Maternal educational attainment in pregnancy and epigenome-wide DNA methylation changes in the offspring from birth until adolescence.

Maternal educational attainment (MEA) shapes offspring health through multiple potential pathways. Differential DNA methylation may provide a mechanistic understanding of these long-term associations. We aimed to quantify the associations of MEA with offspring DNA methylation levels at birth, in childhood and in adolescence. Using 37 studies from high-income countries, we performed meta-analysis of epigenome-wide association studies (EWAS) to quantify the associations of completed years of MEA at the time of pregnancy with offspring DNA methylation levels at birth (n = 9 881), in childhood (n = 2 017), and adolescence (n = 2 740), adjusting for relevant covariates. MEA was found to be associated with DNA methylation at 473 cytosine-phosphate-guanine sites at birth, one in childhood, and four in adolescence. We observed enrichment for findings from previous EWAS on maternal folate, vitamin-B12 concentrations, maternal smoking, and pre-pregnancy BMI. The associations were directionally consistent with MEA being inversely associated with behaviours including smoking and BMI. Our findings form a bridge between socio-economic factors and biology and highlight potential pathways underlying effects of maternal education. The results broaden our understanding of bio-social associations linked to differential DNA methylation in multiple early stages of life. The data generated also offers an important resource to help a more precise understanding of the social determinants of health.

Prenatal and Childhood Smoke Exposure Associations with Cognition, Language, and Attention-Deficit/Hyperactivity Disorder.

OBJECTIVE: To assess the relationships of prenatal and childhood smoke exposure with specific neurodevelopmental and behavioral problems during early childhood. STUDY DESIGN: A subsample (n = 386) of mother-child dyads from the Newborn Epigenetic Study (NEST) prebirth cohort participated in the study. Cotinine concentrations were used to objectively measure prenatal and childhood smoke exposure when youth were aged 3-13 years. Multivariable regression models were used to estimate associations of prenatal and childhood cotinine concentrations with performance on the National Institutes of Health (NIH) Toolbox and attention-deficit/hyperactivity disorder and behavioral symptoms, measured using the Behavior Assessment System for Children, 2nd edition (BASC-2). RESULTS: After adjusting for confounders, childhood cotinine concentrations were associated with poorer cognitive performance on tasks measuring cognitive flexibility (B = -1.29; P = .03), episodic memory (B = -0.97; P = .02), receptive language development (B = -0.58; P = .01), and inhibitory control and attention (B = -1.59; P = .006). Although childhood cotinine concentration was associated with higher levels of attention problems (B = 0.83; P = .004) on the BASC-2, after adjustment for confounders, the association is nonsignificant. Although associations for maternal cotinine concentrations were null, an interaction was detected between prenatal and childhood cotinine concentrations on the NIH Toolbox Picture Vocabulary Task (P = .02). CONCLUSIONS: Our findings suggest that childhood tobacco smoke exposure may lead to poorer attention regulation and language acquisition, complex visual processing ability, and attention problems.

Epigenome-wide methylation and progression to high-grade cervical intraepithelial neoplasia (CIN2+): a prospective cohort study in the United States.

BACKGROUND: Methylation levels may be associated with and serve as markers to predict risk of progression of precancerous cervical lesions. We conducted an epigenome-wide association study (EWAS) of CpG methylation and progression to high-grade cervical intraepithelial neoplasia (CIN2 +) following an abnormal screening test. METHODS: A prospective US cohort of 289 colposcopy patients with normal or CIN1 enrollment histology was assessed. Baseline cervical sample DNA was analyzed using Illumina HumanMethylation 450K (n = 76) or EPIC 850K (n = 213) arrays. Participants returned at provider-recommended intervals and were followed up to 5 years via medical records. We assessed continuous CpG M values for 9 cervical cancer-associated genes and time-to-progression to CIN2+. We estimated CpG-specific time-to-event ratios (TTER) and hazard ratios using adjusted, interval-censored Weibull accelerated failure time models. We also conducted an exploratory EWAS to identify novel CpGs with false discovery rate (FDR) < 0.05. RESULTS: At enrollment, median age was 29.2 years; 64.0% were high-risk HPV-positive, and 54.3% were non-white. During follow-up (median 24.4 months), 15 participants progressed to CIN2+. Greater methylation levels were associated with a shorter time-to-CIN2+ for CADM1 cg03505501 (TTER = 0.28; 95%CI 0.12, 0.63; FDR = 0.03) and RARB Cluster 1 (TTER = 0.46; 95% CI 0.29, 0.71; FDR = 0.01). There was evidence of similar trends for DAPK1 cg14286732, PAX1 cg07213060, and PAX1 Cluster 1. The EWAS detected 336 novel progression-associated CpGs, including those located in CpG islands associated with genes FGF22, TOX, COL18A1, GPM6A, XAB2, TIMP2, GSPT1, NR4A2, and APBB1IP. CONCLUSIONS: Using prospective time-to-event data, we detected associations between CADM1-, DAPK1-, PAX1-, and RARB-related CpGs and cervical disease progression, and we identified novel progression-associated CpGs. IMPACT: Methylation levels at novel CpG sites may help identify individuals with ≤CIN1 histology at higher risk of progression to CIN2+ and inform risk-based cervical cancer screening guidelines.

