Background Prenatal alcohol exposure (PAE) is associated with a range of adverse offspring neurodevelopmental outcomes. Several studies suggest that PAE modifies DNA methylation in offspring cells and tissues, providing evidence for a potential mechanistic link to Fetal Alcohol Spectrum Disorder (FASD). We systematically reviewed existing evidence on the extent to which maternal alcohol use during pregnancy is associated with offspring DNA methylation. Methods A systematic literature search was conducted across five online databases according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed, Web of Science, EMBASE, Google Scholar and CINAHL Databases were searched for articles relating to PAE in placental mammals. Data were extracted from each study and the Risk of Bias in Non-Randomized Studies of Interventions (ROBINS-I) was used to assess the potential for bias in human studies. Results Forty-three articles were identified for inclusion. Twenty-six animal studies and 16 human studies measured offspring DNA methylation in various tissues using candidate gene analysis, methylome-wide association studies (MWAS), or total nuclear DNA methylation content. PAE dose and timing varied between studies. Risk of bias was deemed high in nearly all human studies. There was insufficient evidence in human and animal studies to support global disruption of DNA methylation from PAE. Inconclusive evidence was found for hypomethylation at IGF2/H19 regions within somatic tissues. MWAS assessing PAE effects on offspring DNA methylation showed inconsistent evidence. There was some consistency in the relatively small number of MWAS conducted in populations with FASD. Meta-analyses could not be conducted due to significant heterogeneity between studies. Conclusion Considering heterogeneity in study design and potential for bias, evidence for an association between PAE and offspring DNA methylation was inconclusive. Some reproducible associations were observed in populations with FASD although the limited number of these studies warrants further research. Trail Registration: This review is registered with PROSPERO (registration number: CRD42020167686).

Prenatal alcohol exposure (PAE) is associated with changes to offspring DNA methylation and adverse neurodevelopmental outcomes. Prior studies have utilised candidate gene or microarray approaches resulting in biased representation of the methylome. We employed an established experimental murine model of moderate PAE until gestational days 8-10, with genome-wide DNA methylation sequencing of neonatal brain and liver. Although global DNA methylation levels were generally preserved, we detected 78 alcohol-sensitive regions in neonatal brain, and 759 alcohol-sensitive regions in neonatal liver (p < 0.05 and delta beta > 0.05). Affected coding regions were significantly enriched in genes involved in neurodevelopmental pathways, but behavioural outcomes were unaltered in adult littermates. Seven alcohol-sensitive regions identified in mice were statistically replicated in a human cohort of adolescents with fetal alcohol spectrum disorder (FASD) and known to be linked with traits related to facial morphology, intelligence, educational attainment, autism, schizophrenia, ageing and haematological disorders in the GWAS catalogue. The high methyl donor diet (HMD) across pregnancy partially protected against the effects of alcohol on DNA methylation in offspring tissues. Collectively this study provides a comprehensive evaluation of PAE on offspring DNA methylation and suggests prenatal dietary intervention may be a warranted mitigation strategy.