Background Maternal nutrition in pregnancy has been linked to offspring health in early and later life, with changes to DNA methylation (DNAm) proposed as a mediating mechanism. Objective We investigated intervention-associated DNAm changes in children whose mothers participated in 2 randomized controlled trials of micronutrient supplementation before and during pregnancy, as part of the EMPHASIS (Epigenetic Mechanisms linking Preconceptional nutrition and Health Assessed in India and sub-Saharan Africa) study (ISRCTN14266771). Design We conducted epigenome-wide association studies with blood samples from Indian (n = 698) and Gambian (n = 293) children using the Illumina EPIC array and a targeted study of selected loci not on the array. The Indian micronutrient intervention was food based, whereas the Gambian intervention was a micronutrient tablet. Results We identified 6 differentially methylated CpGs in Gambians [2.5–5.0% reduction in intervention group, all false discovery rate (FDR) <5%], the majority mapping to ESM1, which also represented a strong signal in regional analysis. One CpG passed FDR <5% in the Indian cohort, but overall effect sizes were small (<1%) and did not have the characteristics of a robust signature. We also found strong evidence for enrichment of metastable epialleles among subthreshold signals in the Gambian analysis. This supports the notion that multiple methylation loci are influenced by micronutrient supplementation in the early embryo. Conclusions Maternal preconceptional and pregnancy micronutrient supplementation may alter DNAm in children measured at 7–9 y. Multiple factors, including differences between the nature of the intervention, participants, and settings, are likely to have contributed to the lack of replication in the Indian cohort. Potential links to phenotypic outcomes will be explored in the next stage of the EMPHASIS study.

In humans, DNA methylation marks inherited from gametes are largely erased following fertilisation, prior to construction of the embryonic methylome. Exploiting a natural experiment of seasonal variation including changes in diet and nutritional status in rural Gambia, we analysed three datasets covering two independent child cohorts and identified 259 CpGs showing consistent associations between season of conception (SoC) and DNA methylation. SoC effects were most apparent in early infancy, with evidence of attenuation by mid-childhood. SoC-associated CpGs were enriched for metastable epialleles, parent-of-origin specific methylation and germline DMRs, supporting a periconceptional environmental influence. Many SoC-associated CpGs overlapped enhancers or sites of active transcription in H1 ESCs and fetal tissues. Half were influenced but not determined by measured genetic variants that were independent of SoC. Environmental ‘hotspots’ providing a record of environmental influence at periconception constitute a valuable resource for investigating epigenetic mechanisms linking early exposures to lifelong health and disease.

In humans, DNA methylation marks inherited from sperm and egg are largely erased immediately following conception, prior to construction of the embryonic methylome. Exploiting a natural experiment of cyclical seasonal variation including changes in diet and nutritional status in rural Gambia, we replicated 134 loci with a common season-ofconception methylation signature in two independent child cohorts. These robust candidates for sensitivity to early environment were highly enriched for metastable epialleles, parent-of-origin specific methylation and regions hypomethylated in sperm. They tended to co-locate with endogenous retroviral (ERV1, ERVK) elements. Identified loci were influenced but not determined by measured genetic variation, notably through geneenvironment interactions. To the extent that early methylation changes impact gene expression, environmental sensitivity during genomic remethylation in the very early embryo could thus constitute a sense-record-adapt mechanism linking early environment to later phenotype. RESULTS Association of DNA methylation with Gambian season of conception

Size at birth is known to be influenced by various fetal and maternal factors including genetic effects. South Asians have a high burden of low birthweight and cardiometabolic diseases, yet studies of common genetic variations underpinning these phenotypes are lacking. We generated independent, weighted fetal genetic score (fGS) and maternal genetic score (mGS) from 196 birthweight-associated variants identified in Europeans and conducted association analysis with various fetal birth parameters and anthropometric and cardiometabolic traits measured at different follow-up stages (5-6 years' intervals) from seven Indian and Bangladeshi cohorts of South Asian ancestry. The results from above cohorts were compared with South Asians in UK BioBank and The Exeter Family Study of Childhood Health, a European ancestry cohort. Birthweight increased by 50.7g and 33.6g per standard deviation of fGS (p = 9.1×10-11) and mGS (p = 0.003) respectively in South Asians. A relatively weaker maternal genetic score effect compared to Europeans indicates possible different intrauterine exposures between Europeans and South Asians. Birthweight was strongly associated with body size in both childhood and adolescence (p = 3×10-5 - 1.9×10-51), however, fetal genetic score was associated with body size in childhood only (p < 0.01) and with head circumference, fasting glucose and triglycerides in adults (p < 0.01). The substantially smaller newborn size in South Asians with comparable fetal genetic effect to Europeans on birthweight suggests a significant role of factors related to fetal growth that were not captured by the present genetic scores. These factors may include different environmental exposures, maternal body size, health and nutritional status etc. Persistent influence of genetic loci on size at birth and adult metabolic syndrome in our study supports a common genetic mechanism partly explaining associations between early development and later cardiometabolic health in various populations, despite marked differences in phenotypic and environmental factors in South Asians.