Association of per- and polyfluoroalkyl substances with the antioxidant bilirubin across pregnancy.

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 (ß = 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 (ß = 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 (ß = 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.

Epigenetic associations with neonatal age in infants born very preterm, particularly among genes involved in neurodevelopment.

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.

Transcriptome- and DNA methylation-based cell-type deconvolutions produce similar estimates of differential gene expression and differential methylation.

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.

Examining the Biological Impacts of Parent-Child Relationship Dynamics on Preschool-Aged Children who have Experienced Adversity.

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 1D and longer telomeres, and (2) higher parent-child disengagement was associated with higher levels of methylation of NR3C1 exon 1D and shorter telomeres. Results suggest that parent-child relationship dynamics may have distinct biological effects on children.

Differential impact of prenatal PTSD symptoms and preconception trauma exposure on placental NR3C1 and FKBP5 methylation.

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 NR3C1 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.

Transcriptome- and DNA methylation-based cell-type deconvolutions produce similar estimates of differential gene expression and differential methylation.

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.

Epigenetic associations in HPA axis genes related to bronchopulmonary dysplasia and antenatal steroids.

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.

Epigenetic landscape of 5-hydroxymethylcytosine and associations with gene expression in placenta.

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.

Placental transcriptome variation associated with season, location, and urinary prenatal pyrethroid metabolites of Thai farm-working women.

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.

Toward a more holistic approach to the study of exposures and child outcomes.

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.

Epigenome-wide association study identifies neonatal DNA methylation associated with two-year attention problems in children born very preterm.

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.

Contributions of prenatal risk factors and neonatal epigenetics to cognitive outcome in children born very preterm.

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).

Two-Year Autism Risk Screening and 3-Year Developmental Outcomes in Very Preterm Infants.

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.