The link between neurodevelopment in infants exposed to maternal gestational diabetes mellitus (GDM) and fetal DNA methylation remains unexplored. We conducted this hypothesis-generating study to investigate the association between fetal DNA methylation and neurodevelopmental outcomes in children of mothers with GDM. We carried out a prospective, observational pilot cohort study comparing infants exposed to maternal GDM with an unexposed control group. Umbilical cord blood DNA methylation was assessed using targeted methylome sequencing covering 3.34 million CpG sites. Infant neurodevelopment was evaluated at age two years using the Bayley-III Scales. Bioinformatics processing identified differentially methylated regions (DMRs), followed by multiple enrichment analyses of DMR-associated genes and partial correlation analyses. Multi-dimensional enrichment analysis of the 1053 identified DMR-associated genes revealed a significant convergence of pathways related to neurogenesis, synaptic components, and axonal guidance. Infants born to mothers with GDM exhibited lower scores in cognitive, language, and motor domains, which were associated with identifiable DNA methylation signatures at birth. Significant correlations were observed in genes essential for brain scaffolding and synaptic circuitry, most notably WNT4, the PCDHG alpha/beta clusters, and PALM. Additionally, methylation patterns in FOXF2 and CHFR suggest a potential impact on blood–brain barrier integrity, while associations with FSTL3 and H6PD highlight a systemic metabolic ‘cross-talk’ influencing neurodevelopment. Although these pilot findings are hypothesis-generating and require further functional validation, this study provides pioneering evidence that neurodevelopmental alterations in the offspring of mothers with GDM are potentially associated with intrauterine epigenetic modifications detectable at birth.

Rising reported Autism Spectrum Disorder (ASD) prevalence underscores the need to explore early perinatal biological markers. To conduct an exploratory pilot investigation of cord blood DNA methylation patterns as potential early epigenetic candidate loci associated with ASD in infants born to mothers with obesity and gestational diabetes. This prospective pilot study analyzed 14 mother-infant pairs comprising infants born to mothers with obesity and gestational diabetes later diagnosed with ASD (n = 2); healthy infants born to mothers with obesity and diabetes (IODM, n = 6); and healthy controls (IHM, n = 6). Targeted methyl-capture sequencing (covering 3.34 million CpG sites) identified differentially methylated region (DMR)-associated genes. Neurodevelopment was evaluated at 24–26 months using Bayley-III scales. ASD infants showed lower Bayley-III scores across all domains. In this small cohort (n = 2 ASD cases), we identified 249 DMR-associated genes common to both ASD vs. IHM and ASD vs. IODM comparisons, representing preliminary candidate loci. Enrichment analysis identified homophilic cell–cell adhesion as the most significant pathway (fold enrichment = 8.28; FDR = 6.31 × 10⁻⁶), driven by the PCDH1–PCDHA8 cluster, alongside morphogenesis in neuron differentiation (fold enrichment = 3.45; FDR = 2.08 × 10⁻²). Although prospective validation in larger independent cohorts is required, these pilot results suggest that cord blood PCDHA1–PCDHA8 cluster methylation alterations represent preliminary candidate loci for hypothesis generation and future biomarker evaluation, regardless of maternal metabolic status.