Genome-wide DNA methylation analysis of the liver in adult guinea pigs born preterm and term highlights the importance Cry1 and circadian rhythm in males

Background: Preterm birth is associated with increased lifetime risk of cardiometabolic and neurodevelopmental disease, yet the underlying organ-specific epigenetic mechanisms remain poorly understood. While altered DNA methylation profiles have been reported in accessible tissues such as cord blood and placenta, the long-term epigenetic consequences within metabolically active organs remain largely unexplored Results: We performed DNA methylation profiling (reduced representation bisulphite sequencing) in adult (9‑month) livers from guinea pigs born preterm (gestational age, 62 days) versus term (gestational age 69 days) using an established clinically relevant precocial model. In males we identified 743 significant differentially methylated (5mC) CpGs and 24 significant differentially methylated regions (DMRs) between term born control and preterm born cases. These DMRs were enriched in genes involved in key metabolic and regulatory pathways including circadian rhythm, lipid metabolism, ferroptosis, autophagy, mitochondrial function, vascular signalling, immune regulation, and chromatin modification. Notably, CRY1 emerged as a potential convergent mechanistic node, exhibiting increased DNA methylation and reduced mRNA expression in preterm-born animals, consistent with epigenetic repression of gene regulation. This observation aligns with the established role of CRY1 in circadian regulation and hepatic gluconeogenesis, suggesting disruption of circadian-metabolic integration following pre-term birth. Additional DMRs mapped to genes governing lipid oxidation ( PPARA ), ferroptosis and lipid peroxidation control ( GPX4 ), autophagy/mitochondrial quality control ( UBXN6, VPS13D ), and chromatin repression programs ( KDM2B ), and were enriched for genes linked to metabolic and inflammatory phenotypes associated with later life phenotypes of preterm birth. These observations suggest that durable epigenetic reprogramming of hepatic metabolic networks, potentially increase susceptibility to later-life metabolic disease. Conclusions: Preterm birth in males is associated with persistent, organ-specific DNA methylation alterations in the adult liver that converge across interconnected pathways governing circadian control, lipid homeostasis, mitochondrial quality control, vascular–immune signalling, and chromatin regulation. These findings provide the first evidence of long-term hepatic epigenetic programming following preterm birth and identify CRY1 as a potential mechanistic link between early-life adversity and later-life metabolic disease risk. This work establishes a foundation for future multi-omic and cell-type–resolved studies aimed at identifying targets to mitigate long-term health consequences of preterm birth.