Epigenetic age acceleration in follicular fluid and markers of ovarian response among women undergoing IVF.

STUDY QUESTION: Are markers of epigenetic age acceleration in follicular fluid associated with outcomes of ovarian stimulation? SUMMARY ANSWER: Increased epigenetic age acceleration of follicular fluid using the Horvath clock, but not other epigenetic clocks (GrimAge and Granulosa Cell), was associated with lower peak estradiol levels and decreased number of total and mature oocytes. WHAT IS KNOWN ALREADY: In granulosa cells, there are inconsistent findings between epigenetic age acceleration and ovarian response outcomes. STUDY DESIGN, SIZE, DURATION: Our study included 61 women undergoing IVF at an academic fertility clinic in the New England area who were part of the Environment and Reproductive Health Study (2006-2016). PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants provided a follicular fluid sample during oocyte retrieval. DNA methylation of follicular fluid was assessed using a genome-wide methylation screening tool. Three established epigenetic clocks (Horvath, GrimAge, and Granulosa Cell) were used to predict DNA-methylation-based epigenetic age. To calculate the age acceleration, we regressed epigenetic age on chronological age and extracted the residuals. The association between epigenetic age acceleration and ovarian response outcomes (peak estradiol levels, follicle stimulation hormone, number of total, and mature oocytes) was assessed using linear and Poisson regression adjusted for chronological age, three surrogate variables (to account for cellular heterogeneity), race, smoking status, initial infertility diagnosis, and stimulation protocol. MAIN RESULTS AND ROLE OF CHANCE: Compared to the median chronological age of our participants (34 years), the Horvath clock predicted, on an average, a younger epigenetic age (median: 24.2 years) while the GrimAge (median: 38.6 years) and Granulosa Cell (median: 39.0 years) clocks predicted, on an average, an older epigenetic age. Age acceleration based on the Horvath clock was associated with lower peak estradiol levels (-819.4 unit decrease in peak estradiol levels per standard deviation increase; 95% CI: -1265.7, -373.1) and fewer total (% change in total oocytes retrieved per standard deviation increase: -21.8%; 95% CI: -37.1%, -2.8%) and mature oocytes retrieved (% change in mature oocytes retrieved per standard deviation increase: -23.8%; 95% CI: -39.9%, -3.4%). The age acceleration based on the two other epigenetic clocks was not associated with markers of ovarian response. LIMITATIONS, REASONS FOR CAUTION: Our sample size was small and we did not specifically isolate granulosa cells from follicular fluid samples so our samples could have included mixed cell types. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight that certain epigenetic clocks may be predictive of ovarian stimulation outcomes when applied to follicular fluid; however, the inconsistent findings for specific clocks across studies indicate a need for further research to better understand the clinical utility of epigenetic clocks to improve IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by grants ES009718, ES022955, ES000002, and ES026648 from the National Institute of Environmental Health Sciences (NIEHS) and a pilot grant from the NIEHS-funded HERCULES Center at Emory University (P30 ES019776). RBH was supported by the Emory University NIH Training Grant (T32-ES012870). TRIAL REGISTRATION NUMBER: N/A.

Placental PFAS concentrations are associated with perturbations of placental DNA methylation at loci with important roles on cardiometabolic health.

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

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.

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.

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).

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.

Former smoking associated with epigenetic modifications in human granulosa cells among women undergoing assisted reproduction.

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

Agreement Between Two Behavioral Rating Scales in Preschoolers Born Very Preterm.

