(P23-09-26) Gene Expression in the Mammary Gland of Lactating Mothers With Low Milk Production Versus High Milk Production

Objectives: Insights into molecular mechanisms of human milk production are limited. We compared milk fat–derived mammary epithelial cell transcriptomes of mothers with low vs high milk production using a validated measure of milk production rate (MPR).

Methods: MPR was assessed at weeks 2 and 6 postpartum in MOM2Child subcohort mothers with exclusive breastfeeding intention. MPR (g/h) is defined as milk yield at h2 following complete breast expression at h 0, 1 and 2. RNA sequencing was performed on h2 milk fat layer at week 2. Reads were mapped to genes with Kallisto. Genes with a median count < 1 were removed. Differentially expressed genes in LOW (MPR < 35 g/h, clinical cutoff, n=25) vs HIGH (MPR ≥ 50 g/h, study median, n=22) MPR groups were identified using DESeq2 (|log2 fold change| ≥ 0.3; FDR ≤ 0.05). Gene set enrichment was performed on ranked genes with fgsea package. Upstream regulators were identified by Ingenuity Pathway Analysis and transcription factor enrichment analysis on ENCODE/ChEA datasets.

Results: The LOW group had 95 up- and 266 down-regulated genes vs HIGH. Upregulated genes, CDK1, MKI67, ORC1, and PLK1 were enriched in cell cycle related pathways: E2F target, G2M checkpoint, and MYC targets. Predicted upstream regulators of these pathways include activators (FOXM1, ERα, and E2F) which promote DNA replication and mitotic spindle; and inhibitors were cell cycle-suppressors (p53, p21, and TGF-β1). Cell-cycle signal in LOW was driven by a subset (n=8), all of whom had MPR >35 g/h by week 6. Downregulated genes, TNFRSF1B, NFKB1, NFKB2, NFKBIA, and CXCL8 were enriched in TNF-alpha signaling via NF-κB and inflammatory response, which was predicted to be regulated by RELA inhibition.

Conclusions: Cell proliferation-enriched subset with low milk production at week 2 may reflect a prolonged secretory activation stage of lactation. The appearance of multiple subsets reveals mixed causes of low milk production. Downregulated gene patterns suggest suppressed recruitment of immune cells to the mammary epithelium in low milk producers; the role of immune cells in lactation remains to be elucidated. Multi-omics integration to reveal protein level functional changes, regulatory mechanism, and cellular heterogeneity will further improve our understanding of low milk production.

Funding Sources: NICHD, NIAID, ISRHML