Background: Supplementing overweight/obese mothers with fish oil (FO) during pregnancy has shown promise in improving infant growth, body composition, and other health outcomes. However, the exact mechanisms by which FO affects infant outcomes remain unclear. We hypothesise that it might be via the extracellular vesicles (EVs) in breast milk (BM). BM EVs are highly stable and have been shown to act as intercellular communicators by transferring the molecular cargo of microRNAs (miRNAs) and lipids to the growing infant. Therefore, we aim to examine the effects of supplementing pregnant mothers who are overweight/obese with FO on their BM EV profiles of miRNAs and lipids.

Methods: Banked BM samples (n=141) collected at 2 weeks (n=81) and 3 months (n=60) postpartum from the FO in Pregnancy Trial (ACTRN12617001078347), a double-blind, randomised controlled trial investigating the effects of FO supplementation on women with overweight/obesity during pregnancy and lactation compared to Olive oil (OO) will be analysed. Small RNA sequencing using the Illumina HiSeq system will quantify EV-miRNAs, and lipidomic analysis will be carried out using chromatographic analysis.

Results: Raw reads will be extracted from FASTQ files, and data will be analysed by mapping the reads to a small RNA transcriptome, followed by differential miRNA expression analysis between the FO and OO EVs. Orthogonal partial least squares discriminant analysis (OPLS-DA) will be employed to analyse differences in the identified lipids in the FO and OO EVs. 

Discussion: The results will provide mechanistic insights into how FO supplementation during pregnancy affects infant health outcomes.