Sleep onset, duration, or regularity: which matters most for child adiposity outcomes?

BACKGROUND/OBJECTIVES: Sleep measures, such as duration and onset timing, are associated with adiposity outcomes among children. Recent research among adults has considered variability in sleep and wake onset times, with the Sleep Regularity Index (SRI) as a comprehensive metric to measure shifts in sleep and wake onset times between days. However, little research has examined regularity and adiposity outcomes among children. This study examined the associations of three sleep measures (i.e., sleep duration, sleep onset time, and SRI) with three measures of adiposity (i.e., body mass index [BMI], waist circumference, and waist-to-height ratio [WHtR]) in a pediatric sample. SUBJECTS/METHODS: Children (ages 4-13 years) who were part of the U.S. Newborn Epigenetic STudy (NEST) participated. Children (N = 144) wore an ActiGraph for 1 week. Sleep measures were estimated from actigraphy data. Weight, height, and waist circumference were measured by trained researchers. BMI and WHtR was calculated with the objectively measured waist and height values. Multiple linear regression models examined associations between child sleep and adiposity outcomes, controlling for race/ethnicity, child sex, age, mothers' BMI and sleep duration. RESULTS: When considering sleep onset timing and duration, along with demographic covariates, sleep onset timing was not significantly associated with any of the three adiposity measures, but a longer duration was significantly associated with a lower BMI Z-score (ß = -0.29, p < 0.001), waist circumference (ß = -0.31, p < 0.001), and WHtR (ß = -0.38, p < 0.001). When considering SRI and duration, duration remained significantly associated with the adiposity measures. The SRI and adiposity associations were in the expected direction, but were non-significant, except the SRI and WHtR association (ß = -0.16, p = 0.077) was marginally non-significant. CONCLUSIONS: Sleep duration was consistently associated with adiposity measures in children 4-13 years of age. Pediatric sleep interventions should focus first on elongating nighttime sleep duration, and examine if this improves child adiposity outcomes.

Tobacco Retail Outlets, Neighborhood Deprivation and the Risk of Prenatal Smoke Exposure.

INTRODUCTION: Smoking and smoke exposure among pregnant women remain persistent public health issues. Recent estimates suggest that approximately one out of four nonsmokers have measurable levels of cotinine, a marker indicating regular exposure to secondhand smoke. Epidemiological research has attempted to pinpoint individual-level and neighborhood-level factors for smoking during pregnancy. However, most of these studies have relied upon self-reported measures of smoking. AIMS AND METHODS: To more accurately assess smoke exposure resulting from both smoking and secondhand exposure in mothers during pregnancy, we used Bayesian regression models to estimate the association of cotinine levels with tobacco retail outlet (TRO) exposure and a neighborhood deprivation index (NDI) in six counties in North Carolina centered on Durham County. RESULTS: Results showed a significant positive association between TRO exposure (ß = 0.008, 95% credible interval (CI) = [0.003, 0.013]) and log cotinine after adjusting for individual covariates (eg, age, race/ethnicity, education, marital status). TRO exposure was not significant after including the NDI, which was significantly associated with log cotinine (ß = 0.143, 95% CI = [0.030, 0.267]). However, in a low cotinine stratum (indicating secondhand smoke exposure), TRO exposure was significantly associated with log cotinine (ß = 0.005, 95% CI = [0.001, 0.009]), while in a high cotinine stratum (indicating active smoking), the NDI was significantly associated with log cotinine (ß = 0.176, 95% CI = [0.005, 0.372]). CONCLUSIONS: In summary, our findings add to the evidence that contextual factors are important for active smoking during pregnancy. IMPLICATIONS: In this study, we found several significant associations that suggest a more nuanced understanding of the potential influence of environmental- and individual-level factors for levels of prenatal smoke exposure. Results suggested a significant positive association between TRO exposure and cotinine levels, after adjusting for the individual factors such as race, education, and marital status. Individually, NDI was similarly positively associated with cotinine levels as well. However, when combining TRO exposure alongside NDI in the same model, TROs were no longer significantly associated with overall cotinine levels.

