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Abstract 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 (

Abstract Smoking exposure during adulthood can disrupt oocyte development in women, contributing to infertility and possibly adverse birth outcomes. Some of these effects may be reflected in epigenome profiles in granulosa cells (GCs) in human follicular fluid. We compared the epigenetic modifications throughout the genome in GCs from women who were former (N = 15) versus never smokers (N = 44) undergoing assisted reproductive technologies (ART). This study included 59 women undergoing ART. Smoking history including time since quitting was determined by questionnaire. GCs were collected during oocyte retrieval and DNA methylation (DNAm) levels were profiled using the Infinium MethylationEPIC BeadChip. We performed an epigenome-wide association study with robust linear models, regressing DNAm level at individual loci on smoking status, adjusting for age, ovarian stimulation protocol, and three surrogate variables. We performed differentially methylated regions (DMRs) analysis and over-representation analysis of the identified CpGs and corresponding gene set. 81 CpGs were differentially methylated among former smokers compared to never smokers (FDR

Abstract 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.

Abstract 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.

ABSTRACTEpigenetic 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 (

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.

Abstract Background Higher exposure to traffic-related air pollution (TRAP) is related to lower fertility, with specific adverse effects on the ovary. Folic acid may attenuate these effects. Our goal was to explore the relation of TRAP exposure and supplemental folic acid intake with epigenetic aging and CpG-specific DNA methylation (DNAm) in granulosa cells (GC). Our study included 61 women undergoing ovarian stimulation at a fertility center (2005–2015). DNAm levels were profiled in GC using the Infinium MethylationEPIC BeadChip. TRAP was defined using a spatiotemporal model to estimate residence-based nitrogen dioxide (NO2) exposure. Supplemental folic acid intake was measured with a validated food frequency questionnaire. We used linear regression to evaluate whether NO2 or supplemental folic acid was associated with epigenetic age acceleration according to the Pan-tissue, mural GC, and GrimAge clocks or DNAm across the genome adjusting for potential confounders and accounting for multiple testing with a false discovery rate

A meta-analysis of pre-pregnancy maternal body mass index (ppBMI) and placental DNA methylation from 2631 mother-child pairs identifies 27 CpG sites associated with ppBMI, providing insight into how maternal obesity could be associated with metabolic health outcomes in offspring.

Selenium is an important micronutrient for foetal development. MicroRNAs play an important role in the function of the placenta, in communication between the placenta and maternal systems, and their expression can be altered through environmental and nutritional cues. To investigate the associations between placental selenium concentration and microRNA expression in the placenta, our observational study included 393 mother-child pairs from the New Hampshire Birth Cohort Study (NHBCS) and the Rhode Island Child Health Study (RICHS). Placental selenium concentrations were quantified using inductively coupled plasma mass spectrometry, and microRNA transcripts were measured using RNA-seq. We fit negative binomial additive models for assessing the association between selenium and microRNAs. We used the microRNA Data Integration Portal (mirDIP) to predict the target mRNAs of the differentially expressed microRNAs and verified the relationships between miRNA and mRNA targets in a subset of samples using existing whole transcriptome data (N = 199). We identified a non-monotonic association between selenium concentration and the expression of miR-216a-5p/miR-217-5p cluster (effective degrees of freedom, EDF = 2.44 and 2.08; FDR = 3.08 × 10−5) in placenta. Thirty putative target mRNAs of miR-216a-5p and/or miR-217-5p were identified computationally and empirically and were enriched in selenium metabolic pathways (driven by selenoprotein coding genes, TXNRD2 and SELENON). Our findings suggest that selenium influences placental microRNA expression. Further, miR-216a-5p and its putative target mRNAs could be the potential mechanistic targets of the health effect of selenium.

Summary: 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.

Maternal smoking during pregnancy contributes to poor birth outcomes. Here the authors perform a meta-analysis of the associations between maternal smoking during pregnancy and placental DNA methylation and identify links between these and poor birth outcomes, which may better inform the mechanisms through which smoking impacts placental function and fetal growth.

