SLC6A4 Gene Methylation in Premature Infants Undergoing Kangaroo Mother Care: A Prospective Longitudinal Study

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.