Neighborhood Deprivation is Associated with Increased Risk of Prenatal Smoke Exposure.

Despite years of advisories against the behavior, smoking among pregnant women remains a persistent public health issue in the USA. Recent estimates suggest that 9.4% of women smoke before pregnancy and 7.1% during pregnancy in the USA. Epidemiological research has attempted to pinpoint individual-level and neighborhood-level factors for smoking during pregnancy, including educational attainment, employment status, housing conditions, poverty, and racial demographics. However, most of these studies have relied upon self-reported measures of smoking, which are subject to reporting bias. To more accurately and objectively assess smoke exposure in mothers during pregnancy, we used Bayesian index models to estimate a neighborhood deprivation index (NDI) for block groups in Durham County, North Carolina, and its association with cotinine, a marker of smoke exposure, in pregnant mothers (n = 887 enrolled 2005-2011). Results showed a significant positive association between NDI and log cotinine (beta = 0.20, 95% credible interval = [0.11, 0.29]) after adjusting for individual covariates (e.g., race/ethnicity and education). The two most important variables in the NDI according to the estimated index weights were percent females without a high school degree and percent Black population. At the individual level, Hispanic and other race/ethnicity were associated with lowered cotinine compared with non-Hispanic Whites. Higher education levels were also associated with lowered cotinine. In summary, our findings provide stronger evidence that the socio-geographic variables of educational attainment and neighborhood racial composition are important factors for smoking and secondhand smoke exposure during pregnancy and can be used to target intervention efforts.

Clomifene and Assisted Reproductive Technology in Humans Are Associated with Sex-Specific Offspring Epigenetic Alterations in Imprinted Control Regions.

Children conceived with assisted reproductive technology (ART) have an increased risk of adverse outcomes, including congenital malformations and imprinted gene disorders. In a retrospective North Carolina-based-birth-cohort, we examined the effect of ovulation drugs and ART on CpG methylation in differentially methylated CpGs in known imprint control regions (ICRs). Nine ICRs containing 48 CpGs were assessed for methylation status by pyrosequencing in mixed leukocytes from cord blood. After restricting to non-smoking, college-educated participants who agreed to follow-up, ART-exposed (n = 27), clomifene-only-exposed (n = 22), and non-exposed (n = 516) groups were defined. Associations of clomifene and ART with ICR CpG methylation were assessed with linear regression and stratifying by offspring sex. In males, ART was associated with hypomethylation of the PEG3 ICR [ß(95% CI) = -1.46 (-2.81, -0.12)] and hypermethylation of the MEG3 ICR [3.71 (0.01, 7.40)]; clomifene-only was associated with hypomethylation of the NNAT ICR [-5.25 (-10.12, -0.38)]. In female offspring, ART was associated with hypomethylation of the IGF2 ICR [-3.67 (-6.79, -0.55)]. Aberrant methylation of these ICRs has been associated with cardiovascular disease and metabolic and behavioral outcomes in children. The results suggest that the increased risk of adverse outcomes in offspring conceived through ART may be due in part to altered methylation of ICRs. Larger studies utilizing epigenome-wide interrogation are warranted.

Identifying the Best Questions for Rapid Screening of Secondhand Smoke Exposure Among Children.

