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© Houseman et al.Background: The impact of cell-composition effects in analysis of DNA methylation data is now widely appreciated. With the availability of a reference data set consisting of DNA methylation measurements on isolated cell types, it is possib

Background: There has been a long-standing need in biomedical research for a method that quantifies the normally mixed composition of leukocytes beyond what is possible by simple histological or flow cytometric assessments. The latter is restricted by the

Background: Cigarette smoking is a well-established risk factor for bladder cancer. The effects of smoking duration, intensity (cigarettes per day), and total exposure (pack-years); smoking cessation; exposure to environmental tobacco smoke; and changes in the composition of tobacco and cigarette design over time on risk of bladder cancer are unclear.Methods: We examined bladder cancer risk in relation to smoking practices based on interview data from a large, population-based case-control study conducted in Maine, New Hampshire, and Vermont from 2001 to 2004 (N = 1170 urothelial carcinoma case patients and 1413 control subjects). We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression. To examine changes in smoking-induced bladder cancer risk over time, we compared odds ratios from New Hampshire residents in this study (305 case patients and 335 control subjects) with those from two case-control studies conducted in New Hampshire in 1994-1998 and in 1998-2001 (843 case patients and 1183 control subjects).Results: Regular and current cigarette smokers had higher risks of bladder cancer than never-smokers (for regular smokers, OR = 3.0, 95% CI = 2.4 to 3.6; for current smokers, OR = 5.2, 95% CI = 4.0 to 6.6). In New Hampshire, there was a statistically significant increasing trend in smoking-related bladder cancer risk over three consecutive periods (1994-1998, 1998-2001, and 2002-2004) among former smokers (OR = 1.4, 95% CI = 1.0 to 2.0; OR = 2.0, 95% CI = 1.4 to 2.9; and OR = 2.6, 95% CI = 1.7 to 4.0, respectively) and current smokers (OR = 2.9, 95% CI = 2.0 to 4.2; OR = 4.2, 95% CI = 2.8 to 6.3; OR = 5.5, 95% CI = 3.5 to 8.9, respectively) (P for homogeneity of trends over time periods = .04). We also observed that within categories of intensity, odds ratios increased approximately linearly with increasing pack-years smoked, but the slope of the increasing trend declined with increasing intensity.Conclusions: Smoking-related risks of bladder cancer appear to have increased in New Hampshire since the mid-1990s. Based on our modeling of pack-years and intensity, smoking fewer cigarettes over a long time appears more harmful than smoking more cigarettes over a shorter time, for equal total pack-years of cigarettes smoked. [ABSTRACT FROM AUTHOR]

Children born less than 30 weeks gestational age (GA) are at high risk for neurodevelopmental delay compared to term peers. Prenatal risk factors and neonatal epigenetics could help identify preterm children at highest risk for poor cognitive outcomes. We aimed to understand the associations among cumulative prenatal risk, neonatal DNA methylation, and child cognitive ability at age 3 years, including whether DNA methylation mediates the association between prenatal risk and cognitive ability. We studied 379 neonates (54% male) born less than 30 weeks GA who had DNA methylation measured at neonatal intensive care unit discharge along with 3-year follow-up data. Cumulative prenatal risk was calculated from 24 risk factors obtained from maternal report and medical record and epigenome-wide neonatal DNA methylation was assayed from buccal swabs. At 3-year follow-up, child cognitive ability was assessed using the Bayley Scales of Infant and Toddler Development (third edition). Cumulative prenatal risk and DNA methylation at two cytosine-phosphate-guanines (CpGs) were uniquely associated with child cognitive ability. Using high-dimensional mediation analysis, we also identified differential methylation of 309 CpGs that mediated the association between cumulative prenatal risk and child cognitive ability. Many of the associated CpGs were located in genes ( TNS3, TRAPPC4, MAD1L1, APBB2, DIP2C, TRAPPC9, DRD2 ) that have previously been associated with prenatal exposures and/or neurodevelopmental phenotypes. Our findings suggest a role for both prenatal risk factors and DNA methylation in explaining outcomes for children born preterm and suggest we should further study DNA methylation as a potential mechanism underlying the association between prenatal risk and child neurodevelopment. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

The prevalence of pathogenic mutations within mitochondrial (mt) DNA of youth who were perinatally exposed to HIV and ART but remained uninfected (YHEU) were assessed relative to phenotypic clinical indicators of mitochondrial dysfunction (MtD). This was a cross-sectional, nested case-control study. A total of 144 cases met at least one clinical MtD definition and were matched with up to two controls each (n = 287). At least one risk mutation was present in nearly all YHEU (97 %). No differences in mutation frequencies were observed between metabolic or neurodevelopmental cases and respective controls; however, higher frequencies were found in controls versus respective neurologic or growth cases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Gojanovich is now an Asklepios BioPharmaceuticals, Inc employee. None were declared for the remaining authors., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The time from conception through the first year of life is the most dynamic period in human development. This time period is particularly important for infants born very preterm (< 30 weeks gestation; VPT), as they experience a significant disruption in the normal developmental trajectories and are at heightened risk of experiencing developmental impairments and delays. Variations in the epigenetic landscape during this period may reflect this disruption and shed light on the interrelationships between aging, maturation, and the epigenome. We evaluated how gestational age (GA) and age since conception in neonates [post-menstrual age (PMA)], were related to DNA methylation in buccal cells collected at NICU discharge from VPT infants (n = 538). After adjusting for confounders and applying Bonferroni correction, we identified 2,366 individual CpGs associated with GA and 14,979 individual CpGs associated with PMA, as well as multiple differentially methylated regions. Pathway enrichment analysis identified pathways involved in axonogenesis and regulation of neuron projection development, among many other growth and developmental pathways (FDR q < 0.001). Our findings align with prior work, and also identify numerous novel associations, suggesting that genes important in growth and development, particularly neurodevelopment, are subject to substantial epigenetic changes during early development among children born VPT., (© 2024. The Author(s).)

Background: Changing cell-type proportions can confound studies of differential gene expression or DNA methylation (DNAm) from peripheral blood mononuclear cells (PBMCs). We examined how cell-type proportions derived from the transcriptome versus the methylome (DNAm) influence estimates of differentially expressed genes (DEGs) and differentially methylated positions (DMPs)., Methods: Transcriptome and DNAm data were obtained from PBMC RNA and DNA of Kenyan children (n = 8) before, during, and 6 weeks following uncomplicated malaria. DEGs and DMPs between time points were detected using cell-type adjusted modeling with Cibersortx or IDOL, respectively., Results: Most major cell types and principal components had moderate to high correlation between the two deconvolution methods (r = 0.60-0.96). Estimates of cell-type proportions and DEGs or DMPs were largely unaffected by the method, with the greatest discrepancy in the estimation of neutrophils., Conclusion: Variation in cell-type proportions is captured similarly by both transcriptomic and methylome deconvolution methods for most major cell types., (© 2024. The Author(s).)

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., (© 2024. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

5-hydroxymethylcystosine (5hmC), is an intermediate product in the DNA demethylation pathway, but may act as a functional epigenetic modification. We have conducted the largest study of site-specific 5hmC in placenta to date using parallel bisulphite and oxidative bisulphite modification with array-based assessment. Incorporating parallel RNA-sequencing data allowed us to assess associations between 5hmC and gene expression, using expression quantitative trait hydroxymethylation (eQTHM) analysis. We identified ~ 47,000 loci with consistently elevated (systematic) 5hmC proportions. Systematic 5hmC was significantly depleted ( p  < 0.0001) at CpG islands (CGI), and enriched ( p  < 0.0001) in 'open sea' regions (CpG >4 kb from CGI). 5hmC was most and least abundant at CpGs in enhancers and active transcription start sites (TSS), respectively ( p  < 0.05). We identified 499 significant (empirical-p <0.05) eQTHMs within 1 MB of the assayed gene. At most (75.4%) eQTHMs, the proportion of 5hmC was positively correlated with transcript abundance. eQTHMs were significantly enriched among enhancer CpGs and depleted among CpGs in active TSS ( p  < 0.05 for both). Finally, we identified 107 differentially hydroxymethylated regions (DHMRs, p  < 0.05) across 100 genes. Our study provides insight into placental distribution of 5hmC, and sheds light on the functional capacity of this epigenetic modification in placenta.

