group donors was assessed using a food-frequency questionnaire. LC-MS/MS and pyrosequencing were used to measure global and gene specific methylation, respectively. Dietary intake of methyl-groups before and during pregnancy was associated with changes in LEP, DNMT1, and RXRA cord blood methylation. Statistically significant higher cord blood LEP methylation was observed when mothers started folic acid supplementation more than 6 months before conception compared with 3–6 months before conception (34.6 ± 6.3% vs. 30.1 ± 3.6%, P = 0.011, LEP CpG1) or no folic acid used before conception (16.2 ± 4.4% vs. 13.9 ± 3%, P = 0.036 for LEP CpG3 and 24.5 ± 3.5% vs. 22.2 ± 3.5%, P = 0.045 for LEP mean CpG). Taking folic acid supplements during the entire pregnancy resulted in statistically significantly higher cord blood RXRA methylation as compared with stopping supplementation in the second trimester (12.3 ± 1.9% vs. 11.1 ± 2%, P = 0.008 for RXRA mean CpG). To conclude, long-term folic acid use before and during pregnancy was associated with higher LEP and RXRA cord blood methylation, respectively. To date, pregnant women are advised to take a folic acid supplement of 400 µg/day from 4 weeks before until 12 weeks of pregnancy. Our results suggest significant epigenetic modifications when taking a folic acid supplement beyond the current advice.     

Neonatal DNA methylation patterns associate with gestational age

Risk for adverse neonatal outcome increases with declining gestational age (GA), and changes in DNA methylation may contribute to the relationship between GA and adverse health outcomes in offspring. To test this hypothesis, we evaluated the association between GA and more than 27,000 CpG sites in neonatal DNA extracted from umbilical cord blood from two prospectively-characterized cohorts: (1) a discovery cohort consisting of 259 neonates from women with a history of neuropsychiatric disorders and (2) a replication cohort consisting of 194 neonates of uncomplicated mothers. GA was determined by obstetrician report and maternal last menstrual period. The associations between proportion of DNA methylated and GA were evaluated by fitting a separate linear mixed effects model for each CpG site, adjusting for relevant covariates including neonatal sex, race, parity, birth weight percentile and chip effects. CpG sites in 39 genes were associated with GA (false discovery rate &lt; 0.05) in the discovery cohort. The same CpG sites in 25 of these genes replicated in the replication cohort, with each association replicating in the same direction. Notably, these CpG sites were located in genes previously implicated in labor and delivery (e.g., AVP, OXT, CRHBP and ESR1) or that may influence the risk for adverse health outcomes later in life (e.g., DUOX2, TMEM176A and CASP8). All associations were independent of method of delivery or induction of labor. These results suggest neonatal DNA methylation varies with GA even in term deliveries. The potential contribution of these changes to clinically significant postnatal outcomes warrants further investigation.     

SLC9B1 methylation predicts fetal intolerance of labor

Fetal intolerance of labor is a common indication for delivery by Caesarean section. Diagnosis is based on the presence of category III fetal heart rate tracing, which is an abnormal heart tracing associated with increased likelihood of fetal hypoxia and metabolic acidemia. This study analyzed data from 177 unique women who, during their prenatal visits (7-15 weeks and/or 24–32 weeks) to Atlanta area prenatal care clinics, consented to provide blood samples for DNA methylation (HumanMethylation450 BeadChip) and gene expression (Human HT-12 v4 Expression BeadChip) analyses. We focused on 57 women aged 18–36 (mean 25.4), who had DNA methylation data available from their second prenatal visit. DNA methylation patterns at CpG sites across the genome were interrogated for associations with fetal intolerance of labor. Four CpG sites (P value <8.9 × 10^?9, FDR <0.05) in gene SLC9B1, a Na⁺/H⁺ exchanger, were associated with fetal intolerance of labor. DNA methylation and gene expression were negatively associated when examined longitudinally during pregnancy using a linear mixed-effects model. Positive predictive values of methylation of these four sites ranged from 0.80 to 0.89, while negative predictive values ranged from 0.91 to 0.92. The four CpG sites were also associated with fetal intolerance of labor in an independent cohort (the Johns Hopkins Prospective PPD cohort). Therefore, fetal intolerance of labor could be accurately predicted from maternal blood samples obtained between 24–32 weeks gestation. Fetal intolerance of labor may be accurately predicted from maternal blood samples obtained between 24–32 weeks gestation by assessing DNA methylation patterns of SLC9B1. The identification of pregnant women at elevated risk for fetal intolerance of labor may allow for the development of targeted treatments or management plans.     