MicroRNAs are non-coding RNAs that regulate gene expression post-transcriptionally. In the placenta, the master regulator of foetal growth and development, microRNAs shape the basic processes of trophoblast biology and specific microRNA have been associated with foetal growth. To comprehensively assess the role of microRNAs in placental function and foetal development, we have performed small RNA sequencing to profile placental microRNAs from two independent mother-infant cohorts: the Rhode Island Child Health Study (n = 225) and the New Hampshire Birth Cohort Study (n = 317). We modelled microRNA counts on infant birthweight percentile (BWP) in each cohort, while accounting for race, sex, parity, and technical factors, using negative binomial generalized linear models. We identified microRNAs that were differentially expressed (DEmiRs) with BWP at false discovery rate (FDR) less than 0.05 in both cohorts. hsa-miR-532-5p (miR-532) was positively associated with BWP in both cohorts. By integrating parallel whole transcriptome and small RNA sequencing in the RICHS cohort, we identified putative targets of miR-532. These targets are enriched for pathways involved in adipogenesis, adipocytokine signalling, energy metabolism, and hypoxia response, and included Leptin, which we further demonstrated to have a decreasing expression with increasing BWP, particularly in male infants. Overall, we have shown a robust and reproducible association of miR-532 with BWP, which could influence BWP through regulation of adipocytokines Leptin and Adiponectin.

Abstract Background The objective of this study was to identify conditional relationships between multiple metal biomarkers that predict systolic and diastolic blood pressure in the non-institutionalized United States adult population below the age of 60. Methods We used inorganic exposure biomarker data and blood pressure data from three cycles (1999–2004) of the National Health and Nutrition Examination Survey (NHANES) to construct regression trees for blood pressure among adults ages 20–60 (adjusted for age, sex, body mass index, race, and smoking status) to identify predictors of systolic (SBP) and diastolic blood pressure (DBP). We also considered relationships among non-Hispanic black, Mexican-American, and white adults separately. Results The following metal exposure biomarkers were conditionally predictive of SBP and/or DBP in the full sample: antimony (Sb), barium (Ba), cadmium (Cd), cesium (Cs), lead (Pb), tungsten (W) and molybdenum (Mo). The highest average SBP (> 120 mmHg) was observed among those with low Sb (≤ 0.21 μg/dL) high Cd (> 0.22 μg/g creatinine) and high Pb (> 2.55 μg/dL) biomarkers. Those with the highest average DBP had high urinary W levels (> 0.10 μg/g creatinine) in combination with either urinary Sb > 0.17 μg/g creatinine or those with urinary Sb ≤ 0.17 μg/g creatinine, but with high blood Pb levels (> 1.35 μg/dL). Predictors differed by ethnicity, with Cd as the main predictor of SBP among non-Hispanic black adults, and Pb not selected by the algorithm as a predictor of SBP among non-Hispanic white adults. Conclusions Combinations of metal biomarkers have different apparent relationships with blood pressure. Additional research in toxicological experimental models and in epidemiological studies is warranted to evaluate the suggested possible toxicological interactions between Sb, Cd, and Pb; and between W, Sb, and Pb; for cardiovascular (e.g., blood pressure) health. We also think future epidemiological research on inorganic exposure sets in relation to health outcomes like blood pressure might benefit from stratification by race and ethnicity.

Copper is an essential trace nutrient and an enzymatic cofactor necessary for diverse physiological and biological processes. Copper metabolism is uniquely controlled in the placenta and changes to copper metabolism have been linked with adverse birth outcomes. We investigated associations between patterns of DNA methylation (DNAm; measured at >485 k CpG sites) and copper concentration measured from placentae in two independent mother-infant cohorts: the New Hampshire Birth Cohort Study (NHBCS, n = 306) and the Rhode Island Child Health Study (RICHS, n = 141). We identified nine copper-associated differentially methylated regions (DMRs; adjusted P < 0.05) and 15 suggestive CpGs (raw P < 1e-5). One of the most robust variably methylated CpGs associated with the expression of the antioxidant, GSTP1. Our most robust DMR negatively associates with the expression of the zinc-finger gene, ZNF197 (FDR = 4.5e-11). Genes co-expressed with ZNF197, a transcription factor, are enriched for genes that associate with birth weight in RICHS (OR = 2.9, P = 2.6e-6, N = 194), genes that are near a ZNF197 consensus binding motif (OR = 1.34, P = 0.01, N = 194), and for those classified in GO biological processes growth hormone secretion (P = 3.4e-4), multicellular organism growth (P = 3.8e-4), and molecular functions related to lipid biosynthesis (P = 1.9e-4). Further, putative transcriptional targets for ZNF197 include genes involved in copper metabolism and placentation. Our results suggest that copper metabolism is tied to DNAm in the placenta and that copper-associated patterns in DNAm may mediate normal placentation and foetal development.

