Per- and polyfluoroalkyl substances (PFAS) are ubiquitous endocrine-disrupting pollutants that cross the placenta and affect offspring health, but the extent and timing of their transfer to placental and fetal compartments remain poorly understood. We characterized the relationship between trimester-specific prenatal maternal serum PFAS levels and paired placental and cord plasma levels at term. Data came from the Central Arkansas Glowing prospective cohort (n=151, 2010-2014). Four well-detected PFAS were measured using liquid chromatography-tandem mass spectrometry. Regression, elastic net, and parametric g-formula models tested the association between maternal levels in each trimester and placental or cord PFAS levels. In g-formula models, trimester one (T1) or trimester two (T2) measures consistently had the largest effect sizes associated with placental levels (p<0.001-0.05). Similarly, T1 (PFHxS, PFNA, PFOS, PFOA), T2 (PFNA, PFOS, PFOA), and placental PFOA were associated with cord plasma levels (p<0.05-p<0.001). Results were robust to time-varying adjustment for estimated glomerular filtration rate, serum albumin, or maternal weight. Predictive models improved with additional timepoint measures. Our findings suggest PFOA may transfer more efficiently from the placenta to cord plasma and early-to-mid gestation maternal serum PFAS measures may serve as the most robust sentinels of fetoplacental exposure burden, suggesting early exposure prevention should be prioritized.

Child maltreatment is a leading cause of pediatric morbidity and mortality, potentially propagated by DNA methylation (DNAm) changes. We conducted an EWAS meta-analysis (n=175, 554,979 Illumina EPICv1/EPICv2 sites) in buccal swabs from three hospital-based studies of children with traumatic injuries, stratified by study group to include 1) any traumatic injury, 2) fractures, and 3) traumatic brain injuries. Empirical bayes-moderated linear models tested differential DNAm with M-values, followed by near-promoter gene set enrichment analysis. Abuse was associated with methylation at 11 sites (q<0.10), including enhancers of neuroblast differentiation-associated AHNAK, immunomodulators SCGB1A1 and CCL26, exon 5 of LAMP1, essential for lysosomal function and cytotoxicity, and RGS7, a GTPase essential for synaptic transmission. Enriched biological processes included cranial skeletal system and connective tissue development, neural structure and function, immune regulation, gene expression, and metabolism. Our findings suggest that early abuse may epigenetically affect both proximal injury responses and longer-lived systemic biological dysregulation.

Very preterm infants (<30 weeks gestation) are at elevated risk for neurodevelopmental and social-behavioral challenges. DNA methylation (DNAm) may provide a biological link between preterm birth and later behavioral outcomes. We examined associations between DNAm profiles at neonatal intensive care unit (NICU) discharge and at age 5 with Social Responsiveness Scale (SRS) scores which measure social communication, social interaction, and repetitive behaviors at age 5, including sex-specific effects, in the Neonatal Neurobehavior and Outcomes in Very Preterm Infants (NOVI) Study. Epigenome-wide buccal DNAm was profiled at NICU discharge (n=218) and at 5 years (n=188). We identified 38 neonatal and 6 age-5 CpG sites associated with SRS scores (all q<0.05) using epigenome-wide association studies (EWAS) at each time point. Several CpGs mapped to genes involved in neurodevelopment including TCF4 , KLC4 , CAP2 , PTDSS1 , ADAM12 , SENP1 , CHN2 , SH3D19 , and ITGA1 , with sex-specific effects observed for CpGs in CAMTA1 and GABBR1 . Enriched pathways included neurodevelopment, cytoskeletal regulation, stress-response, and metabolic processes. DNAm patterns during early life, particularly the neonatal period, were associated with social-behavioral development in very preterm children. Findings in key genes such as TCF4 and CAMTA1 highlight potential epigenetic mechanisms linking early-life biology to later behavioral challenges.