Quantitative,high-resolution epigenetic profiling of CpG loci identifies associations with cord blood plasma homocysteine and birth weight in humans

Supplementation with folic acid during pregnancy is known to reduce the risk of neural tube defects and low birth weight. It is thought that folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. We examined the effects of folate on the human methylome using quantitative interrogation of 27,578 CpG loci associated with 14,496 genes at single-nucleotide resolution across 12 fetal cord blood samples. Consistent with previous studies, the majority of CpG dinucleotides located within CpG islands exhibited hypo-methylation while those outside CpG islands showed mid-high methylation. However, for the first time in human samples, unbiased analysis of methylation across samples revealed a significant correlation of methylation patterns with plasma homocysteine, LINE-1 methylation and birth weight centile. Additionally, CpG methylation significantly correlated with either birth weight or LINE-1 methylation were predominantly located in CpG islands. These data indicate that levels of folate-associated intermediates in cord blood reflect their influence and consequences for the fetal epigenome and potentially on pregnancy outcome. In these cases, their influence might be exerted during late gestation or reflect those present during the peri-conceptual period.     

The fetal programming effect of prenatal smoking on Igf1r and Igf1 methylation is organ- and sex-specific

The impact of prenatal smoke exposure (PSE) on DNA methylation has been demonstrated in blood samples from children of smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. As the identified differentially methylated genes can be associated with developmental processes, and insulin-like growth factors (IGFs) play a critical role in prenatal tissue growth, we hypothesized that PSE induces fetal programming of Igf1r and Igf1. Using a mouse model of smoking during pregnancy, we show that PSE alters promoter methylation of Igf1r and Igf1 and deregulates their gene expression in lung and liver of fetal (E17.5) and neonatal (D3) mouse offspring. By further comparing female versus male, lung versus liver, or fetal versus neonatal time point, our results demonstrate that CpG site-specific aberrant methylation patterns sex-dependently vary per organ and time point. Moreover, PSE reduces gene expression of Igf1r and Igf1, dependent on organ, sex, and offspring's age. Our results indicate that PSE may be a source of organ-specific rather than general systemic fetal programming. This is exemplified here by gene promoter methylation and mRNA levels of Igf1r and Igf1, together with a sex- and organ-specific naturally established correlation of both parameters that is affected by prenatal smoke exposure. Moreover, the comparison of fetuses with neonates suggests a CpG site-dependent reversibility/persistence of PSE-induced differential methylation patterns.     

Quantitative,high-resolution epigenetic profiling of CpG loci identifies associations with cord blood plasma homocysteine and birth weight in humans

Supplementation with folic acid during pregnancy is known to reduce the risk of neural tube defects and low birth weight. It is thought that folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. We examined the effects of folate on the human methylome using quantitative interrogation of 27,578 CpG loci associated with 14,496 genes at single-nucleotide resolution across 12 fetal cord blood samples. Consistent with previous studies, the majority of CpG dinucleotides located within CpG islands exhibited hypomethylation while those outside CpG islands showed mid-high methylation. However, for the first time in human samples, unbiased analysis of methylation across samples revealed a significant correlation of methylation patterns with plasma homocysteine, LINE-1 methylation and birth weight centile. Additionally, CpG methylation significantly correlated with either birth weight or LINE-1 methylation were predominantly located in CpG islands. These data indicate that levels of folate-associated intermediates in cord blood reflect their influence and consequences for the fetal epigenome and potentially on pregnancy outcome. In these cases, their influence might be exerted during late gestation or reflect those present during the peri-conceptual period.Key words: cord blood, birth weight, folic acid, homocysteine, BeadArray, hierarchical clustering, Illumina     

Identification of sex-specific DNA methylation changes driven by specific chemicals in cord blood in a Faroese birth cohort

Faroe islanders consume marine foods contaminated with methylmercury (MeHg), polychlorinated biphenyls (PCBs), and other toxicants associated with chronic disease risks. Differential DNA methylation at specific CpG sites in cord blood may serve as a surrogate biomarker of health impacts from chemical exposures. We aimed to identify key environmental chemicals in cord blood associated with DNA methylation changes in a population with elevated exposure to chemical mixtures. We studied 72 participants of a Faroese birth cohort recruited between 1986 and 1987 and followed until adulthood. The cord blood DNA methylome was profiled using Infinium HumanMethylation450 BeadChips. We determined the associations of CpG site changes with concentrations of MeHg, major PCBs, other organochlorine compounds [hexachlorobenzene (HCB), p,p’-dichlorodiphenyldichloroethylene (p,p’-DDE) and p,p’-dichlorodiphenyltrichloroethane], and perfluoroalkyl substances. In a combined sex analysis, among the 16 chemicals studied, PCB congener 105 (CB-105) exposure was associated with the majority of differentially methylated CpG sites (214 out of a total of 250). In female-only analysis, only 73 CB-105 associated CpG sites were detected, 44 of which were mapped to genes in the ELAV1-associated cancer network. In males-only, methylation changes were seen for perfluorooctane sulfonate, HCB, and p,p’-DDE in 10,598, 1,238, and 1,473 CpG sites, respectively, 15% of which were enriched in cytobands of the X-chromosome associated with neurological disorders. In this multiple-pollutant and genome-wide study, we identified key epigenetic toxicants. The significant enrichment of specific X-chromosome sites in males implies potential sex-specific epigenome responses to prenatal chemical exposures.