Human Milk-Derived Levels of let-7g-5p May Serve as a Diagnostic and Prognostic Marker of Low Milk Supply in Breastfeeding Women

Abstract

Low milk supply (LMS) is associated with early breastfeeding cessation; however, the biological underpinnings in the mammary gland are not understood. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally downregulate gene expression, and we hypothesized the profile of miRNAs secreted into milk reflects lactation performance. Longitudinal changes in milk miRNAs were measured using RNAseq in women with LMS (n = 47) and adequate milk supply (AMS; n = 123). Relationships between milk miRNAs, milk supply, breastfeeding outcomes, and infant weight gain were assessed, and interactions between milk miRNAs, maternal diet, smoking status, and BMI were determined. Women with LMS had lower milk volume (p = 0.003), were more likely to have ceased breast feeding by 24 wks (p = 0.0003) and had infants with a lower mean weight-for-length z-score (p = 0.013). Milk production was significantly associated with milk levels of miR-16-5p (R = -0.14, adj p = 0.044), miR-22-3p (R = 0.13, adj p = 0.044), and let-7g-5p (R = 0.12, adj p = 0.046). Early milk levels of let-7g-5p were significantly higher in mothers with LMS (adj p = 0.0025), displayed an interaction between lactation stage and milk supply (p < 0.001), and were negatively related to fruit intake (p = 0.015). Putative targets of let-7g-5p include genes important to hormone signaling, RNA regulation, ion transport, and the extracellular matrix, and down-regulation of two targets (PRLR and IGF2BP1/IMP1) was confirmed in mammary cells overexpressing let-7g-5p in vitro. Our data provide evidence that milk-derived miRNAs reflect lactation performance in women and warrant further investigation to assess their utility for predicting LMS risk and early breastfeeding cessation.

Keywords: breastfeeding; human milk; lactation; low milk supply; miRNAs.

Figure 1

CONSORT Diagram. Research staff screened 2487 mother–infant dyads, approached 359 eligible dyads, and obtained consent from 221 dyads. There were 180 mothers that provided at least one milk sample and completed sufficient longitudinal surveys to determine whether they experienced low milk supply (LMS) or adequate milk supply (AMS). There were 4 or 6 mothers excluded from each group for excessive infant weight gain or weight loss (defined as a change in weight-for-length (WfL) Z-score > 2.0), which suggested milk production may have been over- or under-estimated. This left 47 mothers with LMS, and 123 mothers with AMS. Of the 123 mothers with AMS, 48 introduced formula into their infant’s diet prior to 12 months for reasons other than concerns about milk supply.

Figure 2

Levels of miRNAs in breast milk differ among women with low milk supply. (A) Results of a two-dimensional partial least squares discriminant analysis (PLSDA) employing levels of 30 miRNAs with the most robust concentrations in maternal breast milk in the first week after delivery. Note that total milk miRNA profiles do not differ between mothers with adequate milk supply (AMS; green) and mothers with low milk supply (LMS; red). However, milk levels of five miRNAs did differ in the milk of mothers with LMS. In the first week after delivery, levels of (B) let-7a-5p (V = 2194, p = 0.015, adj p = 0.090), (C) let-7g-5p (V = 1765, p = 8.5 × 10⁻⁵, adj p = 0.0025), and (D) miR-22-3p (V = 2135, p = 0.0080, adj p = 0.080) were higher in the milk of mothers with LMS, whereas levels of (E) miR-16-5p (V = 0.3595, p = 0.013 adj p = 0.090) and (F) miR-151a-3p (V = 3670, p = 0.0063, adj p = 0.080) were lower.

Figure 3

Milk levels of let-7g-5p differ over time among mothers with low milk supply and are related to maternal fruit intake. The effects plot displays normalized concentrations of let-7g-5p in 453 samples from 170 lactating mothers across three time points: 1 wk, 4 wks, and 16 wks after delivery (A). Mean concentrations are shown for mothers with low milk supply (LMS, green), and mothers with adequate milk supply (AMS, blue). There was a significant interaction effect (p < 0.001) between LMS/AMS group and time (F = 7.5), with let-7g-5p levels increasing between 1 wk and 4 wks post-delivery in the AMS group only. (B) A second mixed effects model also revealed a significant effect of maternal fruit consumption on milk levels of let-7g-5p (F = 5.99, p = 0.015). Higher levels of let-7g-5p were associated with lower fruit consumption, as reported longitudinally on the Dietary Screener Questionnaire.

Figure 4

Physiologic pathways targeted by the five milk miRNAs implicated in low milk supply. The heatmap displays the relative level of messenger RNA targets for 13 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, where red represents the highest number of targets. These 13 pathways all displayed a significantly greater number of targets than would be expected by chance alone on Fisher Exact Testing. Note that pathways and miRNAs have been clustered using the Ward method, and the dendrograms display relatedness of each pathway or miRNA.

Figure 5

Let-7g-5p reduced IGF2BP1/IMP1 and prolactin receptor (PRLR) expression in mammary epithelial (HC11) cells. (A) Representative immunoblot of IGF2BP1/IMP1 in protein lysates (20 μg protein/well) from HC11 cells transfected with 10 nM or 30 nM of let-7g-5p mimic compared with non-transfected (NC) cells. Caco-2 (C) and SW480 (SW) protein lysates were used as positive controls. Membranes were striped and re-probed for β-actin. (B) Relative expression of the long form of IGF2BP1/IMP1 (~70 kDa) after normalization to β-actin. Results represent the mean ratio ± SD (n = 2–3 samples/group). ** p < 0.01 (C) Relative expression of the short form of IGF2BP1/IMP1 (~42 kDa) after normalization to β-actin. Results represent the mean ratio ± SD (n = 2–3 samples/group). * p < 0.05, ** p < 0.01 (D) Representative immunoblot of PRLR in protein lysates (20 μg protein/well) from HC11 cells transfected with 10 nM or 30 nM of let-7g-5p mimic compared with non-transfected (NC€ells. (E) Relative PRLR protein expression of after normalization to β-actin. Results represent the mean ratio ± SD (n = 3 samples/group) from two separate experiments. * p < 0.05.