The Developmental Origins of Health and Disease (DOHaD) hypothesis proposes that the perinatal environment shapes susceptibility to complex traits across life [1]. The placenta, a transient organ mediating maternal-fetal exchange, plays a central role in this process and has emerged as a key molecular archive in utero [2-4]. Placental DNA methylation (DNAm) is a unique mediator between prenatal exposures, fetal genetics and later-life outcomes [5-9]. DNAm quantitative trait loci (mQTL) have helped disentangling causal mechanisms underlying GWAS loci for complex diseases [10-15]. Despite growing evidence that placental genomic regulation has broad and profound effects on the developmental programming of early- and later-life health outcomes [17], existing placental studies remain limited in scale and largely focused on growth- and neuro-related traits [12-16]. Here, we construct a high-resolution placental mQTL resource and systematically investigate how placental DNAm relates to early- and later-life traits, and to shared vulnerability and complex interactions among them.

BACKGROUND:Prenatal air pollution exposure (PAPE) may have programming effects on offspring health. Placental epigenetic modifications are hypothesised to mediate this relationship, but evidence remains limited. We investigated the mediating role of placental DNA methylation (DNAm) in the association between prenatal exposure to NO2, PM2.5 and PM2.5 oxidative potential (OP) and newborn lung function. METHODS:Placental DNAm was measured using the Infinium HumanMethylationEPIC BeadChip in 395 participants from the French cohort SEPAGES. PAPE was estimated via personal sensors. Lung function (6 parameters) was measured at two months through tidal breathing analysis and nitrogen multiple breath washout test. We conducted adjusted epigenome-wide mediation analyses to identify genomic loci (CpGs and aggregated methylated regions; AMRs) that explain the association between PAPE and offspring lung function. FINDINGS:Thirty CpGs and 166 AMRs significantly mediated the PAPE-lung function relationship. Most mediators were involved in the effect of PM2.5 OP on the functional residual capacity (FRC) and the lung clearance index (LCI). While most DNAm changes mediated adverse effects, several loci-especially within imprinted genes-were associated with potentially adaptive responses. Mediating loci mapped to genes involved in lung development, immunity, inflammation and oxidative stress. Expression quantitative trait methylation (eQTM) analyses revealed that nearly one quarter of the mediating CpGs and one half of the AMRs may regulate gene expression. INTERPRETATION:Our findings provide insights into the epigenetic pathways linking PAPE to early-life lung function. They highlight the placenta as a critical interface, where both detrimental and compensatory epigenetic modifications may shape newborn lung function. FUNDING:French Agency for National Research.

Children born preterm are at higher risk of attention problems compared to children born at term. The purpose of this study was to identify epigenetic predictors of early childhood attention problems among children born very preterm and to examine the joint role of epigenetic and environmental factors in predicting attention problems in this population. We studied 242 participants from a multi-site study of infants born < 30 weeks gestational age. Neonatal buccal swabs were assayed for DNA methylation levels at over 850,000 CpG sites, dimension reduction steps allowed us to focus on 450,000 loci, and age acceleration metrics were calculated using existing epigenetic clocks. Cumulative postnatal environmental adversity was calculated using maternal reported risk factors. Attention problems were assessed in early childhood (mean age 6.58 years) using the Conner’s Kiddie Continuous Performance Test 2nd Edition. After adjustment for multiple testing, DNA methylation at 9 loci were associated with childhood attention problems. Several CpGs were located in genes previously linked to neurodevelopmental traits and inflammation in prior epigenome-wide and genome-wide association studies. Greater environmental adversity was also associated with increased attention problems. When tested together, DNA methylation and environmental adversity independently predicted attention problems. This study is the first to show associations between DNA methylation, environmental adversity, and objectively measured attention problems in school-age children born very preterm. These results could shed light on the etiology of attention problems in this population and may help us identify at birth preterm children at highest risk for later ADHD diagnosis.