OBJECTIVE: Broadband parent rating scales are commonly used to assess behavioral problems in children. Multiple rating scales are available, yet agreement between them is not well-understood. The objective of this study was to evaluate agreement between the Behavior Assessment System for Children, Third Edition (BASC-3), and Child Behavior Checklist 1.5 to 5 years (CBCL) in a sample of children born very preterm. METHOD: We assessed 73 children born < 30 weeks' gestational age whose caregivers completed the BASC-3 and CBCL at age 4. We examined correlations, within-person differences, and agreement in clinical categorization for all corresponding subscales and composites. RESULTS: Comparable subscales on the BASC-3 and CBCL were significantly correlated, albeit to differing magnitudes. Subscales indexing hyperactivity and attention problems were the most comparable across the 2 measures, evidenced by strong correlations and few to no differences in mean T-scores. Composite scores indexing internalizing, externalizing, and total problems were also strongly correlated, and there were no differences in the mean T-scores for externalizing or total problems across measures. Agreement in clinical classifications were weak to moderate, though again, the highest agreement was found for hyperactivity, attention, externalizing, and total problems. CONCLUSION: Agreement between BASC-3 and CBCL subscales was weak to moderate, with the exception of subscales related to attention and hyperactivity, as well as composite scores indicating overall behavior problems. Researchers and clinicians should consider these discrepancies when interpreting the results of behavior rating scales with preschool children because conclusions could differ based on the assessment that is used.

Epigenetic age acceleration, neonatal morbidities, and neurobehavioral profiles in infants born very preterm.

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.

Evaluation of pediatric epigenetic clocks across multiple tissues.

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.

Developing a National-Scale Exposure Index for Combined Environmental Hazards and Social Stressors and Applications to the Environmental Influences on Child Health Outcomes (ECHO) Cohort.

Tools for assessing multiple exposures across several domains (e.g., physical, chemical, and social) are of growing importance in social and environmental epidemiology because of their value in uncovering disparities and their impact on health outcomes. Here we describe work done within the Environmental influences on Child Health Outcomes (ECHO)-wide Cohort Study to build a combined exposure index. Our index considered both environmental hazards and social stressors simultaneously with national coverage for a 10-year period. Our goal was to build this index and demonstrate its utility for assessing differences in exposure for pregnancies enrolled in the ECHO-wide Cohort Study. Our unitless combined exposure index, which collapses census-tract level data into a single relative measure of exposure ranging from 0-1 (where higher values indicate higher exposure to hazards), includes indicators for major air pollutants and air toxics, features of the built environment, traffic exposures, and social determinants of health (e.g., lower educational attainment) drawn from existing data sources. We observed temporal and geographic variations in index values, with exposures being highest among participants living in the West and Northeast regions. Pregnant people who identified as Black or Hispanic (of any race) were at higher risk of living in a "high" exposure census tract (defined as an index value above 0.5) relative to those who identified as White or non-Hispanic. Index values were also higher for pregnant people with lower educational attainment. Several recommendations follow from our work, including that environmental and social stressor datasets with higher spatial and temporal resolutions are needed to ensure index-based tools fully capture the total environmental context.

Traffic-related air pollution and supplemental folic acid intake in relation to DNA methylation in granulosa cells.

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 < 0.1. RESULTS: There were no associations between NO2 or supplemental folic acid intake and epigenetic age acceleration of GC. NO2 and supplemental folic acid were associated with 9 and 11 differentially methylated CpG sites. Among these CpGs, only cg07287107 exhibited a significant interaction (p-value = 0.037). In women with low supplemental folic acid, high NO2 exposure was associated with 1.7% higher DNAm. There was no association between NO2 and DNAm in women with high supplemental folic acid. The genes annotated to the top 250 NO2-associated CpGs were enriched for carbohydrate and protein metabolism, postsynaptic potential and dendrite development, and membrane components and exocytosis. The genes annotated to the top 250 supplemental folic acid-associated CpGs were enriched for estrous cycle, learning, cognition, synaptic organization and transmission, and size and composition of neuronal cell bodies. CONCLUSIONS: We found no associations between NO2, supplemental folic acid, and DNAm age acceleration of GC. However, there were 20 differentially methylated CpGs and multiple enriched GO terms associated with both exposures suggesting that differences in GC DNAm could be a plausible mechanism underlying the effects of TRAP and supplemental folic acid on ovarian function.

