Background/Objectives: Prematurity is a significant global health concern, often associated with neurodevelopmental challenges. Solute Carrier Family 6 Member 4 (SLC6A4), the gene encoding the serotonin transporter, a key component in serotonin reuptake in the synaptic cleft, plays a key role in stress response and neurodevelopment. Epigenetic regulation of stress-related genes, such as SLC6A4, influences neonatal stress adaptation and developmental outcomes. This study aimed to quantify and compare DNA methylation levels at 13 CpG sites in the promoter region of the SLC6A4 gene between preterm and term neonates at three time points. Methods: A cohort of 46 preterm infants and a cohort of 49 full-term infants were analyzed. Blood samples collected at birth (D0), the fifth day (D5), and the thirtieth day (D30) were used to analyze DNA methylation, using bisulfite conversion and pyrosequencing. Results: Significant differences in SLC6A4 methylation were observed. At D0, CpGs 12 and 13 showed higher methylation in preterm infants. CpG 9 showed lower methylation in preterm infants at D5. Extremely preterm infants had the highest values of methylation at the three time points. Longitudinal mixed-effects analysis revealed distinct temporal patterns between groups. Total and site-specific methylation at CpGs 2, 8, and 9 increased over time in full-term infants, while methylation remained stable over time in very preterm and extremely preterm infants. Conclusions: This study reveals significant differences in SLC6A4 methylation between very preterm, extremely preterm, and full-term infants, highlighting the impact of prematurity and early-life stress on the epigenome. These findings contribute to improving our understanding of the epigenetic mechanisms shaping neurodevelopment and stress adaptation in neonates.

Aim: Promoter methylation of LINE-1 may be affected by prematurity, but there is little evidence in the literature.Materials & methods: Blood from premature and full-term neonates on days 0, 5, 30 and 90 was analyzed for DNA methylation percentage in a promoter region of the LINE-1, after bisulfite conversion and pyrosequencing.Results: Premature infants, as a whole, showed significantly lower methylation percentage at birth, but this difference diminished over time. However, the subgroup of extremely premature (<28 weeks gestational age) had higher methylation percentages, similar to full-term newborns.Conclusion: This research underscores the critical role of prematurity on the methylation pattern of LINE-1. These findings underline the complexity of epigenetic regulation in prematurity and emphasize the need for further studies.

Background/Objectives: Kangaroo Mother Care (KMC) has been proposed as a protective intervention that may modulate the epigenetic regulation of stress-related genes, such as SLC6A4, which encodes the serotonin transporter. Few studies have explored this association in humans. This study aimed to evaluate whether KMC affects the methylation status of SLC6A4 in preterm newborns. Methods: This longitudinal observational study included preterm infants with birth weight ≤ 1800 g and gestational age between 25 and 34 weeks. Blood samples were collected at birth, Neonatal Intensive Care Unit discharge, and hospital discharge. Methylation levels at 13 CpG sites within the SLC6A4 promoter region were quantified by bisulfite conversion and pyrosequencing. Methylation dynamics were analyzed using linear mixed-effects models adjusted for clinical covariates. Results: 75 preterm infants were analyzed (51 KMC; 24 non-KMC). Methylation levels ranged from 0.78% to 10.76% across all CpG sites and remained stable over time. At hospital discharge, the KMC group exhibited lower methylation at CpG6 than the non-KMC group (median = 0.96% vs. 1.21%, p = 0.021), but this difference was not statistically significant after correction for multiple testing. No significant differences were observed at other sites or in longitudinal methylation trajectories between groups. Conclusions: KMC was not associated with major longitudinal changes in SLC6A4 methylation during the neonatal period. The nominal difference at CpG6 should be interpreted as exploratory and warrants further investigation. Larger, multicenter studies with long-term follow-up are needed to clarify the epigenetic mechanisms linking early caregiving experiences with stress regulation and neurodevelopmental outcomes in preterm infants.