INTRODUCTION: Many children suffer from secondhand smoke exposure (SHSe), which leads to a variety of negative health consequences. However, there is no consensus on how clinicians can best query parents for possible SHSe among children. We employed a data-driven approach to create an efficient screening tool for clinicians to quickly and correctly identify children at risk for SHSe. METHODS: Survey data from mothers and biospecimens from children were ascertained from the Neurodevelopment and Improving Children's Health following Environmental Tobacco Smoke Exposure (NICHES) study. Included were mothers and their children whose saliva were assayed for cotinine (n = 351 pairs, mean child age = 5.6 years). Elastic net regression predicting SHSe, as indicated from cotinine concentration, was conducted on available smoking-related questions and cross-validated with 2015-2016 National Health and Nutrition Examination Survey (NHANES) data to select the most predictive items of SHSe among children (n = 1670, mean child age = 8.4 years). RESULTS: Answering positively to at least one of the two final items ("During the past 30 days, did you smoke cigarettes at all?" and "Has anyone, including yourself, smoked tobacco in your home in the past 7 days?") showed area under the curve = .82, and good specificity (.88) and sensitivity (.74). These results were validated with similar items in the nationally representative NHANES sample, area under the curve = .82, specificity = .78, and sensitivity = .77. CONCLUSIONS: Our data-driven approach identified and validated two items that may be useful as a screening tool for a speedy and accurate assessment of SHSe among children. IMPLICATIONS: The current study used a rigorous data-driven approach to identify questions that could reliably predict SHSe among children. Using saliva cotinine concentration levels as a gold standard for determining SHSe, our analysis employing elastic net regression identified two questions that served as good classifier for distinguishing children who might be at risk for SHSe. The two items that we validated in the current study can be readily used by clinicians, such as pediatricians, as part of screening procedures to quickly identify whether children might be at risk for SHSe.

Epigenome-wide meta-analysis of DNA methylation and childhood asthma.

BACKGROUND: Epigenetic mechanisms, including methylation, can contribute to childhood asthma. Identifying DNA methylation profiles in asthmatic patients can inform disease pathogenesis. OBJECTIVE: We sought to identify differential DNA methylation in newborns and children related to childhood asthma. METHODS: Within the Pregnancy And Childhood Epigenetics consortium, we performed epigenome-wide meta-analyses of school-age asthma in relation to CpG methylation (Illumina450K) in blood measured either in newborns, in prospective analyses, or cross-sectionally in school-aged children. We also identified differentially methylated regions. RESULTS: In newborns (8 cohorts, 668 cases), 9 CpGs (and 35 regions) were differentially methylated (epigenome-wide significance, false discovery rate < 0.05) in relation to asthma development. In a cross-sectional meta-analysis of asthma and methylation in children (9 cohorts, 631 cases), we identified 179 CpGs (false discovery rate < 0.05) and 36 differentially methylated regions. In replication studies of methylation in other tissues, most of the 179 CpGs discovered in blood replicated, despite smaller sample sizes, in studies of nasal respiratory epithelium or eosinophils. Pathway analyses highlighted enrichment for asthma-relevant immune processes and overlap in pathways enriched both in newborns and children. Gene expression correlated with methylation at most loci. Functional annotation supports a regulatory effect on gene expression at many asthma-associated CpGs. Several implicated genes are targets for approved or experimental drugs, including IL5RA and KCNH2. CONCLUSION: Novel loci differentially methylated in newborns represent potential biomarkers of risk of asthma by school age. Cross-sectional associations in children can reflect both risk for and effects of disease. Asthma-related differential methylation in blood in children was substantially replicated in eosinophils and respiratory epithelium.

Snacking frequency and dietary intake in toddlers and preschool children.

Understanding the relationship between snacking and dietary intake in early life years is one key but understudied area. In this study, we examined snacking patterns in toddlers and preschool children and the associations between snacking frequency and daily energy intake. We analyzed data from children aged 12-72 months (N = 1186) in the Newborn Epigenetic STudy (NEST). We used Bonferroni multiple comparison methods to examine the differences in snacking patterns across subgroups. Linear and quantile regression models were fit to investigate the association between dietary intake and snacking frequency. Our estimates suggest that Non-Hispanic blacks had the highest total daily energy intake from snacks (334 kcal/day) compared to non-Hispanic whites (270 kcal/day) and Hispanics (274 kcal/day) in 12-to-24-month-olds. In 2-to-6-year-olds, mean energy intake from snacks was 296 kcal/day without a significant racial/ethnic difference. Carbohydrate, fat and protein from snacks contributed about 17%, 9% and 4% respectively of the total energy intake in 12-to-24-month-olds while they contributed about 15%, 7% and 2% respectively of the total energy intake in the other age group. Snacking frequency was positively and significantly associated with total daily energy intake in both 12-to-24-month-olds and 2-to-6-year-olds as indicated by regression coefficient estimates of snacking frequency (ß = 31.3 kcal/day with P = 0.027 and ß = 175.4 kcal/day with P < 0.0001, respectively, indicating a higher snacking frequency was associated with a greater total daily energy intake). In conclusion, snacking frequency was positively associated with daily energy intake. Carbohydrates and fats from snacks are significant energy contributors. Age differentiation was apparent regarding the relationship between snacking frequency and dietary intake. Differentiated interventions that are age-specific and focus on the dietary quality of snacks instead of quantity are needed.