Background: In mechanistic and preliminary human studies, prenatal exposure to per- and polyfluoroalkyl substances (PFAS) is associated with oxidative stress, a potential contributor to maternal liver disease. Bilirubin is an endogenous antioxidant abundant in the liver that may serve as a physiological modulator of oxidative stress in pregnant people. Hence, our objective was to estimate the association between repeated measures of PFAS and bilirubin during pregnancy., Methods: The study population included 332 participants in the Atlanta African American Maternal-Child Cohort between 2014 and 2020. Serum samples were collected up to two times (early pregnancy: 6-18 gestational weeks; late pregnancy: 21-36 gestational weeks) for the measurement of perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and total bilirubin. We analyzed single PFAS with linear mixed effect regression and a mixture of the four PFAS with quantile g-computation. Models were repeated with a multiplicative interaction term to explore effect modification by study visit., Results: Overall, PFHxS was positively associated with bilirubin (β = 0.08, 95 % CI = 0.01, 0.15). We also found during late pregnancy, there was a positive association of PFHxS and the PFAS mixture with bilirubin (β = 0.12, 95 % CI = 0.02, 0.22; ψ = 0.19, 95 % CI = 0.03, 0.34, respectively). Finally, study visit modified the PFOA-bilirubin association (interaction p-value = 0.09), which was greater during early pregnancy (β = 0.08, 95 % CI = 0.01, 0.15)., Conclusion: In a prospective cohort of pregnant African Americans, an increase in PFOA, PFHxS, and the PFAS mixture was associated with an increase in bilirubin. Our results suggest that, depending on pregnancy stage, prenatal PFAS exposure disrupts the maternal liver antioxidant capacity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Prenatal exposure to pyrethroids is linked to adverse health effects in early life and proper placental function is critical to fetal development. This study explores the impact of prenatal pyrethroid exposure, as well as factors impacting exposure and effect, on the placental transcriptome, to understand pyrethroid exposures' relationship to placental function. The study of Asian Women and their Offspring's Development and Environmental Exposures (SAWASDEE) recruited pregnant farm-working women from two agricultural districts in the Chiang Mai province of Thailand between 2017 and 2019. This cohort was predominantly exposed to cypermethrin (type II), alongside pyrethroids such as cyfluthrin (type II) and permethrin (type I). In 253 participants, maternal urinary pyrethroid metabolites, 3-phenoxybenzoic acid (PBA), cis-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (CDCCA), and trans-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (TDCCA) were measured in early, middle, and late pregnancy and adjusted for urinary creatinine. The placental transcriptome was analyzed using RNA-Seq. Using generalized linear regression, we identified differentially expressed genes (DEGs) associated with the sum of each metabolite across pregnancy, as well as those associated with location of residence and season of birth. Pathway and upstream transcription factor analyses were performed to examine potential mechanisms associated with DEGs. Notably, TDCCA and CDCCA levels peaked in late pregnancy, with significant regional differences, particularly higher levels in the Fang region. Placental gene expression analysis showed no DEGs associated with individual metabolites at FDR<0.05. However, 251 DEGs by location, implicating immune response and oxidative phosphorylation pathways, were identified, while season of birth was associated with 2585 DEGs, over-represented in fibrosis signaling and metabolism pathways. Finally, transcription factor analysis identified 226 and 282 transcription factors associated with location and season, respectively, related to cell proliferation, differentiation, and the immune system. These alterations may have significant implications for fetal development and other pathologic processes, highlighting the importance of monitoring environmental exposures during pregnancy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Carmen Marsit reports financial support was provided by National Institute of Environmental Health Sciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aim: The current work was designed to demonstrate the application of the exposome framework in examining associations between exposures and children's long-term neurodevelopmental and behavioral outcomes. Methods: Longitudinal data were collected from birth through age 6 from 402 preterm infants. Three statistical methods were utilized to demonstrate the exposome framework: exposome-wide association study, cumulative exposure and machine learning models, with and without epigenetic data. Results: Each statistical approach answered a distinct research question regarding the impact of exposures on longitudinal child outcomes. Findings highlight associations between exposures, epigenetics and executive function. Conclusion: Findings demonstrate how an exposome-based approach can be utilized to understand relationships between internal (e.g., DNA methylation) and external (e.g., prenatal risk) exposures and long-term developmental outcomes in preterm children.

Prior research has identified epigenetic predictors of attention problems in school-aged children but has not yet investigated these in young children, or children at elevated risk of attention problems due to preterm birth. The current study evaluated epigenome-wide associations between neonatal DNA methylation and attention problems at age 2 years in children born very preterm. Participants included 441 children from the Neonatal Neurobehavior and Outcomes in Very Preterm Infants (NOVI) Study, a multi-site study of infants born < 30 weeks gestational age. DNA methylation was measured from buccal swabs collected at NICU discharge using the Illumina MethylationEPIC Bead Array. Attention problems were assessed at 2 years of adjusted age using the attention problems subscale of the Child Behavior Checklist (CBCL). After adjustment for multiple testing, DNA methylation at 33 CpG sites was associated with child attention problems. Differentially methylated CpG sites were located in genes previously linked to physical and mental health, including several genes associated with ADHD in prior epigenome-wide and genome-wide association studies. Several CpG sites were located in genes previously linked to exposure to prenatal risk factors in the NOVI sample. Neonatal epigenetics measured at NICU discharge could be useful in identifying preterm children at risk for long-term attention problems and related psychiatric disorders, who could benefit from early prevention and intervention efforts., (© 2024. The Author(s).)

Parent-child relationship dynamics have been shown to predict socioemotional and behavioral outcomes for children, but little is known about how they may affect biological development. The aim of this study was to test if observational assessments of parent-child relationship dynamics (cohesion, enmeshment, and disengagement) were associated with three biological indices of early life adversity and downstream health risk: (1) methylation of the glucocorticoid receptor gene ( NR3C1 ), (2) telomere attrition, and (3) mitochondrial biogenesis, indexed by mitochondrial DNA copy number (mtDNAcn), all of which were measured in children's saliva. We tested hypotheses using a sample of 254 preschool-aged children ( M age = 51.04 months) with and without child welfare-substantiated maltreatment (52% with documented case of moderate-severe maltreatment) who were racially and ethnically diverse (17% Black, 40% White, 23% biracial, and 20% other races; 45% Hispanic) and from primarily low-income backgrounds (91% qualified for public assistance). Results of path analyses revealed that: (1) higher parent-child cohesion was associated with lower levels of methylation of NR3C1 exon 1 D and longer telomeres, and (2) higher parent-child disengagement was associated with higher levels of methylation of NR3C1 exon 1 D and shorter telomeres. Results suggest that parent-child relationship dynamics may have distinct biological effects on children., Competing Interests: Conflicts of Interest: The authors have no relevant financial or non-financial interests to disclose.