Abstract Background To identify novel epigenetic markers of adolescent asthma and replicate findings in an independent cohort, then explore whether such markers are detectable at birth, predictive of early-life wheeze, and associated with gene expression in cord blood. Methods We performed epigenome-wide screening with recursive random forest feature selection and internal validation in the IOW birth cohort. We then tested whether we could replicate these findings in the independent cohort ALSPAC and followed-up our top finding with children of the IOW cohort. Results We identified 10 CpG sites associated with adolescent asthma at a 5% false discovery rate (IOW, n = 370), five of which exhibited evidence of associations in the replication study (ALSPAC, n = 720). One site, cg16658191, within HK1 displayed particularly strong associations after cellular heterogeneity adjustments in both cohorts (ORIOW = 0.17, 95% CI 0.04–0.57) (ORALSPAC = 0.57, 95% CI 0.38–0.87). Additionally, higher expression of HK1 (OR = 3.81, 95% CI 1.41–11.77) in cord blood was predictive of wheezing in infancy (n = 82). Conclusion We identified novel associations between asthma and wheeze with methylation at cg16658191 and the expression of HK1, which may serve as markers of, predictors of, and potentially etiologic factors involved in asthma and early life wheeze.

Birthweight has been found to associate with later-life health outcomes. Here the authors perform a meta-analysis of epigenome-wide association studies of 8,825 neonates from 24 birth cohorts in the Pregnancy And Childhood Epigenetics Consortium, identifying differentially methylated CpGs in neonatal blood that associate with birthweight.

Background/Aim: Selenium (Se) levels in pregnancy have been linked to neurobehavioral development of the offspring. DNA methylation is a potential mechanism underlying the impacts of environmental exposures on fetal development; however, very few studies have been done elucidating the role of DNA methylation linking prenatal Se and child neurobehavior. We aimed to investigate the associations between placental Se concentration and epigenome-wide DNA methylation in two U.S. cohorts, and to assess the association between Se-related DNA methylation modifications and newborns’ neurobehavior. Methods: We measured placental Se concentrations in 343 newborns enrolled in the New Hampshire Birth Cohort Study and in 141 newborns in the Rhode Island Child Health Study. Genome-wide placental DNA methylation was measured by HumanMethylation450 BeadChip, and newborn neurobehavioral development was assessed by the NICU Network Neurobehavioral Scales (NNNS). We meta-analyzed the associations between placental Se concentration and DNA methylation in each cohort, adjusting for covariates. We also fit multiple linear regression and ordinal logistic regression for methylation and newborn NNNS summary scores. Results: We identified five Se-related differentially methylated CpG sites. Among them was cg09674502 (GFI1), where selenium concentration was positively associated with methylation (β-coefficient = 1.11, FDR-adjusted p-value = 0.045), and where we observed that a one percent methylation level increase was associated with a 15% reduced odds of higher muscle tone in the arms, legs and trunk of newborns, (OR [95% Confidence Interval, CI] = 0.85 [0.77, 0.95]). We also observed for each interquartile range (IQR) increase in selenium concentration in the placenta, there was 1.76 times greater odds of higher hypotonicity (OR [95% CI] = 1.76 [1.12, 2.82]). Conclusions: Placental selenium concentration was inversely associated with muscle tone of newborns, and hypermethylation of GFI1 could be a potential mechanism underlying this association. Keywords: Selenium, DNA methylation, Placenta, Neurobehavior, NNNS, Muscle tone

