Human Breast Milk miRNA, Maternal Probiotic Supplementation and Atopic Dermatitis in Offspring

Abstract

Background: Perinatal probiotic ingestion has been shown to prevent atopic dermatitis (AD) in infancy in a number of randomised trials. The Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT) trial involved a probiotic supplementation regime given solely to mothers in the perinatal period and demonstrated a ~40% relative risk reduction in the cumulative incidence of AD at 2 years of age. However, the mechanisms behind this effect are incompletely understood. Micro-RNAs (miRNA) are abundant in mammalian milk and may influence the developing gastrointestinal and immune systems of newborn infants. The objectives of this study were to describe the miRNA profile of human breast milk, and to investigate breast milk miRNAs as possible mediators of the observed preventative effect of probiotics.

Methods: Small RNA sequencing was conducted on samples collected 3 months postpartum from 54 women participating in the ProPACT trial. Differential expression of miRNA was assessed for the probiotic vs placebo and AD vs non-AD groups. The results were further analysed using functional prediction techniques.

Results: Human breast milk samples contain a relatively stable core group of highly expressed miRNAs, including miR-148a-3p, miR-22-3p, miR-30d-5p, let-7b-5p and miR-200a-3p. Functional analysis of these miRNAs revealed enrichment in a broad range of biological processes and molecular functions. Although several miRNAs were found to be differentially expressed on comparison of the probiotic vs placebo and AD vs non-AD groups, none had an acceptable false discovery rate and their biological significance in the development of AD is not immediately apparent from their predicted functional consequences.

Conclusion: Whilst breast milk miRNAs have the potential to be active in a diverse range of tissues and biological process, individual miRNAs in breast milk 3 months postpartum are unlikely to play a major role in the prevention of atopic dermatitis in infancy by probiotics ingestion in the perinatal period.

Trial registration: ClinicalTrials.gov NCT00159523.

Fig 1. Patient flow and sample selection for ProPACT trial and miRNA sequencing project.

^aMissing 1 or more of the following biological samples collected at 3 months post-partum: breast milk and stool samples from mother, blood and stool samples from the infant.

Fig 2. Overview of RNA profile and relative abundance of highly expressed miRNAs from breast milk samples.

(A) Bioanalyzer 6000 Nano gel from a representative sample showing abundant short RNAs up to 1000nt; (B) closer review of small RNAs using Agilent’s Small RNA kit demonstrating peaks at 22-23nt, 29-30nt, 33-34nt, 53-54nt, 90nt and approximately 140nt and 170nt; (C) bar graph demonstrating average percentage of small RNA sequences aligned to different RNA species with 95% confidence intervals; (D) proportion of reads accounted for by the top 5, 10, 20 and 125 miRNAs along with the number of predicted target genes (excluding repeated target prediction for alternate transcripts of the same gene); (E) boxplot of counts per million (CPM) mature miRNA of the 20 most abundant miRNAs in the 54 samples.