Importance: Use of the Modified Checklist for Autism in Toddlers, Revised With Follow-Up, a 2-stage parent-report autism risk screening tool, has been questioned due to reports of poor sensitivity and specificity. How this measure captures developmental delays for very preterm infants may provide support for continued use in pediatric care settings., Objective: To determine whether autism risk screening with the 2-stage parent-report autism risk screening tool at age 2 years is associated with behavioral and developmental outcomes at age 3 in very preterm infants., Design, Setting, and Participants: Neonatal Neurobehavior and Outcomes for Very Preterm Infants was a longitudinal, multisite cohort study. Enrollment occurred April 2014 to June 2016, and analyses were conducted from November 2022 to May 2023. Data were collected across 9 university-affiliated neonatal intensive care units (NICUs). Inclusion criteria were infants born less than 30 weeks' gestational age, a parent who could read and speak English and/or Spanish, and residence within 3 hours of the NICU and follow-up clinic., Exposures: Prematurity and use of the 2-stage parent-report autism risk screening tool at age 2 years., Main Outcomes and Measures: Outcomes include cognitive, language, motor composites on Bayley Scales for Infant and Toddler Development, third edition (Bayley-III) and internalizing, externalizing, total problems, and pervasive developmental disorder (PDD) subscale on the Child Behavior Checklist (CBCL) at age 3 years. Generalized estimating equations tested associations between the 2-stage parent-report autism risk screening tool and outcomes, adjusting for covariates., Results: A total of 467 children (mean [SD] gestational age, 27.1 [1.8] weeks; 243 male [52%]) were screened with the 2-stage parent-report autism risk screening tool at age 2 years, and outcome data at age 3 years were included in analyses. Mean (SD) maternal age at birth was 29 (6) years. A total of 51 children (10.9%) screened positive on the 2-stage parent-report autism risk screening tool at age 2 years. Children with positive screening results were more likely to have Bayley-III composites of 84 or less on cognitive (adjusted odds ratio [aOR], 4.03; 95% CI, 1.65-9.81), language (aOR, 5.38; 95% CI, 2.43-11.93), and motor (aOR, 4.74; 95% CI, 2.19-10.25) composites and more likely to have CBCL scores of 64 or higher on internalizing (aOR, 4.83; 95% CI, 1.88-12.44), externalizing (aOR, 2.69; 95% CI, 1.09-6.61), and PDD (aOR, 3.77; 95% CI, 1.72-8.28) scales., Conclusions and Relevance: Results suggest that the 2-stage parent-report autism risk screening tool administered at age 2 years was a meaningful screen for developmental delays in very preterm infants, with serious delays detected at age 3 years.

Perinatal stress is associated with altered placental methylation, which plays a critical role in fetal development and infant outcomes. This proof-of-concept pilot study investigated the impact of lifetime trauma exposure and perinatal PTSD symptoms on epigenetic regulation of placenta glucocorticoid signaling genes ( NR3C1 and FKBP5). Lifetime trauma exposure and PTSD symptoms during pregnancy were assessed in a racially/ethnically diverse sample of pregnant women ( N  = 198). Participants were categorized into three groups: (1) No Trauma (-T); (2) Trauma, No Symptoms (T - S); and (3) Trauma and Symptoms (T + S). Placental tissue was analyzed via bisulfite pyrosequencing for degree of methylation at the NR3C1 promoter and FKBP5 regulatory regions. Analyses of covariance were used to test group differences in percentages of NR3C1 and FKBP5 methylation overall and at each CpG site. We found a significant impact of PTSD symptoms on placental NR3C1 methylation. Compared to the -T group, the T + S group had greater NR3C1 methylation overall and at CpG6, CpG8, CpG9, and CpG13, but lower methylation at CpG5. The T + S group had significantly higher N R3C1 methylation overall and at CpG8 compared to the T - S group. There were no differences between the T - S group and - T group. Additionally, no group differences emerged for FKBP5 methylation. Pregnant trauma survivors with PTSD symptoms exhibited differential patterns of placental NR3C1 methylation compared to trauma survivors without PTSD symptoms and pregnant women unexposed to trauma. Results highlight the critical importance of interventions to address the mental health of pregnant trauma survivors.

Background: Very preterm infants are at high risk for neurodevelopmental impairments. We used a child-centered approach (latent profile analysis [LPA]) to describe 2-year neurobehavioral profiles for very preterm infants based on cognitive, motor, and behavioral outcomes. We hypothesized that distinct outcome profiles would differ in the severity and co-occurrence of neurodevelopmental and behavioral impairment., Methods: We studied children born <33 weeks' gestation from the Environmental influences on Child Health Outcomes Program with at least one neurobehavioral assessment at age 2 (Bayley Scales of Infant and Toddler Development, Child Behavior Checklist, Modified Checklist for Autism in Toddlers, cerebral palsy diagnosis). We applied LPA to identify subgroups of children with different patterns of outcomes., Results: In 2036 children (52% male; 48% female), we found four distinct neurobehavioral profiles. Most children (~85%) were categorized into one of two profiles characterized by no/mild neurodevelopmental delay and a low prevalence of behavioral problems. Fewer children (~15%) fell into one of two profiles characterized by severe neurodevelopmental impairments. One profile consisted of children (5%) with co-occurring neurodevelopmental impairment and behavioral problems., Conclusion: Child-centered approaches provide a comprehensive, parsimonious description of neurodevelopment following preterm birth and can be useful for clinical and research purposes., Impact: Most research on outcomes for children born very preterm have reported rates of impairment in single domains. Child-centered approaches describe profiles of children with unique combinations of cognitive, motor, and behavioral strengths and weaknesses. We capitalized on data from the nationwide Environmental influences on Child Health Outcomes Program to examine these profiles in a large sample of children born <33 weeks gestational age. We found four distinct neurobehavioral profiles consisting of different combinations of cognitive, motor, and behavioral characteristics. This information could aid in the development of clinical interventions that target different profiles of children with unique developmental needs., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Epigenetic age acceleration is a risk factor for chronic diseases of ageing and may reflect aspects of biological ageing. However, few studies have examined epigenetic ageing during the early neonatal period in preterm infants, who are at heightened risk of developmental problems. We examined relationships between neonatal age acceleration, neonatal morbidities, and neurobehavioral domains among very preterm (<30 weeks gestation) infants to characterize whether infants with early morbidities or different neurobehavioral characteristics had accelerated or decelerated epigenetic ageing. This study uses data from the Neonatal Neurobehavior and Outcomes in Very Preterm Infants (NOVI) study, restricted to infants with data on variables assessed ( n  = 519). We used generalized estimating equations to test for differences in age acceleration associated with severe neonatal medical morbidities and neurobehavioral characteristics. We found that infants with neonatal morbidities, in particular, bronchopulmonary dysplasia (BPD), had accelerated epigenetic age - and some evidence that infants with hypertonicity and asymmetric reflexes had increased and decreased age acceleration, respectively. Adjustment for gestational age attenuated some associations, suggesting that the relationships observed may be driven by the duration of gestation. Our most robust finding shows that very preterm infants with neonatal morbidities (BPD in particular) exhibit age acceleration, but most neonatal neurobehavioral characteristics and morbidities are not associated with early life age acceleration. Lower gestational age at birth may be an upstream factor driving these associations.

The placenta undergoes many changes throughout gestation to support the evolving needs of the foetus. There is also a growing appreciation that male and female foetuses develop differently in utero , with unique epigenetic changes in placental tissue. Here, we report meta-analysed sex-specific associations between gestational age and placental DNA methylation from four cohorts in the National Institutes of Health (NIH) Environmental influences on Child Health Outcomes (ECHO) Programme (355 females/419 males, gestational ages 23-42 weeks). We identified 407 cytosine-guanine dinucleotides (CpGs) in females and 794 in males where placental methylation levels were associated with gestational age. After cell-type adjustment, 55 CpGs in females and 826 in males were significant. These were enriched for biological processes critical to the immune system in females and transmembrane transport in males. Our findings are distinct between the sexes: in females, associations with gestational age are largely explained by differences in placental cellular composition, whereas in males, gestational age is directly associated with numerous alterations in methylation levels.

