Breast Milk Extracellular Vesicle miRNAs from a Population-Based Cohort in the Faroe Islands

Human breast milk (BM) plays a critical role in infant development and health, particularly in cognitive, immune, and cardiometabolic functions. BM contains extracellular vesicles (EVs) that can transport biologically relevant cargo from mother to infant, including microRNAs (miRNAs). However, to date, most studies on BMEVs have been limited in sample size. We aimed to investigate BMEV-miRNA profiles in a human population cohort, characterize BMEV-miRNAs patterns, and assess potential pathways and ontology. We thus conducted the first study to describe the EV miRNA profile of BM in 364 mothers from a population-based mother-infant cohort in the Faroe Islands using small RNA sequencing. We detected 1,523 miRNAs with = one read in 70% of samples, and 447 miRNAs with = one read in 100% of samples. Using hierarchical clustering, we determined five BMEV-miRNA clusters, the top three consisting of 15, 27 and 67 miRNAs. Correlation coefficients indicated that the expression of many miRNAs within the top three clusters was highly correlated. Top-cluster BMEV-miRNAs were involved in pathways enriched for the endocrine system, cellular community, neurodevelopment, and cancers. Future studies investigating determinants of BMEV-miRNAs and associated health outcomes are needed to elucidate the role of BMEV-miRNAs in health and disease. Overall design: We leveraged 364 breast milk samples collected at two weeks postpartum from the Faroe Islands Birth Cohort, which recruited women between 1997 and 2000 from the National Hospital in Torshavn, Faroe Islands. Samples were centrifuged at 200g for 10 mins to remove fat globules, then 3,000g for 15 mins to pellet cellular debris. Extracellular vesicle (EV) RNA was isolated with the ExoEasy Maxi Kit and the miRNeasy RNA Isolation Kit (Qiagen) according to manufacturer instructions. EV-miRNA libraries were prepared with the HTG Molecular Whole Transcriptome miRNA Assay (HTG Molecular, Tucson, AZ), which profiles 2,083 mature miRNAs via a targeted library prep method, and sequenced in four lanes on a HiSeq4000 (Illumina) with 50 nucleotide single-end reads. Three replicate EV-miRNA samples and three human brain positive control samples were included in each sequencing run.