DNA methylation of imprinted gene control regions in the regression of low-grade cervical lesions.

The role of host epigenetic mechanisms in the natural history of low-grade cervical intraepithelial neoplasia (CIN1) is not well characterized. We explored differential methylation of imprinted gene regulatory regions as predictors of the risk of CIN1 regression. A total of 164 patients with CIN1 were recruited from 10 Duke University clinics for the CIN Cohort Study. Participants had colposcopies at enrollment and up to five follow-up visits over 3 years. DNA was extracted from exfoliated cervical cells for methylation quantitation at CpG (cytosine-phosphate-guanine) sites and human papillomavirus (HPV) genotyping. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression to quantify the effect of methylation on CIN1 regression over two consecutive visits, compared to non-regression (persistent CIN1; progression to CIN2+; or CIN1 regression at a single time-point), adjusting for age, race, high-risk HPV (hrHPV), parity, oral contraceptive and smoking status. Median participant age was 26.6 years (range: 21.0-64.4 years), 39% were African-American, and 11% were current smokers. Most participants were hrHPV-positive at enrollment (80.5%). Over one-third of cases regressed (n = 53, 35.1%). Median time-to-regression was 12.6 months (range: 4.5-24.0 months). Probability of CIN1 regression was negatively correlated with methylation at IGF2AS CpG 5 (HR = 0.41; 95% CI = 0.23-0.77) and PEG10 DMR (HR = 0.80; 95% CI = 0.65-0.98). Altered methylation of imprinted IGF2AS and PEG10 DMRs may play a role in the natural history of CIN1. If confirmed in larger studies, further research on imprinted gene DMR methylation is warranted to determine its efficacy as a biomarker for cervical cancer screening.

Maternal blood cadmium, lead and arsenic levels, nutrient combinations, and offspring birthweight.

BACKGROUND: Cadmium (Cd), lead (Pb) and arsenic (As) are common environmental contaminants that have been associated with lower birthweight. Although some essential metals may mitigate exposure, data are inconsistent. This study sought to evaluate the relationship between toxic metals, nutrient combinations and birthweight among 275 mother-child pairs. METHODS: Non-essential metals, Cd, Pb, As, and essential metals, iron (Fe), zinc (Zn), selenium (Se), copper (Cu), calcium (Ca), magnesium (Mg), and manganese (Mn) were measured in maternal whole blood obtained during the first trimester using inductively coupled plasma mass spectrometry. Folate concentrations were measured by microbial assay. Birthweight was obtained from medical records. We used quantile regression to evaluate the association between toxic metals and nutrients due to their underlying wedge-shaped relationship. Ordinary linear regression was used to evaluate associations between birth weight and toxic metals. RESULTS: After multivariate adjustment, the negative association between Pb or Cd and a combination of Fe, Se, Ca and folate was robust, persistent and dose-dependent (p < 0.05). However, a combination of Zn, Cu, Mn and Mg was positively associated with Pb and Cd levels. While prenatal blood Cd and Pb were also associated with lower birthweight. Fe, Se, Ca and folate did not modify these associations. CONCLUSION: Small sample size and cross-sectional design notwithstanding, the robust and persistent negative associations between some, but not all, nutrient combinations with these ubiquitous environmental contaminants suggest that only some recommended nutrient combinations may mitigate toxic metal exposure in chronically exposed populations. Larger longitudinal studies are required to confirm these findings.

Geographic clustering of elevated blood heavy metal levels in pregnant women.