Background: 'Epigenetic clocks' have been developed to accurately predict chronologic gestational age and have been associated with child health outcomes in prior work. Methods: We meta-analysed results from four prospective U.S cohorts investigating the association between epigenetic age acceleration estimated using blood DNA methylation collected at birth and preschool age Childhood Behavior Checklist (CBCL) scores. Results: Epigenetic ageing was not significantly associated with CBCL total problem scores (β = 0.33, 95% CI: -0.95, 0.28) and DSM-oriented pervasive development problem scores (β = -0.23, 95% CI: -0.61, 0.15). No associations were observed for other DSM-oriented subscales. Conclusions: The meta-analysis results suggest that epigenetic gestational age acceleration is not associated with child emotional and behavioural functioning for preschool age group. These findings may relate to our study population, which includes two cohorts enriched for ASD and one preterm birth cohort.; future work should address the role of epigenetic age in child health in other study populations. Abbreviations: DNAm: DNA methylation; CBCL: Child Behavioral Checklist; ECHO: Environmental Influences on Child Health Outcomes; EARLI: Early Autism Risk Longitudinal Investigation; MARBLES: Markers of Autism Risk in Babies - Learning Early Signs; ELGAN: Extremely Low Gestational Age Newborns; ASD: autism spectrum disorder; BMI: body mass index; DSM: Diagnostic and Statistical Manual of Mental Disorders.

Background: Epigenetic clocks are promising tools for assessing biological age. We assessed the accuracy of pediatric epigenetic clocks in gestational and chronological age determination., Results: Our study used data from seven tissue types on three DNA methylation profiling microarrays and found that the Knight and Bohlin clocks performed similarly for blood cells, while the Lee clock was superior for placental samples. The pediatric-buccal-epigenetic clock performed the best for pediatric buccal samples, while the Horvath clock is recommended for children's blood cell samples. The NeoAge clock stands out for its unique ability to predict post-menstrual age with high correlation with the observed age in infant buccal cell samples., Conclusions: Our findings provide valuable guidance for future research and development of epigenetic clocks in pediatric samples, enabling more accurate assessments of biological age., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Health outcomes among children born prematurely are known to be sexually dimorphic, with male infants often more affected, yet the mechanism behind this observation is not clear. CpG methylation levels in the placenta and blood also differ by sex and are associated with adverse health outcomes. We contrasted CpG methylation levels in the placenta and neonatal blood (n = 358) from the Extremely Low Gestational Age Newborn (ELGAN) cohort based on the EPIC array, which assays over 850,000 CpG sites across the epigenome. Sex-specific epigenome-wide association analyses were conducted for the placenta and neonatal blood samples independently, and the results were compared to determine tissue-specific differences between the methylation patterns in males and females. All models were adjusted for cell type heterogeneity. Enrichment pathway analysis was performed to identify the biological functions of genes related to the sexually dimorphic CpG sites., Results: Approximately 11,500 CpG sites were differentially methylated in relation to sex. Of these, 5949 were placenta-specific and 5361 were blood-specific, with only 233 CpG sites overlapping in both tissues. For placenta-specific CpG sites, 90% were hypermethylated in males. For blood-specific CpG sites, 95% were hypermethylated in females. In the placenta, keratinocyte differentiation biological pathways were enriched among the differentially methylated genes. No enrichment pathways were observed for blood., Conclusions: Distinct methylation patterns were observed between male and female children born extremely premature, and keratinocyte differentiation pathways were enriched in the placenta. These findings provide new insights into the epigenetic mechanisms underlying sexually dimorphic health outcomes among extremely premature infants., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Single-cohort studies have identified distinct neurobehavioral profiles that are associated with prenatal and neonatal factors based on the NICU Network Neurobehavioral Scale (NNNS). We examined socioeconomic, medical, and substance use variables as predictors of NNNS profiles in a multi-cohort study of preterm and term-born infants with different perinatal exposures., Methods: We studied 1112 infants with a neonatal NNNS exam from the Environmental influences on Child Health Outcomes (ECHO) consortium. We used latent profile analysis to characterize infant neurobehavioral profiles and generalized estimating equations to determine predictors of NNNS profiles., Results: Six distinct neonatal neurobehavioral profiles were identified, including two dysregulated profiles: a hypo-aroused profile (16%) characterized by lethargy, hypotonicity, and nonoptimal reflexes; and a hyper-aroused profile (6%) characterized by high arousal, excitability, and stress, with low regulation and poor movement quality. Infants in the hypo-aroused profile were more likely to be male, have younger mothers, and have mothers who were depressed prenatally. Infants in the hyper-aroused profile were more likely to be Hispanic/Latino and have mothers who were depressed or used tobacco prenatally., Conclusions: We identified two dysregulated neurobehavioral profiles with distinct perinatal antecedents. Further understanding of their etiology could inform targeted interventions to promote positive developmental outcomes., Impact: Prior research on predictors of neonatal neurobehavior have included single-cohort studies, which limits generalizability of findings. In a multi-cohort study of preterm and term-born infants, we found six distinct neonatal neurobehavioral profiles, with two profiles being identified as dysregulated. Hypo- and hyper-aroused neurobehavioral profiles had distinct perinatal antecedents. Understanding perinatal factors associated with dysregulated neurobehavior could help promote positive developmental outcomes., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Background: Poor placental function is a common cause of intrauterine growth restriction, which in turn is associated with increased risks of adverse health outcomes. Our prior work suggests that birthweight and childhood obesity-associated genetic variants functionally impact placental function and that placental microRNA are associated with birthweight. To address the influence of the placenta beyond birth, we assessed the relationship between placental microRNAs and early childhood growth., Methods: Using the SITAR package, we generated two parameters that describe individual weight trajectories of children (0-5 years) in the New Hampshire Birth Cohort Study (NHBCS, n = 238). Using negative binomial generalized linear models, we identified placental microRNAs that relate to growth parameters (FDR < 0.1), while accounting for sex, gestational age at birth, and maternal parity., Results: Genes targeted by the six growth trajectory-associated microRNAs are enriched (FDR < 0.05) in growth factor signaling (TGF/beta: miR-876; EGF/R: miR-155, Let-7c; FGF/R: miR-155; IGF/R: Let-7c, miR-155), calmodulin signaling (miR-216a), and NOTCH signaling (miR-629)., Conclusions: Growth-trajectory microRNAs target pathways affecting placental proliferation, differentiation and function. Our results suggest a role for microRNAs in regulating placental cellular dynamics and supports the Developmental Origins of Health and Disease hypothesis that fetal environment can have impacts beyond birth., Impact: We found that growth trajectory associated placenta microRNAs target genes involved in signaling pathways central to the formation, maintenance and function of placenta; suggesting that placental cellular dynamics remain critical to infant growth to term and are under the control of microRNAs. Our results contribute to the existing body of research suggesting that the placenta plays a key role in programming health in the offspring. This is the first study to relate molecular patterns in placenta, specifically microRNAs, to early childhood growth trajectory., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

