Genomic characterization of normal and aberrant human milk production [bulkRNA-seq]

Breastfeeding is vital for reducing morbidity and mortality, yet exclusive breastfeeding rates are low, with insufficient milk supply being a major weaning factor whose molecular causes remain largely unknown. In this study, we collected fresh milk samples from 30 lactating individuals, classified as low, normal, or high milk producers at multiple postpartum stages, and conducted extensive genomic and microbiome analysis. Using bulk RNA sequencing on human milk fat globules (MFG), milk cells, and breast tissue, we found that MFG-derived RNA closely resembles RNA from milk luminal cells. Furthermore, bulk and single-cell RNA-seq revealed changes in the transcriptome and cellular content linked to milk production. We identified specific genes and cell-type proportions differing in low and high milk production. Infant microbiome diversity was affected by feeding type, but not by maternal milk supply. This study provides a comprehensive human milk transcriptomic catalog, identifies genes associated with milk production, and highlights MFG as a useful biomarker for milk transcriptome analysis. Human milk samples from donors with low, normal, and high milk production were collected fresh and centrifuged at 800g for 20 minutes in 4°C to pellet the cells and separate the fat layer. Fat layer was removed using a sterile spoon and mixed with RLT lysis buffer for RNA extraction using the RNeasy kit (Qiagen) then sequenced with RNA-seq. For select samples, pelleted cells were lysed and sequenced as well.