Human milk contains a heterogeneous population of EVs and microRNAs that resist simulated digestion

Abstract Mother’s milk is known for its crucial roles in infant health and development. Probably the most commonly studied effects of milk are those on an infant’s intestinal barrier, metabolism, and immunity. While these functions of milk were mostly attributed to its protein and fat content, recent evidence points to a potential role of milk microRNAs in these processes. MicroRNAs are small non-coding RNAs that can fine-tune gene expression at the post-transcriptional level. Human milk (HM) is rich in microRNAs, which are mainly found associated with milk extracellular vesicles (EVs). HM microRNAs are proposed to transfer from mother to infant via breastfeeding and execute gene regulatory functions in infant cells. For microRNAs to be able to act as “genetic programmers” rather than mere nutritional molecules, they should resist digestion in the infant’s gastrointestinal tract. Milk EVs are believed to protect microRNAs against degradation and facilitate their delivery to the cells. Here, we used two lots of pasteurized HM that were originally destined for human milk banks. We showed that HM contains different populations of EVs with different physicochemical properties, similar to those previously identified in commercial bovine milk. We also showed that these EVs, which are often discarded, contain the majority of HM microRNAs. Finally, we showed that three highly abundant milk microRNAs resisted differentially to infant’s simulated digestion conditions, with a relatively small number of microRNAs surviving a two-hour digestion. Milk microRNA copy numbers surviving digestion may be too low to influence gene expression in infant cells. Highlights Pasteurized HM contains heterogeneous populations of EVs. These EVs associate with the majority of HM microRNAs. Different microRNAs show varying stability during infant digestion. The copy number of milk miR-148a-3p surviving digestion might be too low to influence gene expression in infant cells.