The intrauterine environment and early life stress regulation are widely recognized as an early foundation for lifelong physical and mental health. Methylation of CpG sites in the placenta represents an epigenetic modification that can potentially affect placental function, influence fetal development, and ultimately impact the health of offspring by programming the hypothalamic-pituitary-adrenal (HPA) axis stress response during prenatal development. Leptin, an adipokine produced by the placenta, is essential for energy homeostasis. It is also epigenetically regulated by promoter DNA methylation. Mounting evidence suggests that leptin also affects the stress response system. Though heterogeneity in the early stress response system may influence life-long mental and physical health, few studies explicitly examine the heterogeneity in the newborn stress response system. Less is known about leptin's association with the human hypothalamic-pituitary-adrenocortical (HPA) axis early in life. This study sought to serve as a proof of concept study investigating the relationship between newborn cortisol output trajectories and placental leptin DNA methylation in 117 healthy newborns from socioeconomically and racially- and ethnically-diverse families. We characterized heterogeneity in newborn cortisol output during the NICU Network Neurobehavioral Scales exam in the first week of life with latent growth mixture models. We then evaluated whether leptin promoter (LEP) methylation in placental samples was associated with newborn cortisol trajectories. Our findings suggest that increased placental LEP methylation, which corresponds to decreased leptin production, is associated with infant cortisol trajectories marked by increased cortisol output in the NNNS exam. These results provide important insights into the role of placental leptin DNA methylation in human newborn HPA axis development and subsequent developmental origins of health and disease processes., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to disclose., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Descriptive epidemiological data on incidence rates (IRs) of asthma with recurrent exacerbations (ARE) are sparse., Objectives: This study hypothesized that IRs for ARE would vary by time, geography, age, and race and ethnicity, irrespective of parental asthma history., Methods: The investigators leveraged data from 17,246 children born after 1990 enrolled in 59 US with 1 Puerto Rican cohort in the Environmental Influences on Child Health Outcomes (ECHO) consortium to estimate IRs for ARE., Results: The overall crude IR for ARE was 6.07 per 1000 person-years (95% CI: 5.63-6.51) and was highest for children aged 2-4 years, for Hispanic Black and non-Hispanic Black children, and for those with a parental history of asthma. ARE IRs were higher for 2- to 4-year-olds in each race and ethnicity category and for both sexes. Multivariable analysis confirmed higher adjusted ARE IRs (aIRRs) for children born 2000-2009 compared with those born 1990-1999 and 2010-2017, 2-4 versus 10-19 years old (aIRR = 15.36; 95% CI: 12.09-19.52), and for males versus females (aIRR = 1.34; 95% CI 1.16-1.55). Black children (non-Hispanic and Hispanic) had higher rates than non-Hispanic White children (aIRR = 2.51; 95% CI 2.10-2.99; and aIRR = 2.04; 95% CI: 1.22-3.39, respectively). Children born in the Midwest, Northeast and South had higher rates than those born in the West (P < .01 for each comparison). Children with a parental history of asthma had rates nearly 3 times higher than those without such history (aIRR = 2.90; 95% CI: 2.43-3.46)., Conclusions: Factors associated with time, geography, age, race and ethnicity, sex, and parental history appear to influence the inception of ARE among children and adolescents., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Introduction: Traffic-related air pollution (TRAP), a common exposure, potentially impacts pregnancy through altered placental function. We investigated associations between prenatal TRAP exposure and placental gene expression., Methods: Whole transcriptome sequencing was performed on placental samples from CANDLE (Memphis, TN) (n = 776) and GAPPS (Seattle and Yakima, WA) (n = 205), cohorts of the ECHO-PATHWAYS Consortium. Residential NO 2 exposures were computed via spatiotemporal models for full-pregnancy, each trimester, and the first/last months of pregnancy. Individual cohort-specific, covariate-adjusted linear models were fit for 10,855 genes and respective exposures (NO 2 or roadway proximity [≤150 m]). Infant-sex/exposure interactions on placental gene expression were tested with interaction terms in separate models. Significance was based on false discovery rate (FDR<0.10)., Results: In GAPPS, final-month NO 2 exposure was positively associated with MAP1LC3C expression (FDR p-value = 0.094). Infant-sex interacted with second-trimester NO 2 on STRIP2 expression (FDR interaction p-value = 0.011, inverse and positive associations among male and female infants, respectively) and roadway proximity on CEBPA expression (FDR interaction p-value = 0.045, inverse among females). In CANDLE, infant-sex interacted with first-trimester and full-pregnancy NO 2 on RASSF7 expression (FDR interaction p-values = 0.067 and 0.013, respectively, positive among male infants and inverse among female infants)., Discussion: Overall, pregnancy NO 2 exposure and placental gene expression associations were primarily null, with exception of final month NO 2 exposure and placental MAP1LC3C association. We found several interactions of infant sex and TRAP exposures on placental expression of STRIP2, CEBPA, and RASSF7. These highlighted genes suggest influence of TRAP on placental cell proliferation, autophagy, and growth, though additional replication and functional studies are required for validation., Competing Interests: Declaration of competing interest All authors have read and approved the submission of the manuscript. The Institutional Review Boards of all participating institutions approved study protocols. Neither the authors nor the institutions with which they are affiliated have any direct or indirect financial interest in the subject matter of our research. All authors report no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Marginalized populations experience disproportionate rates of preterm birth and early term birth. Exposure to per- and polyfluoroalkyl substances (PFAS) has been reported to reduce length of gestation, but the underlying mechanisms are unknown. In the present study, we characterized the molecular signatures of prenatal PFAS exposure and gestational age at birth outcomes in the newborn dried blood spot metabolome among 267 African American dyads in Atlanta, Georgia between 2016 and 2020. Pregnant people with higher serum perfluorooctanoic acid and perfluorohexane sulfonic acid concentrations had increased odds of an early birth. After false discovery rate correction, the effect of prenatal PFAS exposure on reduced length of gestation was associated with 8 metabolomic pathways and 52 metabolites in newborn dried blood spots, which suggested perturbed tissue neogenesis, neuroendocrine function, and redox homeostasis. These mechanisms explain how prenatal PFAS exposure gives rise to the leading cause of infant death in the United States., (© 2023. The Author(s).)

Our earlier work has shown that genomic risk for schizophrenia converges with early life complications in affecting risk for the disorder and sex-biased neurodevelopmental trajectories. Here, we identify specific genes and potential mechanisms that, in placenta, may mediate such outcomes. We performed TWAS in healthy term placentae (N = 147) to derive candidate placental causal genes that we confirmed with SMR; to search for placenta and schizophrenia-specific associations, we performed an analogous analysis in fetal brain (N = 166) and additional placenta TWAS for other disorders/traits. The analyses in the whole sample and stratifying by sex ultimately highlight 139 placenta and schizophrenia-specific risk genes, many being sex-biased; the candidate molecular mechanisms converge on the nutrient-sensing capabilities of placenta and trophoblast invasiveness. These genes also implicate the Coronavirus-pathogenesis pathway and showed increased expression in placentae from a small sample of SARS-CoV-2-positive pregnancies. Investigating placental risk genes for schizophrenia and candidate mechanisms may lead to opportunities for prevention that would not be suggested by study of the brain alone., (© 2023. The Author(s).)