BACKGROUND: Cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As) exposure is ubiquitous and has been associated with higher risk of growth restriction and cardiometabolic and neurodevelopmental disorders. However, cost-efficient strategies to identify at-risk populations and potential sources of exposure to inform mitigation efforts are limited. The objective of this study was to describe the spatial distribution and identify factors associated with Cd, Pb, Hg, and As concentrations in peripheral blood of pregnant women. METHODS: Heavy metals were measured in whole peripheral blood of 310 pregnant women obtained at gestational age ~12 weeks. Prenatal residential addresses were geocoded and geospatial analysis (Getis-Ord Gi* statistics) was used to determine if elevated blood concentrations were geographically clustered. Logistic regression models were used to identify factors associated with elevated blood metal levels and cluster membership. RESULTS: Geospatial clusters for Cd and Pb were identified with high confidence (p-value for Gi* statistic <0.01). The Cd and Pb clusters comprised 10.5 and 9.2 % of Durham County residents, respectively. Medians and interquartile ranges of blood concentrations (µg/dL) for all participants were Cd 0.02 (0.01-0.04), Hg 0.03 (0.01-0.07), Pb 0.34 (0.16-0.83), and As 0.04 (0.04-0.05). In the Cd cluster, medians and interquartile ranges of blood concentrations (µg/dL) were Cd 0.06 (0.02-0.16), Hg 0.02 (0.00-0.05), Pb 0.54 (0.23-1.23), and As 0.05 (0.04-0.05). In the Pb cluster, medians and interquartile ranges of blood concentrations (µg/dL) were Cd 0.03 (0.02-0.15), Hg 0.01 (0.01-0.05), Pb 0.39 (0.24-0.74), and As 0.04 (0.04-0.05). Co-exposure with Pb and Cd was also clustered, the p-values for the Gi* statistic for Pb and Cd was <0.01. Cluster membership was associated with lower education levels and higher pre-pregnancy BMI. CONCLUSIONS: Our data support that elevated blood concentrations of Cd and Pb are spatially clustered in this urban environment compared to the surrounding areas. Spatial analysis of metals concentrations in peripheral blood or urine obtained routinely during prenatal care can be useful in surveillance of heavy metal exposure.

Association between F2-isoprostanes and self-reported stressors in pregnant americans of African and European ancestry.

Background: Poor birth outcomes such as preterm birth/delivery disproportionately affect African Americans compared to White individuals. Reasons for this disparity are likely multifactorial, and include prenatal psychosocial stressors, and attendant increased lipid peroxidation; however, empirical data linking psychosocial stressors during pregnancy to oxidative status are limited. Methods: We used established scales to measure five psychosocial stressors. Maternal adverse childhood experiences, financial stress, social support, anxiety, and depression were measured among 50 African American and White pregnant women enrolled in the Stress and Health in Pregnancy cohort. Liquid chromatography-tandem mass spectrometry was used to measure biomarkers of oxidative stress (four urinary F2-isoprostane isomers), to estimate oxidative status. Linear regression models were used to evaluate associations between psychosocial stressors, prenatal oxidative status and preterm birth. Results: After adjusting for maternal obesity, gestational diabetes, and cigarette smoking, African American women with higher oxidative status were more likely to report higher maternal adverse childhood experience scores (ß = 0.16, se = 1.07, p-value = 0.024) and depression scores (ß = 0.05, se = 0.02, p = 0.014). Higher oxidative status was also associated with lower gestational age at birth (ß = -0.13, se = 0.06, p = 0.04) in this population. These associations were not apparent in Whites. However, none of the cross-product terms for race/ethnicity and social stressors reached statistical significance (p > 0.05). Conclusion: While the small sample size limits inference, our novel data suggest that psychosocial stressors may contribute significantly to oxidative stress during pregnancy, and preterm birth or delivery African Americans. If replicated in larger studies, these findings would support oxidative stress reduction using established dietary or pharmacological approaches present a potential avenue to mitigate adverse effects of psychosocial stressors on birth outcomes.

Duration of exposure to epidural anesthesia at delivery, DNA methylation in umbilical cord blood and their association with offspring asthma in Non-Hispanic Black women.

Epidural anesthesia is an effective pain relief modality, widely used for labor analgesia. Childhood asthma is one of the commonest chronic medical illnesses in the USA which places a significant burden on the health-care system. We recently demonstrated a negative association between the duration of epidural anesthesia and the development of childhood asthma; however, the underlying molecular mechanisms still remain unclear. In this study of 127 mother-child pairs comprised of 75 Non-Hispanic Black (NHB) and 52 Non-Hispanic White (NHW) from the Newborn Epigenetic Study, we tested the hypothesis that umbilical cord blood DNA methylation mediates the association between the duration of exposure to epidural anesthesia at delivery and the development of childhood asthma and whether this differed by race/ethnicity. In the mother-child pairs of NHB ancestry, the duration of exposure to epidural anesthesia was associated with a marginally lower risk of asthma (odds ratio = 0.88, 95% confidence interval = 0.76-1.01) for each 1-h increase in exposure to epidural anesthesia. Of the 20 CpGs in the NHB population showing the strongest mediation effect, 50% demonstrated an average mediation proportion of 52%, with directional consistency of direct and indirect effects. These top 20 CpGs mapped to 21 genes enriched for pathways engaged in antigen processing, antigen presentation, protein ubiquitination and regulatory networks related to the Major Histocompatibility Complex (MHC) class I complex and Nuclear Factor Kappa-B (NFkB) complex. Our findings suggest that DNA methylation in immune-related pathways contributes to the effects of the duration of exposure to epidural anesthesia on childhood asthma risk in NHB offspring.