BACKGROUND:Cannabis and tobacco are contaminated with insecticides and used during pregnancy in the U.S., raising concerns for co-exposures and compounded neurodevelopmental effects. However, these cumulative effects remain unexplored. We examine the associations of prenatal cannabis, tobacco, pyrethroid, and organophosphate insecticides co-exposures with early childhood neurobehaviors. METHODS:Among 197 mother-child pairs from a birth cohort in Atlanta, Georgia, cannabis (THCCOOH), tobacco (COT and 3OH-COT), pyrethroids (3PBA), and organophosphates (TCPY) metabolite levels were quantified in maternal urine sampled at 8-14 and 24-30 weeks' gestation. Infant arousal and attention were evaluated 2 weeks postnatally using the NICU Network Neurobehavioral Assessment Scale. Externalizing and internalizing behaviors were assessed annually using the Child Behavior Checklist and averaged across ages 2-5 years. We examined individual associations using linear regression; cumulative associations using quantile g-computation and Bayesian kernel machine regression (BKMR); and whether THCCOOH modified the cumulative effect of tobacco and insecticides. RESULTS:Of the prenatal exposures, only insecticides were associated with child neurobehavior. For example, a doubling in 3PBA was positively related to internalizing behaviors (β = 18.1 %; 95 % confidence interval [CI] = 0.0 %, 39.5 %), and TCPY was negatively associated with externalizing behaviors (β = -12.9 %; 95 % CI = -27.8 %, 5.0 %). These were modified by THCCOOH and sex. The prenatal 3PBA, TCPY, COT, and 3OH-COT mixture was associated with lower externalizing behaviors among females with detectable THCCOOH (quantile g-computation β = -46.8 %; 95 % CI = -70.4 %, -4.1 %). BKMR showed no interactions and dose-responses. DISCUSSION:Prenatally, 3PBA and TCPY were associated with child neurobehaviors, and effects differed by THCCOOH and sex. Further studies on the neurodevelopmental burden of cannabis, tobacco, and insecticide co-exposures are needed.

e22576 Background: The biological mechanisms underlying air pollution-induced lung carcinogenesis remained underexplored. We assessed individual and joint effects of six air pollutants on human proteome and their associations with lung cancer risk, aiming to identify key proteins and pathways involved in mediating this process. Methods: We included 95 matched case-control pairs of elderly with pre-diagnosis blood samples from the Cancer Prevention Study-II Nutrition Cohort (1998-2001). Plasma levels of 484 proteins were measured. Annual individual exposures to fine and coarse particulate matter (PM 10 ), nitrogen dioxide, ozone, sulfur dioxide, and carbon monoxide in the year of blood draw were retrieved. We used linear regression for individual pollutant effects and quantile g-computation for joint effects on proteins, and conditional logistic regression for proteins and lung cancer risk, adjusting for confounders and multiple testing. We then conducted pathway analysis to identify overlapping proteins and pathways associated with both exposures and lung cancer risk, followed by an exploratory high-dimensional mediation analysis to investigate the potential mediation roles of proteins. Results: Fifteen proteins were associated with air pollution and six with lung cancer risk (FDR < 0.2). Pathway analysis revealed five pathways related to air pollution and two to lung cancer risk, closely linked to immunity and signaling. Seventeen proteins were associated with both exposures and lung cancer risk ( P < 0.05 or FDR < 0.2), including ADAM15, ATRN, and CX3CL1. The PI3K-Akt signaling pathway was associated with both PM 10 and lung cancer risk. Twenty-two proteins were identified as significant mediators of the association between air pollution and lung cancer risk, including several ILs, CCL20, and IFN-gamma. Conclusions: Various immunity and signaling related proteins and pathways may play significant roles in the association between air pollution exposures and lung cancer. The findings provide novel insights into understanding the underlying etiology and contribute to future biomarker development for preventive and therapeutic strategies.

Per- and polyfluoroalkyl substances (PFAS) are endocrine-disrupting chemicals and widespread environmental contaminants. PFAS cross the placental barrier and reach the developing fetus with potential impacts on many organ systems. There are no studies of PFAS in residents of central Arkansas despite reports of environmental contamination in the region. We aimed to quantify PFAS concentrations in repeated serum samples from participants living in central Arkansas and to investigate relationships with maternal cardiometabolic health during pregnancy. Participants were enrolled during early pregnancy in a longitudinal study (NCT01131117) from 2010 to 2014. PFAS concentrations were measured in serum from each trimester (first trimester n = 282, second trimester n = 217, and third trimester n = 195). PFAS were compared across pregnancy. Linear and linear-mixed effects models were used to investigate relationships between trimester-specific PFAS levels, as single exposures, and maternal outcomes. Effects of PFAS as an exposure mixture were estimated using quantile g-computation. Six PFAS were detected in more than 70