Organophosphate and pyrethroid pesticides are among the most extensively used insecticides worldwide. Prenatal exposures to both classes of pesticides have been linked to a wide range of neurobehavioral deficits in the offspring. The placenta is a neuroendocrine organ and the crucial regulator of the intrauterine environment; early-life toxicant exposures could impact neurobehavior by disrupting placental processes. Female C57BL/6 J mice were exposed via oral gavage to an organophosphate, chlorpyrifos (CPF) at 5 mg/kg, a pyrethroid, deltamethrin (DM), at 3 mg/kg, or vehicle only control (CTL). Exposure began two weeks before breeding and continued every three days until euthanasia at gestational day 17. The transcriptomes of fetal brain (CTL n = 18, CPF n = 6, DM n = 8) and placenta (CTL n = 19, CPF n = 16, DM n = 12) were obtained through RNA sequencing, and resulting data was evaluated using weighted gene co-expression networks, differential expression, and pathway analyses. Fourteen brain gene co-expression modules were identified; CPF exposure disrupted the module related to ribosome and oxidative phosphorylation, whereas DM disrupted the modules related to extracellular matrix and calcium signaling. In the placenta, network analyses revealed 12 gene co-expression modules. While CPF exposure disrupted modules related to endocytosis, Notch and Mapk signaling, DM exposure dysregulated modules linked to spliceosome, lysosome and Mapk signaling pathways. Overall, in both tissues, CPF exposure impacted oxidative phosphorylation, while DM was linked to genes involved in spliceosome and cell cycle. The transcription factor Max involved in cell proliferation was overexpressed by both pesticides in both tissues. In summary, gestational exposure to two different classes of pesticide can induce similar pathway-level transcriptome changes in the placenta and the brain; further studies should investigate if these changes are linked to neurobehavioral impairments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Exposure to organophosphate (OP) pesticides during pregnancy has been linked to deficiencies of neurobehavioral development in childhood; however, the molecular mechanisms underlying this association remain elusive. The placenta plays a crucial role in protecting the fetus from environmental insults and safeguarding proper fetal development including neurodevelopment. The aim of our study is to evaluate changes in the placental transcriptome associated with prenatal OP exposure., Methods: Pregnant farm workers from two agricultural districts in northern Thailand were recruited for the Study of Asian Women and Offspring's Development and Environmental Exposures (SAWASDEE) from 2017 to 2019. For 254 participants, we measured maternal urinary concentrations of six nonspecific dialkyl phosphates (DAP) metabolites in early, middle, and late pregnancy. In parallel, we profiled the term placental transcriptome from the same participants using RNA-Sequencing and performed Weighted Gene co-expression Network Analysis (WGCNA). Generalized linear regression modeling was used to examine associations of urinary OP metabolites and placental co-expression module eigenvalues., Results: We identified 21 gene co-expression modules in the placenta. From the six DAP metabolites assayed, diethylphosphate (DEP) and diethylthiophosphate (DETP) were detected in more than 70% of the urine samples. Significant associations between DEP at multiple time points and two specific placental gene modules were observed. The 'black' module, enriched in genes involved in epithelial-to-mesenchymal transition (EMT) and hypoxia, was negatively associated with DEP in early (p = 0.034), and late pregnancies (p = 0.016). The 'lightgreen' module, enriched in genes involved in myogenesis and EMT, was negatively associated with DEP in late pregnancy (p = 0.010). We observed 2 hub genes (CELSR1 and PYCR1) of the 'black' module to be negatively associated with DEP in early and late pregnancies., Conclusions: Our results suggest that prenatal OP exposure may disrupt placental gene networks in a time-dependent manner. Such transcriptomic effects may lead to down-stream changes in placental function that ultimately affect the developing fetus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Children born preterm are at heightened risk of neurodevelopmental impairments, including Autism Spectrum Disorder (ASD). The placenta is a key regulator of neurodevelopmental processes, though the precise underlying molecular mechanisms remain unclear. Here, we employed a multi-omic approach to identify placental transcriptomic and epigenetic modifications related to ASD diagnosis at age 10, among children born preterm. Working with the extremely low gestational age (ELGAN) cohort, we hypothesized that a pro-inflammatory placental environment would be predictive of ASD diagnosis at age 10. Placental messenger RNA (mRNA) expression, CpG methylation, and microRNA (miRNA) expression were compared among 368 ELGANs (28 children diagnosed with ASD and 340 children without ASD). A total of 111 genes displayed expression levels in the placenta that were associated with ASD. Within these ASD-associated genes is an ASD regulatory complex comprising key genes that predicted ASD case status. Genes with expression that predicted ASD case status included Ewing Sarcoma Breakpoint Region 1 (EWSR1) (OR: 6.57 (95% CI: 2.34, 23.58)) and Bromodomain Adjacent To Zinc Finger Domain 2A (BAZ2A) (OR: 0.12 (95% CI: 0.03, 0.35)). Moreover, of the 111 ASD-associated genes, nine (8.1%) displayed associations with CpG methylation levels, while 14 (12.6%) displayed associations with miRNA expression levels. Among these, LRR Binding FLII Interacting Protein 1 (LRRFIP1) was identified as being under the control of both CpG methylation and miRNAs, displaying an OR of 0.42 (95% CI: 0.17, 0.95). This gene, as well as others identified as having functional epimutations, plays a critical role in immune system regulation and inflammatory response. In summary, a multi-omic approach was used to identify functional epimutations in the placenta that are associated with the development of ASD in children born preterm, highlighting future avenues for intervention., (© 2023 International Society for Autism Research and Wiley Periodicals LLC.)

