Maternal Sedentary Behavior and Breast Milk Extracellular Vesicle MicroRNA Across Pregnancy: A Pilot Study

Background/Objectives: Human breast milk (HM) is the gold-standard nutritional source for infants, delivering not only macro- and micronutrients but also bioactive components, including extracellular vesicles (EVs) enriched with microRNAs (miRNAs) that regulate immune, metabolic, and neurodevelopmental programming. Maternal lifestyle, including nutrition, is known to shape milk composition, yet it remains unknown whether maternal physical activity modulates the bioactive miRNA cargo of HM EVs. Methods: This pilot study examined associations between moderate-to-vigorous physical activity (MVPA; ≥3.0 metabolic equivalents) and prolonged sedentary behavior (SED60+; total sitting/lying minutes of bouts ≥60 min) with EV-enriched miRNA expression in HM collected in the early postnatal period (n = 11). Maternal activity was measured using a thigh-worn activPAL monitor across pregnancy trimesters and days 10–18 postnatal; HM was collected at day 18 postnatal. The EV-enriched fraction from skim milk was isolated and small RNA sequencing identified 691 unique miRNAs. Results: Principal component analysis showed that EV-enriched miRNA profiles were nominally most associated with prolonged sedentary behavior during the first two trimesters, though these associations did not survive correction for multiple testing. Differential expression analysis identified nine sedentary-associated miRNAs in trimester two, with smaller signals in trimesters one and three. Pathway enrichment revealed overrepresentation of genes involved in RNA regulation and post-transcriptional control, cell adhesion and cytoskeletal organization, and growth and nutrient signaling. Conclusions: Together, these findings suggest that maternal sedentary behavior during early and mid-pregnancy may be associated with candidate variation in HM EV-miRNA composition, offering a preliminary molecular link between prenatal behaviors and early-life programming that warrants replication in larger, independent cohorts.