AHRR Hypomethylation mediates the association between maternal smoking and metabolic profiles in children.

BACKGROUND: Tobacco smoking during pregnancy is associated with metabolic dysfunction in children, but mechanistic insights remain limited. Hypomethylation of cg05575921 in the aryl hydrocarbon receptor repressor (AHRR) gene is associated with in utero tobacco smoke exposure. In this study, we evaluated whether AHRR hypomethylation mediates the association between maternal smoking and metabolic dysfunction in children. METHODS: We assessed metabolic dysfunction using liver fat content (LFC), serum, and clinical data in children aged 7-12 years (n=78) followed since birth. Maternal smoking was self-reported at 12 weeks gestation. Methylation was measured by means of pyrosequencing at 3 sequential CpG sites, including cg05575921, at birth and at ages 7-12. Regression models were used to evaluate whether AHRR methylation mediated the association between maternal smoking and child metabolic dysfunction. RESULTS: Average AHRR methylation at birth was significantly higher among children of nonsmoking mothers compared with children of mothers who smoked (69.8% ± 4.4% vs. 63.5% ± 5.5, p=0.0006). AHRR hypomethylation at birth was associated with higher liver fat content (p=0.01), triglycerides (p=0.01), and alanine aminotransferase levels (p=0.03), and lower HDL cholesterol (p=0.01) in childhood. AHRR hypomethylation significantly mediated associations between maternal smoking and liver fat content (indirect effect=0.213, p=0.018), triglycerides (indirect effect=0.297, p=0.044), and HDL cholesterol (indirect effect = -0.413, p=0.007). AHRR methylation in childhood (n=78) was no longer significantly associated with prenatal smoke exposure or child metabolic parameters (p>0.05). CONCLUSIONS: AHRR hypomethylation significantly mediates the association between prenatal tobacco smoke exposure and features of childhood metabolic dysfunction, despite the lack of persistent hypomethylation of AHRR into childhood. Further studies are needed to replicate these findings and to explore their causal and long-term significance.

Maternal Mediterranean Diet Adherence and Its Associations with Maternal Prenatal Stressors and Child Growth.

Background: Psychosocial and physiologic stressors, such as depression and obesity, during pregnancy can have negative consequences, such as increased systemic inflammation, contributing to chronic disease for both mothers and their unborn children. These conditions disproportionately affect racial/ethnic minorities. The effects of recommended dietary patterns in mitigating the effects of these stressors remain understudied. Objectives: We aimed to evaluate the relations between maternal Mediterranean diet adherence (MDA) and maternal and offspring outcomes during the first decade of life in African Americans, Hispanics, and Whites. Methods: This study included 929 mother-child dyads from the NEST (Newborn Epigenetics STudy), a prospective cohort study. FFQs were used to estimate MDA in pregnant women. Weight and height were measured in children between birth and age 8 y. Multivariable linear regression models were used to examine associations between maternal MDA, inflammatory cytokines, and pregnancy and postnatal outcomes. Results: More than 55% of White women reported high MDA during the periconceptional period compared with 22% of Hispanic and 18% of African American women (P < 0.05). Higher MDA was associated with lower likelihood of depressive mood (ß = -0.45; 95% CI: -0.90, -0.18; P = 0.02) and prepregnancy obesity (ß = -0.29; 95% CI: -0.57, -0.0002; P = 0.05). Higher MDA was also associated with lower body size at birth, which was maintained to ages 3-5 and 6-8 y-this association was most apparent in White children (3-5 y: ß = -2.9, P = 0.02; 6-8 y: ß = -3.99, P = 0.01). Conclusions: If replicated in larger studies, our data suggest that MDA provides a potent avenue by which effects of prenatal stressors on maternal and fetal outcomes can be mitigated to reduce ethnic disparities in childhood obesity.

Parenting Stress, Child Weight-Related Behaviors, and Child Weight Status.