The fate of environmental chemicals in maternal and fetal tissues might be affected by pregnancy-related hemodynamic changes that occur across gestation. Specifically, hemodilution and renal function are hypothesized to confound associations between per- and polyfluoroalkyl substances (PFAS) exposure measures in late pregnancy with gestational length and fetal growth. We sought to analyze two pregnancy-related hemodynamic biomarkers, creatinine and estimated glomerular filtration rate (eGFR), as confounders of the trimester-specific relationships between maternal serum PFAS concentrations and adverse birth outcomes. Participants were enrolled in the Atlanta African American Maternal-Child Cohort between 2014 and 2020. Biospecimens were collected at up to two timepoints, which were categorized into the 1st trimester (N = 278; 11 mean weeks gestation), 2nd trimester (N = 162; 24 mean weeks gestation), and 3rd trimester (N = 110; 29 mean weeks gestation). We quantified six PFAS in serum, creatinine in serum and urine, and eGFR using the Cockroft-Gault equation. Multivariable regression models estimated the associations between single PFAS and their sum with gestational age at delivery (weeks), preterm birth (PTB, <37 gestational weeks), birthweight z-scores, and small for gestational age (SGA). Primary models were adjusted for sociodemographics. We additionally adjusted for serum creatinine, urinary creatinine, or eGFR in the confounding assessments. An interquartile range increase in perfluorooctanoic acid (PFOA) produced a non-significant reduction in birthweight z-score during the 1st and 2nd trimesters (β = -0.01 g [95% CI = -0.14, 0.12] and β = -0.07 g [95% CI = -0.19, 0.06], respectively) whereas the relationship was significant and positive during the 3rd trimester (β = 0.15 g; 95% CI = 0.01, 0.29). Trimester-specific effects were similar for the other PFAS and adverse birth outcomes, which persisted after adjusting for creatinine or eGFR. The relationships between prenatal PFAS exposure and adverse birth outcomes were not strongly confounded by renal function or hemodilution. However, 3rd trimester samples consistently exhibited different effects than those collected during the 1st and 2nd trimesters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Prenatal cadmium (Cd) exposure has been implicated in both placental toxicity and adverse neurobehavioral outcomes. Placental microRNAs (miRNAs) may function to developmentally program adverse pregnancy and newborn health outcomes in response to gestational Cd exposure., Methods: In a subset of the Rhode Island Child Health Study (RICHS, n = 115) and the New Hampshire Birth Cohort Study (NHBCS, = 281), we used small RNA sequencing and trace metal analysis to identify Cd-associated expression of placental miRNAs using negative binomial generalized linear models. We predicted mRNAs targeted by Cd-associated miRNAs and relate them to neurobehavioral outcomes at birth through the integration of transcriptomic data and summary scores from the NICU Network Neurobehavioral Scale (NNNS)., Results: Placental Cd concentrations are significantly associated with the expression level of five placental miRNAs in NHBCS, with similar effect sizes in RICHS. These miRNA target genes overrepresented in nervous system development, and their expression is correlated with NNNS metrics suggestive of atypical neurobehavioral outcomes at birth., Conclusions: Gestational Cd exposure is associated with the expression of placental miRNAs. Predicted targets of these miRNAs are involved in nervous system development and may also regulate placental physiology, allowing their dysregulation to modify developmental programming of early life health outcomes., Impact: This research aims to address the poor understanding of the molecular mechanisms governing adverse pregnancy and newborn health outcomes in response to Gestational cadmium (Cd) exposure. Our results outline a robust relationship between Cd-associated placental microRNA expression and NICU Network Neurobehavioral Scales (NNNS) at birth indicative of atypical neurobehavior. This study utilized healthy mother-infant cohorts to describe the role of Cd-associated dysregulation of placental microRNAs as a potential mechanism by which adverse neurobehavioral outcomes are developmentally programmed., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Background: Infants born extremely premature are at increased risk for health complications later in life for which neonatal inflammation may be a contributing biological driver. Placental CpG methylation provides mechanistic information regarding the relationship between prenatal epigenetic programming, prematurity, neonatal inflammation, and later-in-life health., Methods: We contrasted CpG methylation in the placenta and neonatal blood spots in relation to neonatal inflammation in the Extremely Low Gestational Age Newborn (ELGAN) cohort. Neonatal inflammation status was based on the expression of six inflammation-related proteins, assessed as (1) day-one inflammation (DOI) or (2) intermittent or sustained systemic inflammation (ISSI, inflammation on ≥2 days in the first 2 postnatal weeks). Epigenome-wide CpG methylation was assessed in 354 placental samples and 318 neonatal blood samples., Results: Placental CpG methylation displayed the strongest association with ISSI (48 CpG sites) but was not associated with DOI. This was in contrast to CpG methylation in blood spots, which was associated with DOI (111 CpG sites) and not with ISSI (one CpG site)., Conclusions: Placental CpG methylation was strongly associated with ISSI, a measure of inflammation previously linked to later-in-life cognitive impairment, while day-one neonatal blood methylation was associated with DOI., Impact: Neonatal inflammation increases the risk of adverse later-life outcomes, especially in infants born extremely preterm. CpG methylation in the placenta and neonatal blood spots were evaluated in relation to neonatal inflammation assessed via circulating proteins as either (i) day-one inflammation (DOI) or (ii) intermittent or sustained systemic inflammation (ISSI, inflammation on ≥2 days in the first 2 weeks). Tissue specificity was observed in epigenetic-inflammatory relationships: placental CpG methylation was associated with ISSI, neonatal blood CpG methylation was associated with DOI. Supporting the placental origins of disease framework, placental epigenetic patterns are associated with a propensity for ISSI in neonates., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Purpose of Review: To summarize recent literature relating early-life environmental exposures on DNA methylation in the placenta, to identify how variation in placental methylation is regulated in an exposure-specific manner, and to encourage additional work in this area., Recent Findings: Multiple studies have evaluated associations between prenatal environmental exposures and placental methylation in both gene-specific and epigenome-wide frameworks. Specific exposures lead to unique variability in methylation, and cross-exposure assessments have uncovered certain genes that demonstrate consistency in differential placental methylation. Exposure studies that assess methylation effects in a trimester-specific approach tend to find larger effects during the 1st trimester exposure. Earlier studies have more targeted gene-specific approaches to methylation, while later studies have shifted towards epigenome-wide, array-based approaches. Studies focusing on exposures such as air pollution, maternal smoking, environmental contaminants, and trace metals appear to be more abundant, while studies of socioeconomic adversity and circadian disruption are scarce but demonstrate remarkable effects. Understanding the impacts of early-life environmental exposures on placental methylation is critical to establishing the link between the maternal environment, epigenetic variation, and long-term health. Future studies into this field should incorporate repeated measures of exposure throughout pregnancy, in order to determine the critical windows in which placental methylation is most heavily affected. Additionally, the use of methylation-based scores and sequencing technology could provide important insights into epigenetic gestational age and uncovering more genomic regions where methylation is affected. Studies examining the impact of other exposures on methylation, including pesticides, alcohol, and other chemicals are also warranted., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Importance: Acoustic cry characteristics have been associated with severe medical problems in newborns. However, little is known about the utility of neonatal acoustic cry characteristics in the prediction of long-term outcomes of very preterm infants., Objectives: To evaluate whether acoustic characteristics of infant cry at neonatal intensive care unit (NICU) discharge are associated with behavioral and developmental outcomes at age 2 years in infants born very preterm., Design, Setting, and Participants: Infants born less than 30 weeks postmenstrual age (PMA) were enrolled from April 2014 through June 2016 as part of a multicenter (9 US university affiliated NICUs) cohort study and followed to adjusted age 2 years. Reported analyses began on September 2021. Data were analyzed from September 2021 to September 2022., Exposures: The primary exposure was premature birth (<30 weeks PMA)., Main Outcomes and Measures: Cries were recorded during a neurobehavioral examination administered during the week of NICU discharge. Cry episodes were analyzed using a previously published computerized system to characterize cry acoustics. Year-2 outcomes included the Bayley-III Composite scores, Child Behavior Checklist (CBCL) and the Modified Checklist for Autism in Toddlers (M-CHAT R/F), dichotomized using clinically significant cutoffs (<85 on Bayley Language, Cognitive and/or Motor Composite scores, T-score >63 on the CBCL Internalizing, Externalizing and/or Total Problem Scales and total M-CHAT R/F score >2)., Results: Analyzed infants (363 participants) were primarily male (202 participants [55.65%]) and had a mean [SD] gestational age of 27.08 [1.95] weeks). Cross-validated random forest models revealed that cry acoustics were associated with 2-year outcomes. Tests of diagnostic odds ratios (DOR) revealed that infants who exhibited total problem behavior CBCL scores greater than 63 at age 2 years were 3.3 times more likely (95% CI, 1.44-7.49) to be identified as so by random forest model estimates relative to other infants (scores ≤63); this association was robust to adjustment for family-wise type-I error rates and covariate measures. Similar associations were observed for internalizing (DOR, 2.39; 95% CI, 1.04-5.47) and externalizing (DOR, 2.25; 95% CI, 1.12-4.54) scores on the CBCL, clinically significant language (DOR, 1.71; 95% CI, 1.10-2.67) and cognitive (DOR, 1.70; 95% CI, 1.00-2.88) scores on the Bayley-III, and a positive autism screen on the M-CHAT (DOR, 1.91; 95% CI, 1.05-3.44)., Conclusions and Relevance: In this cohort study of preterm infants, findings pointed to the potential use of acoustic cry characteristics in the early identification of risk for long-term developmental and behavioral deficits.

In the United States, cardiovascular disease is the leading cause of death and the rate of maternal mortality remains among the highest of any industrialized nation. Maternal cardiometabolic health throughout gestation and postpartum is representative of placental health and physiology. Both proper placental functionality and placental microRNA expression are essential to successful pregnancy outcomes, and both are highly sensitive to genetic and environmental sources of variation. Placental pathologies, such as preeclampsia, are associated with maternal cardiovascular health but may also contribute to the developmental programming of chronic disease in offspring. However, the role of more subtle alterations to placental function and microRNA expression in this developmental programming remains poorly understood. We performed small RNA sequencing to investigate microRNA in placentae from the Rhode Island Child Health Study ( n = 230). MicroRNA counts were modeled on maternal family history of cardiovascular disease using negative binomial generalized linear models. MicroRNAs were considered to be differentially expressed at a false discovery rate (FDR) less than 0.10. Parallel mRNA sequencing data and bioinformatic target prediction software were then used to identify potential mRNA targets of differentially expressed microRNAs. Nine differentially expressed microRNAs were identified (FDR < 0.1). Bioinformatic target prediction revealed 66 potential mRNA targets of these microRNAs, many of which are implicated in TGFβ signaling pathway but also in pathways involving cellular metabolism and immunomodulation. A robust association exists between familial cardiovascular disease and placental microRNA expression which may be implicated in both placental insufficiencies and the developmental programming of chronic disease.

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., (© 2022. The Author(s).)

During infancy, the interplay between the developing immune system and the microbiome is critical. We examined whether blood immune cell composition at birth in the umbilical cord (inferred by DNA methylation profiling) related to the early infant gut microbiome (assessed by 16S rRNA gene sequencing) among 73 infants in the New Hampshire Birth Cohort Study. We used generalized estimating equations and controlled for false discovery rate to select microbial taxa associated with immune cells. We found associations between the infant gut microbiome and immune cells, including a positive association between B cells and Enterobacter , a negative association between natural killer cells and Bifidobacterium , and a positive association between granulocytes and Bifidobacterium . Our findings give clues that immune profiles at the time of birth as measured in umbilical cord blood are associated with the development of the gut microbiome in early life., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Prenatal lead (Pb) exposure is associated with adverse developmental outcomes and to epigenetic alterations such as DNA methylation and hydroxymethylation in animal models and in newborn blood. Given the importance of the placenta in foetal development, we sought to examine how prenatal Pb exposure was associated with differential placental DNA methylation and hydroxymethylation and to identify affected biological pathways linked to developmental outcomes. Maternal (n = 167) and infant (n = 172) toenail and placenta (n = 115) samples for prenatal Pb exposure were obtained from participants in a US birth cohort, and methylation and hydroxymethylation data were quantified using the Illumina Infinium MethylationEPIC BeadChip. An epigenome-wide association study was applied to identify differential methylation and hydroxymethylation associated with Pb exposure. Biological functions of the Pb-associated genes were determined by overrepresentation analysis through ConsensusPathDB. Prenatal Pb quantified from maternal toenail, infant toenail, and placenta was associated with 480, 27, and 2 differentially methylated sites (q < 0.05), respectively, with both increases and decreases associated with exposure. Alternatively, we identified 2, 1, and 14 differentially hydroxymethylated site(s) associated with maternal toenail, infant toenail, and placental Pb, respectively, with most showing increases in hydroxymethylation with exposure. Significantly overrepresented pathways amongst genes associated with differential methylation and hydroxymethylation (q < 0.10) included mechanisms pertaining to nervous system and organ development, calcium transport and regulation, and signalling activities. Our results suggest that both methylation and hydroxymethylation in the placenta can be variable based on Pb exposure and that the pathways impacted could affect placental function.

