RNA sequencing of the human milk fat layer transcriptome reveals distinct gene expression profiles at three stages of lactation.

Danielle G Lemay,Olivia A Ballard,Nelson D Horseman,Maria A Hughes,Laurie A Nommsen-Rivers,Ardythe L Morrow,Kartik Shankar

doi:10.1371/journal.pone.0067531

Abstract

Aware of the important benefits of human milk, most U.S. women initiate breastfeeding but difficulties with milk supply lead some to quit earlier than intended. Yet, the contribution of maternal physiology to lactation difficulties remains poorly understood. Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA-sequencing to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. The resulting transcriptomes paint an exquisite portrait of human lactation. The resulting transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during similar postpartum time frames could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (105-fold) in transcript abundances not previously observed with microarray technology. We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding β-casein (CSN2) and α-lactalbumin (LALBA), make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, protein tyrosine phosphatase, receptor type, F (PTPRF) may serve as a biomarker linking insulin resistance with insufficient milk supply. This study provides the methodology and reference data set to enable future targeted research on the physiological contributors of sub-optimal lactation in humans.

Highlights

Breastfeeding provides numerous benefits for both mother and infant [1,2,3]
Factors Affecting Milk Fat Layer RNA Quality Because the milk fat layer is an unconventional extracellular source of mRNA and its membrane is more fragile than cell membranes [16], we examined the vulnerability of this RNA to degradation (Table S1)
Non-invasive sampling of the transcriptome of milk-producing cells via RNA secreted into human milk provides a powerful window into the biology of human lactation

Summary

Introduction

Breastfeeding provides numerous benefits for both mother and infant [1,2,3]. The U.S Surgeon General [4], Institute of Medicine [5], and American Academy of Pediatrics [6] all recommend that infants be exclusively breastfed for the first 6 months with continued breastfeeding for at least 1 year. There is an intriguing workaround–human milk secreted during lactation is a rich source of mammary epithelial cell RNA. About 3–8% of human milk fat globules contain mammary epithelial cell cytoplasmic remnants, including RNA, captured during milk fat globule formation and secretion [9]. Maningat et al demonstrated that the microarray-generated human milk fat layer transcriptome includes genes uniquely expressed in the lactating mammary epithelial cell [10]. Their ground-breaking work established the human milk fat layer as a potential window into mammary epithelial cell gene expression during lactation without invasive tissue biopsy

Methods

Results

Conclusion