Placental gestational age acceleration (GAA) is the difference between the actual gestational age (GA) at birth and their estimated epigenetic gestational age (EGA), which is calculated from placental DNA methylation. Understanding the role of placental GAA in postnatal growth trajectories is crucial for early identification of infants at risk of altered growth patterns and associated long-term health outcomes. The objective of this study is to investigate the association between placental GAA and longitudinal growth trajectories specifically weight, height, fat mass, and lean mass gain in early childhood. This study uses placental DNA methylation at birth to calculate epigenetic GAA and longitudinal measures of weight, height, fat mass, and lean mass to generate growth trajectory characteristics. Higher placental GAA was significantly associated with slower weight gain (95% CI [- 0.03, - 0.001]) and fat mass (95% CI [- 0.08, - 0.02]) gain, as well as reduced average fat mass (95% CI [add this]) over the follow-up period. However, no significant associations were found between GAA and height or lean mass gain. Placental GAA can give early insights into altered postnatal growth trajectories, particularly for weight and fat mass where an increase in GAA is associated with decreased weight and fatmass gain over time while we observed no effect on height and lean mass. Understanding these associations offers insights into early developmental patterns and long-term health outcomes in children, highlighting the importance of perinatal factors in shaping growth trajectories in early childhood.

The impact of air pollution exposure on circulating proteins remains underexplored, particularly in vulnerable elderly populations. This study investigated the individual and joint effects of air pollutants on circulating proteins in 208 elderly participants from the Cancer Prevention Study-II Nutrition Cohort. Prediagnostic plasma samples were collected (1998-2001), and 484 proteins were measured using the Olink platform. Annual average exposures to six air pollutants in the calendar year of blood draw were estimated. We used linear regression for individual pollutants and quantile g-computation for mixture effects, adjusting for confounders, considering multiple comparison correction, and testing interactions with smoking status. Pathway enrichment and protein-protein interaction analyses were conducted for the associated proteins. We identified 167 distinct proteins associated with individual pollutants or mixtures (p < 0.05), including 15 meeting a false discovery rate <0.2. IL32, ADAM15, and IL8 demonstrated consistent negative associations with ≥4 exposure metrics. Twenty proteins were associated with both mixtures and individual air pollutants with consistent effect directions. These proteins were enriched in pathways linked to immunity and signaling. Stratified analyses revealed differing associations with 99 proteins between current and former smokers. The findings offer valuable insight into the chronic biological response in plasma protein levels to air pollution exposure.

Abstract Background Night shift work during pregnancy has been associated with differential DNA methylation in placental tissue, but no studies have explored this association in cord blood. We aimed to examine associations of maternal night shift work with cord blood DNA methylation. Methods A total of 4487 mother–newborn pairs from 7 studies were included. Maternal night shift work during pregnancy was ascertained via questionnaires and harmonized into “any” versus “no”. DNA methylation was measured in cord blood using the Illumina Infinium Methylation arrays. Robust linear regression models adjusted for relevant confounders were run in the individual cohorts, and results were meta-analyzed. Results Maternal night shift work during pregnancy ranged from 3.4% to 26.3%. Three CpGs were differentially methylated in relation to maternal night shift work during pregnancy at a false discovery rate adjusted P < 0.05: cg10945885 (estimate (β) 0.38%, standard error (SE) 0.07), cg00773359 (β 0.25%, SE 0.05), and cg21836426 (β − 0.29%, SE 0.05). Associations of the identified CpGs were found in previous literature for gestational age and childhood and adolescent BMI. In a mouse model of prenatal jet lag exposure, information on offspring DNA methylation of ten homologous genes annotated to the 16 CpGs with P < 1 × 10−5 in our analysis was available, of which eight were associated (enrichment P: 1.62 × 10−11). Conclusion Maternal night shift work during pregnancy was associated with newborn DNA methylation at 3 CpGs. Top findings overlapped with those in a mouse model of gestational jet lag. This work strengthens evidence that DNA methylation could be a marker or mediator of impacts of circadian rhythm disturbances.