Background: There has been limited examination of the association between parenting stress and child weight-related behaviors. We aimed to determine whether parenting stress is associated with child weight-related behaviors, including physical activity, screen time, diet, sedentary time, and eating in the absence of hunger (EAH). Secondarily, we assessed association between parenting stress and child weight status. Methods: Mother-child dyads (N = 291) enrolled in the Newborn Epigenetic STudy (NEST), a longitudinal cohort study, completed surveys to describe parenting stress, and child diet. Children participated in the EAH task and wore accelerometers to assess sedentary time and physical activity. Child weight status was assessed using measured height and weight. Outcomes and exposures were examined using generalized linear models and restricted cubic splines as appropriate based on linear lack-of-fit test. Results: Child sedentary time and vegetable consumption were inversely associated with parenting stress (Total Stress B = -0.78; 95% confidence interval [CI]: -1.35 to -0.20; p = 0.017; and Total Stress adjusted odds ratio [aOR] = 0.98; 95% CI: 0.99 to 1.00; p = 0.022, respectively). Child screen time was directly associated with parenting stress (Total Stress = aOR 1.01; 95% CI: 1.00-1.02; p = 0.032). Fast-food intake was nonlinearly associated with parenting stress. There was no evidence of association between parenting stress and child EAH, physical activity, or weight status. Associations between parenting stress and child weight-related behaviors were not moderated by race or family structure. Conclusions: Parenting stress was associated with important child weight-related behaviors but not weight status. Management of parenting stress may represent a reasonable adjunct to family-based behavioral interventions.

Sex-specific DNA methylation and associations with in utero tobacco smoke exposure at nuclear-encoded mitochondrial genes.

Sex-linked differences in mitochondrial ATP production, enzyme activities, and reactive oxygen species generation have been reported in multiple tissue and cell types. While the effects of reproductive hormones underlie many of these differences, regulation of sexually dimorphic mitochondrial function has not been fully characterized. We hypothesized that sex-specific DNA methylation contributes to sex-specific expression of nuclear genes that influence mitochondrial function. Herein, we analysed DNA methylation data specifically focused on nuclear-encoded mitochondrial genes in 191 males and 190 females. We found 596 differentially methylated sites (DMSs) (FDR p < 0.05), corresponding to 324 genes, with at least a 1% difference in methylation between sexes. To investigate the potential functional significance, we utilized gene expression microarray data. Of the 324 genes containing DMSs, 17 showed differences in gene expression by sex. Particularly striking was that ATP5G2, encoding subunit C of ATP synthase, contains seven DMSs and exhibits a sex difference in expression (p = 0.04). Finally, we also found that alterations in DNA methylation associated with in utero tobacco smoke exposure were sex-specific in these nuclear-encoded mitochondrial genes. Interestingly, the level of sex differences in DNA methylation at nuclear-encoded mitochondrial genes and the level of methylation changes associated with smoke exposure were less prominent than that of other genes. This suggests more conservative regulation of DNA methylation at these nuclear-encoded mitochondrial genes as compared to others. Overall, our findings suggest that sex-specific DNA methylation may help establish sex differences in expression and function and that sex-specific alterations in DNA methylation in response to exposures could contribute to sex-variable toxicological responses.

Alterations in DNA methylation associate with fatty liver and metabolic abnormalities in a multi-ethnic cohort of pre-teenage children.

Non-Alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in children. Epigenetic alterations, such as through DNA methylation (DNAm), may link adverse childhood exposures and fatty liver and provide non-invasive methods for identifying children at high risk for NAFLD and associated metabolic dysfunction. We investigated the association between differential DNAm and liver fat content (LFC) and liver injury in pre-adolescent children. Leveraging data from the Newborn Epigenetics Study (NEST), we enrolled 90    mother-child dyads and used linear regression to identify CpG sites and differentially methylated regions (DMRs) in peripheral blood associated with LFC and alanine aminotransferase (ALT) levels in 7-12yo children. DNAm was measured using Infinium HumanMethylationEPIC BeadChips (Illumina). LFC and fibrosis were quantified by magnetic resonance imaging proton density fat fraction and elastography. Median LFC was 1.4% (range, 0.3-13.4%) and MRE was 2.5 kPa (range, 1.5-3.6kPa). Three children had LFC ≥ 5%, while six (7.6%) met our definition of NAFLD (LFC ≥ 3.7%). All children with NAFLD were obese and five were Black. LFC was associated with 88 DMRs and 106 CpGs (FDR<5%). The top two CpGs, cg25474373 and cg07264203, mapped to or near RFTN2 and PRICKLE2 genes. These two CpG sites were also significantly associated with a NAFLD diagnosis. As higher LFC associates with an adverse cardiometabolic profile already in childhood, altered DNAm may identify these children early in disease course for targeted intervention. Larger, longitudinal studies are needed to validate these findings and determine mechanistic relevance.