Impact of lactation stage, maternal age, and parity on mammary epithelial cell populations in fresh milk

Human milk contains viable mammary epithelial cells (MECs), providing a unique, non-invasive source of primary breast cells. As these cells originate from the lactating breast, they enable direct insights into mammary epithelial biology in vivo, and on the maternal influence on epithelial composition and function potential. Yet, such influences remain poorly characterized. We analyzed 127 milk samples to investigate how lactation stage, maternal age, and parity affect milk-derived MECs abundance, phenotype, and functional properties. Cells were characterized by flow cytometry, gene expression profiling, and protein quantification at isolation and after 21 days of in vitro culture. The lactation stage significantly influenced milk's cellular composition: total cell counts declined over time, while epithelial cell proportion and expression of lactation-related genes increased. Advanced maternal age was associated with lower prolactin receptor expression, fewer epithelial cells, and reduced proliferative potential in vitro. MECs from multiparous women exhibited a higher luminal-to-basal ratio, supporting the existence of parity-associated epithelial imprinting. Such shifts in epithelial subtypes may contribute to long-term breast remodeling and disease risk. This study provides a comprehensive characterization of milk-derived MECs. It highlights human milk as a valuable model to study lactation, epithelial plasticity, and the cellular contexts relevant to breast cancer risk.