Prenatal and perinatal factors associated with neonatal neurobehavioral profiles in the ECHO Program.

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.

Acoustic Cry Characteristics in Preterm Infants and Developmental and Behavioral Outcomes at 2 Years of Age.

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.

Sex-based differences in placental DNA methylation profiles related to gestational age: an NIH ECHO meta-analysis.

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.

Umbilical cord blood immune cell profiles in relation to the infant gut microbiome.

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.

Epigenetic differences in stress response gene FKBP5 among children with abusive vs accidental injuries.

BACKGROUND: Survivors of child abuse experience high rates of adverse physical and mental health outcomes. Epigenetic alterations in the stress response system, the FKBP5 gene specifically, have been implicated as one mechanism that may link abuse to lifelong health issues. Prior studies primarily included older individuals with a remote history of maltreatment; our objective was to test for differential methylation of FKBP5 in children with abusive vs accidental injuries at the time of diagnosis. METHODS: We conducted a cross-sectional pilot study of acutely injured children <4 years old at two children's hospitals (n = 82). Research personnel collected injury histories, buccal swabs (n = 65), and blood samples (n = 25) to measure DNA methylation. An expert panel classified the injuries as abusive, accidental, or indeterminate. RESULTS: Children with abusive as compared to accidental injuries had lower methylation of the FKBP5 promoter in buccal and blood cells, even after controlling for injury severity, socioeconomic status, and psychosocial risk factors. CONCLUSION: These findings suggest that epigenetic variation in FKBP5 may occur at the earliest indication of abuse and may be associated with delayed resolution of the HPA axis stress response. Additional testing for epigenetic differences in larger sample sizes is needed to further verify these findings. IMPACT: Children (<4 years old) with abusive compared to accidental injuries showed lower methylation of the FKBP5 promoter in buccal and blood cells at the time of initial diagnosis even after controlling for injury severity, socioeconomic status, and psychosocial risk factors. Early childhood physical abuse may impact the epigenetic regulation of the stress response system, including demethylation within promoters and enhancers of the FKBP5 gene, even at the earliest indication of abuse. The findings are important because unmitigated stress is associated with adverse health outcomes throughout the life-course.

Variation in placental microRNA expression associates with maternal family history of cardiovascular disease.

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 TGFß 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.

Epigenetics of type 2 diabetes and diabetes-related outcomes in the Strong Heart Study.

BACKGROUND: The prevalence of type 2 diabetes has dramatically increased in the past years. Increasing evidence supports that blood DNA methylation, the best studied epigenetic mark, is related to diabetes risk. Few prospective studies, however, are available. We studied the association of blood DNA methylation with diabetes in the Strong Heart Study. We used limma, Iterative Sure Independence Screening and Cox regression to study the association of blood DNA methylation with fasting glucose, HOMA-IR and incident type 2 diabetes among 1312 American Indians from the Strong Heart Study. DNA methylation was measured using Illumina's MethylationEPIC beadchip. We also assessed the biological relevance of our findings using bioinformatics analyses. RESULTS: Among the 358 differentially methylated positions (DMPs) that were cross-sectionally associated either with fasting glucose or HOMA-IR, 49 were prospectively associated with incident type 2 diabetes, although no DMPs remained significant after multiple comparisons correction. Multiple of the top DMPs were annotated to genes with relevant functions for diabetes including SREBF1, associated with obesity, type 2 diabetes and insulin sensitivity; ABCG1, involved in cholesterol and phospholipids transport; and HDAC1, of the HDAC family. (HDAC inhibitors have been proposed as an emerging treatment for diabetes and its complications.) CONCLUSIONS: Our results suggest that differences in peripheral blood DNA methylation are related to cross-sectional markers of glucose metabolism and insulin activity. While some of these DMPs were modestly associated with prospective incident type 2 diabetes, they did not survive multiple testing. Common DMPs with diabetes epigenome-wide association studies from other populations suggest a partially common epigenomic signature of glucose and insulin activity.