Maternal serum per- and polyfluoroalkyl substances (PFAS) are linked to infant neurodevelopment vulnerabilities. However, the impact of placental PFAS exposure, a more proximal in utero exposure estimate, remains unknown. We hypothesize that elevated placental PFAS levels are associated with delayed neurodevelopment at 12 and 24 months.Mother-infant dyads (n = 151) were enrolled in a prospective cohort in Arkansas, US. PFOA, PFOS, PFHxS, PFNA, and PFDA were detected in >65 % of placentas. The Bayley Scales of Infant and Toddler Development (BSID)-III was administered at 12 and 24 months. Individual associations of each placental PFAS on BSID-III scores were examined using linear regressions. Mixture effects were assessed using quantile g-computation and Bayesian Kernel Machine Regression.Placental PFAS were jointly associated with lower cognitive (Ψ = -2.6; 95 % CI = -6.0, 0.9) and motor (Ψ = -2.9; 95 % CI = -6.6, 0.9) scores at 12 months and higher social-emotional scores (Ψ = 3.3; 95 % CI = -1.9, 8.5) at 24 months among males. Among females, higher PFAS mixture levels were associated with higher language (Ψ = 3.3; 95 % CI = -1.2, 7.7) and social-emotional (Ψ = 3.7; 95 % CI = -3.3, 10.6) scores at 24 months. Linear regressions showed PFHxS and PFOS were associated with lower cognitive scores at 12 months and PFDA was associated with lower motor scores at 12 months among males, while PFOS and PFNA were associated with higher social-emotional scores at 24 months among females, although confidence intervals included the null. In a matched subset with maternal serum, cord serum and placental PFAS (n = 98), we observed mostly null associations consistently across biomatrices and noted few associations, such as a negative relation of cord PFOA and PFHxS with language scores at 12 months among females.Overall, placental PFAS levels were modestly associated with performance on neurodevelopmental assessments in early childhood and these relationships were sex- and compound-specific, and most confidence intervals cross the null.

BACKGROUND:Per- and polyfluoroalkyl substances (PFAS) are environmental toxicants associated with adverse neonatal outcomes. The exact mechanisms by which PFAS impairs neonatal health are undefined, but the placenta is a likely target. OBJECTIVE:We applied a systems biology approach to identify placental RNA co-expression modules (gene sets) associated with PFAS exposure and birth weight. METHODS:Placental tissue samples (n = 147) from the GLOWING study underwent RNA-sequencing, and PFAS concentrations were quantified using liquid chromatography-tandem mass spectrometry. We constructed a weighted gene co-expression network using Spearman correlations across 15,028 transcripts, identifying 20 gene modules. Linear regression models were used to examine associations between PFAS and module eigengenes, adjusting for potential confounders. Effect modification by fetal sex was also tested. RESULTS:One module showed a negative association with perfluorononanoic acid (PFNA; β = -0.012, q = 0.009). This association was sex-specific, with the sexes exhibiting varied PFAS associations but similar directional effects. Genes within the PFNA-associated module were involved in histone modification (q ≤ 0.05) and were enriched for targets of the Vitamin D Receptor (VDR), a transcription factor previously linked to PFAS.

The placenta is crucial for fetal development, is affected by PFAS toxicity, and evidence is accumulating that gestational PFAS perturb the epigenetic activity of the placenta. Gestational PFAS exposure is can adversely affect offspring, yet individual and cumulative impacts of PFAS on the placental epigenome remain underexplored. Here, we conducted an epigenome-wide association study (EWAS) to examine the relationships between placental PFAS levels and DNA methylation in a cohort of mother-infant dyads in Arkansas. We measured 17 PFAS in human placental tissues and quantified placental DNA methylation levels via the Illumina EPIC Microarray. We tested for differential DNA methylation with individual PFAS, and with mixtures of multiple PFAS. Our results demonstrated that numerous epigenetic loci were perturbed by PFAS, with PFHxS exhibiting the most abundant effects. Mixture analyses suggested cumulative effects of PFOA and PFOS, while PFHxS may act more independently. We additionally explored whether sex-specific effects may be present and concluded that future large studies should explicitly test for sex-specific effects. The genes that are annotated to our PFAS-associated epigenetic loci are primarily involved in growth processes and cardiometabolic health, while some genes are involved in neurodevelopment. These findings shed light on how prenatal PFAS exposures affect birth outcomes and children's health, emphasizing the importance of understanding PFAS mechanisms in the in-utero environment.