Infertility affects one in 6 couples in developed nations, resulting in an increasing use of assisted reproductive technologies (ART). Both ART and subfertility appear to be linked to lower birth weight outcomes, setting infants up for poor long-term health. Prenatal growth is, in part, regulated via epigenetically-controlled imprinted genes in the placenta. Although differences in DNA methylation between ART and control infants have been found, it remains unclear whether these differences are due to the ART procedures or to the underlying parental subfertility and how these methylation differences affect imprinted gene expression. In this study, we examined the expression of 108 imprinted genes in placental tissues from infants born to subfertile parents (n = 79), matched naturally-conceived controls (n = 158), and infants conceived using in vitro fertilization (IVF, n = 18). Forty-five genes were identified as having significantly different expression between the subfertile infants and controls, whereas no significant differences were identified between the IVF and control groups. The expression of 4 genes—IGF2, NAPIL5, PAX8-AS1, and TUBGCP5—was significantly downregulated in the IVF compared with the subfertile group. Three of the 45 genes significantly dysregulated between subfertile and control placentae—GRB10, NDN, and CD44 —were found to have a significant positive correlation between expression and birth weight. Methylation levels for these 3 genes and 4 others—MKRN3, WRB, DHCR24, and CYR61—were significantly correlated with expression. Our findings indicate that epigenetic differences in placentas resulting from IVF pregnancies may be related to the underlying subfertility in parents using IVF rather than the IVF procedure itself.

Abstract Background In utero arsenic exposure may alter fetal developmental programming by altering DNA methylation, which may result in a higher risk of disease in later life. We evaluated the association between in utero arsenic exposure and DNA methylation (DNAm) in cord blood and its influence in later life. Methods Genome-wide DNA methylation in cord blood from 64 subjects in the Taiwanese maternal infant and birth cohort was analyzed. Robust regressions were applied to assess the association of DNA methylation with in utero arsenic exposure. Multiple testing was adjusted by controlling false discovery rate (FDR) of 0.05. The DAVID bioinformatics tool was implemented for functional annotation analyses on the detected CpGs. The identified CpGs were further tested in an independent cohort. For the CpGs replicated in the independent cohort, linear mixed models were applied to assess the association of DNA methylation with low-density lipoprotein (LDL) at different ages (2, 5, 8, 11 and 14 years). Results In total, 579 out of 385,183 CpGs were identified after adjusting for multiple testing (FDR = 0.05), of which ~60% were positively associated with arsenic exposure. Functional annotation analysis on these CpGs detected 17 KEGG pathways (FDR = 0.05) including pathways for cardiovascular diseases (CVD) and diabetes mellitus. In the independent cohort, about 46% (252 out of 553 CpGs) of the identified CpGs showed associations consistent with those in the study cohort. In total, 11 CpGs replicated in the independent cohort were in the pathways related to CVD and diabetes mellitus. Via longitudinal analyses, we found at 5 out of the 11 CpGs methylation was associated with LDL over time and interactions between DNA methylation and time were observed at 4 of the 5 CpGs, cg25189764 (coeff = 0.157, p-value = 0.047), cg04986899 (coeff. For interaction [coeff.int] = 0.030, p-value = 0.024), cg04903360 (coeff.int = 0.026, p-value = 0.032), cg08198265 (coeff.int = −0.063, p-value = 0.0021), cg10473311 (coeff.int = −0.021, p-value = 0.027). Conclusion In utero arsenic exposure was associated with cord blood DNA methylation at various CpGs. The identified CpGs may help determine pathological epigenetic mechanisms linked to in utero arsenic exposure. Five CpGs (cg25189764, cg04986899, cg04903360, cg08198265 and cg10473311) may serve as epigenetic markers for changes in LDL later in life.

Circadian disruption is a common environmental and occupational exposure with public health consequences, but not much is known about whether circadian disruption affects in utero development. We investigated whether maternal circadian disruption, using night shift work as a proxy, is associated with variations in DNA methylation patterns of placental tissue in an epigenome-wide association study (EWAS) of night shift work. Here, we compared cytosine-guanosine dinucleotide (CpG) specific methylation genome-wide of placental tissue (measured with the Illumina 450K array) from participants (n = 237) in the Rhode Island Child Health Study (RICHS) who did (n = 53) and did not (n = 184) report working the night shift, using robust linear modeling and adjusting for maternal age, pre-pregnancy smoking, infant sex, maternal adversity, and putative cell mixture. Statistical analyses were adjusted for multiple comparisons and results presented with Bonferroni or Benjamini and Hochberg (BH) adjustment for false discovery rate. Night shift work was associated with differential methylation in placental tissue, including CpG sites in the genes NAV1, SMPD1, TAPBP, CLEC16A, DIP2C, FAM172A, and PLEKHG6 (Bonferroni-adjusted p