Higher maternal pre-pregnancy body mass index (ppBMI) is associated with increased neonatal morbidity, as well as with pregnancy complications and metabolic outcomes in offspring later in life. The placenta is a key organ in fetal development and has been proposed to act as a mediator between the mother and different health outcomes in children. The overall aim of the present work is to investigate the association of ppBMI with epigenome-wide placental DNA methylation (DNAm) in 10 studies from the PACE consortium, amounting to 2631 mother-child pairs. We identify 27 CpG sites at which we observe placental DNAm variations of up to 2.0% per 10 ppBMI-unit. The CpGs that are differentially methylated in placenta do not overlap with CpGs identified in previous studies in cord blood DNAm related to ppBMI. Many of the identified CpGs are located in open sea regions, are often close to obesity-related genes such as GPX1 and LGR4 and altogether, are enriched in cancer and oxidative stress pathways. Our findings suggest that placental DNAm could be one of the mechanisms by which maternal obesity is associated with metabolic health outcomes in newborns and children, although further studies will be needed in order to corroborate these findings., (© 2022. The Author(s).)

Background: Melamine, melamine derivatives, and aromatic amines are nitrogen-containing compounds with known toxicity and widespread commercial uses. Nevertheless, biomonitoring of these chemicals is lacking, particularly during pregnancy, a period of increased susceptibility to adverse health effects., Objectives: We aimed to measure melamine, melamine derivatives, and aromatic amine exposure in pregnant women across the United States (U.S.) and evaluate associations with participant and urine sample collection characteristics., Methods: We measured 43 analytes, representing 45 chemicals (i.e., melamine, three melamine derivatives, and 41 aromatic amines), in urine from pregnant women in nine diverse ECHO cohorts during 2008-2020 (N = 171). To assess relations with participant and urine sample collection characteristics, we used generalized estimating equations to estimate prevalence ratios (PRs) for analytes dichotomized at the detection limit, % differences (%Δ) for continuous analytes, and 95% confidence intervals. Multivariable models included age, race/ethnicity, marital status, urinary cotinine, and year of sample collection., Results: Twelve chemicals were detected in >60% of samples, with near ubiquitous detection of cyanuric acid, melamine, aniline, 4,4'-methylenedianiline, and a composite of o-toluidine and m-toluidine (99-100%). In multivariable adjusted models, most chemicals were associated with higher exposures among Hispanic and non-Hispanic Black participants. For example, concentrations of 3,4-dichloroaniline were higher among Hispanic (%Δ: +149, 95% CI: +17, +431) and non-Hispanic Black (%Δ: +136, 95% CI: +35, +311) women compared with non-Hispanic White women. We observed similar results for ammelide, o-/m-toluidine, 4,4'-methylenedianiline, and 4-chloroaniline. Most chemicals were positively associated with urinary cotinine, with strongest associations observed for o-/m-toluidine (%Δ: +23; 95% CI: +16, +31) and 3,4-dichloroaniline (%Δ: +25; 95% CI: +17, +33). Some chemicals exhibited annual trends (e.g., %Δ in melamine per year: -11; 95% CI: -19, -1) or time of day, seasonal, and geographic variability., Discussion: Exposure to melamine, cyanuric acid, and some aromatic amines was ubiquitous in this first investigation of these analytes in pregnant women. Future research should expand biomonitoring, identify sources of exposure disparities by race/ethnicity, and evaluate potential adverse health effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Exposure to metals increases risk for pregnancy complications. Extracellular vesicle (EV) miRNA contribute to maternal-foetal communication and are dysregulated in pregnancy complications. However, metal impacts on maternal circulating EV miRNA during pregnancy are unknown. Our objective was to investigate the impact of multiple metal exposures on EV miRNA in maternal circulation during pregnancy in the MADRES Study. Associations between urinary concentrations of nine metals and 106 EV miRNA in maternal plasma during pregnancy were investigated using robust linear regression (N = 231). Primary analyses focused on metal-miRNA associations in early pregnancy (median: 12.3 weeks gestation). In secondary analyses, we investigated associations with late pregnancy miRNA counts (median: 31.8 weeks gestation) in a subset of participants (N = 184) with paired measures. MiRNA associated with three or more metals (P FDR <0.05) were further investigated using Bayesian Kernel Machine Regression (BKMR), an environmental mixture method. Thirty-five miRNA were associated (P FDR <0.05) with at least one metal in early pregnancy. One association (an inverse association between cobalt and miR-150-5p) remained statistically significant when evaluating late pregnancy miRNA counts. Eight miRNA (miR-302b-3p, miR-199a-5p, miR-188-5p, miR-138-5p, miR-212-3p, miR-608, miR-1272, miR-19b-3p) were associated with three metals (barium, mercury, and thallium) in early pregnancy, and their predicted target genes were enriched in pathways important for placental development. Results were consistent when using BKMR. Early pregnancy exposure to barium, mercury, and thallium may have short-term impacts on a common set of EV miRNA which target pathways important for placental development.

Inhalation of ambient PM2.5, shown to be able to cross the placenta, has been linked to adverse obstetric and postnatal metabolic health outcomes. The placenta regulates fetal growth and influences postnatal development via fetal programming. Placental gene expression may be influenced by intrauterine exposures to PM2.5. Herein, we explore whether maternal PM2.5 exposure during pregnancy alters placental gene expression related to lipid and glucose metabolism in a U.S. birth cohort, the Rhode Island Child Health Study (RICHS). Average PM2.5 exposure level was estimated linking residential addresses and satellite data across the three trimesters using spatio-temporal models. Based on Gene Ontology annotations, we curated a list of 657 lipid and glucose metabolism genes. We conducted a two-staged analysis by leveraging placental RNA-Seq data from 148 subjects to identify top dysregulated metabolic genes associated with PM2.5 (Phase I) and then validated the results in placental samples from 415 participants of the cohort using RT-qPCR (Phase II). Associations between PM2.5 and placental gene expression were explored using multivariable linear regression models in the overall population and in sex-stratified analyses. The average level of PM2.5 exposure across pregnancy was 8.0μg/m 3 , which is below the national standard of 12μg/m 3 . Phase I revealed that expression levels of 32 out of the curated list of 657 genes were significantly associated with PM2.5 exposure (FDR P<0.01), 28 genes showed differential expression modified by sex of the infant. Five of these genes (ABHD3, ATP11A, CLTCL1, ST6GALNAC4 and PSCA) were validated using RT-qPCR. Associations were stronger in placentas from male births compared to females, indicating a sex-dependent effect. These genes are involved in inflammation, lipid transport, cell-cell communication or cell invasion. Our results suggest that gestational PM2.5 exposure may alter placental metabolic function. However, whether it confers long-term programming effects postnatally, especially in a sex-specific matter, warrants further studies., (Copyright © 2022 Elsevier Inc. All rights reserved.)