Smoking exposure during adulthood can disrupt oocyte development in women, contributing to infertility and possibly adverse birth outcomes. Some of these effects may be reflected in epigenome profiles in granulosa cells (GCs) in human follicular fluid. We compared the epigenetic modifications throughout the genome in GCs from women who were former (N = 15) versus never smokers (N = 44) undergoing assisted reproductive technologies (ART). This study included 59 women undergoing ART. Smoking history including time since quitting was determined by questionnaire. GCs were collected during oocyte retrieval and DNA methylation (DNAm) levels were profiled using the Infinium MethylationEPIC BeadChip. We performed an epigenome-wide association study with robust linear models, regressing DNAm level at individual loci on smoking status, adjusting for age, ovarian stimulation protocol, and three surrogate variables. We performed differentially methylated regions (DMRs) analysis and over-representation analysis of the identified CpGs and corresponding gene set. 81 CpGs were differentially methylated among former smokers compared to never smokers (FDR < 0.05). We identified 2 significant DMRs (KCNQ1 and RHBDD2). The former smoking-associated genes were enriched in oxytocin signaling, adrenergic signaling in cardiomyocytes, platelet activation, axon guidance, and chemokine signaling pathway. These epigenetic variations have been associated with inflammatory responses, reproductive outcomes, cancer development, neurodevelopmental disorder, and cardiometabolic health. Secondarily, we examined the relationships between time since quitting and DNAm at significant CpGs. We observed three CpGs in negative associations with the length of quitting smoking (p < 0.05), which were cg04254052 (KCNIP1), cg22875371 (OGDHL), and cg27289628 (LOC148145), while one in positive association, which was cg13487862 (PLXNB1). As a pilot study, we demonstrated epigenetic modifications associated with former smoking in GCs. The study is informative to potential biological pathways underlying the documented association between smoking and female infertility and biomarker discovery for smoking-associated reproductive outcomes.

Background Bronchopulmonary dysplasia (BPD), a common morbidity among very preterm infants, is associated with chronic disease and neurodevelopmental impairments. A hypothesized mechanism for these outcomes lies in altered glucocorticoid (GC) activity. We hypothesized that BPD and its treatments may result in epigenetic differences in the hypothalamic-pituitary-adrenal (HPA) axis, which is modulated by GC, and could be ascertained using an established GC risk score and DNA methylation (DNAm) of HPA axis genes. Methods DNAm was quantified from buccal tissue (ECHO-NOVI) and from neonatal blood spots (ELGAN ECHO) via the EPIC microarray. Prenatal maternal characteristics, pregnancy complication, and neonatal medical complication data were collected from medical record review and maternal interviews. Results The GC score was not associated with steroid exposure or BPD. However, six HPA genes involved in stress response regulation demonstrated differential methylation with antenatal steroid exposure; two CpGs within FKBP5 and POMC were differentially methylated with BPD severity. These findings were sex-specific in both cohorts; males had greater magnitude of differential methylation within these genes. Conclusions These findings suggest that BPD severity and antenatal steroids are associated with DNAm at some HPA genes in very preterm infants and the effects appear to be sex-, tissue-, and age-specific. Impact This study addresses bronchopulmonary dysplasia (BPD), an important health outcome among preterm neonates, and interrogates a commonly studied pathway, the hypothalamic-pituitary-adrenal (HPA) axis. The combination of BPD, the HPA axis, and epigenetic markers has not been previously reported. In this study, we found that BPD itself was not associated with epigenetic responses in the HPA axis in infants born very preterm; however, antenatal treatment with steroids was associated with epigenetic responses.