A compound best known as a metabolic waste product changes in breast milk as infants grow. Researchers investigated what these fluctuations could mean for the developing gut microbiome and how infant formula compares.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Study: Urea is a dynamic and functional component of mammalian breast milk. Image Credit: Nastyaofly / Shutterstock
A recent non-peer-reviewed paper published on the bioRxiv preprint* server suggests that breast milk urea could help shape the infant gut microbiome. The researchers found bacteria with potential urea-metabolizing capacity in maternal stool and breast milk, along with genes involved in this process in metagenomes from milk and infant stool.
Unlike breast milk, infant formulas do not provide urea concentrations that change with lactation, and five of the eight products tested contained higher concentrations than human milk. The mammary gland also appeared to regulate urea transport through mechanisms not explained by the measured maternal protein consumption and blood urea concentrations.
In laboratory tests, urea favored different bacteria at different concentrations: a strain of Bifidobacterium longum grew best at levels typical of early lactation, whereas higher levels supported Klebsiella pneumoniae. Separately, the genus Klebsiella was more common in infants born by Cesarean section.
The research was conducted by scientists from Rutgers University, the University of California, Irvine, Princeton University, Rockefeller University, and other institutions in the United States, in collaboration with researchers from Denmark, Kenya, South Africa, and Canada.
Human milk contains a range of nutrients and protective factors, including proteins, carbohydrates, lipids, antibodies, lysozyme, lactoferrin, and maternal immune cells. Urea is another component, but researchers know relatively little about how its levels change during lactation or how it interacts with bacteria in the infant intestine. Most studies of urea have instead examined its presence in blood, urine, saliva, and sweat.
About the study
In the present study, the researchers combined three human cohorts, a mouse model, and gut microbiome profiling to investigate urea levels in breast milk, how they are regulated during lactation, and their potential effects on the developing infant gut microbiome.
The team measured milk urea concentrations across lactation in a North American cohort (USA-1) of 38 mothers and, separately, in 63 mother-infant pairs from the New Jersey Kids Study (NJKS), sampled between week 4 and week 20 postpartum. Milk samples from 23 mothers in South Africa nursing four-week-old infants were also analyzed. To examine whether these patterns were conserved, they measured milk urea levels in control, germ-free, and antibiotic-treated mice. Paired serum and milk samples from 34 NJKS mothers were assessed to identify factors influencing milk urea concentrations.
To investigate mammary urea production and transport, the researchers analyzed publicly available single-cell ribonucleic acid sequencing (scRNA-seq) datasets and performed bulk mammary RNA sequencing in mice. They used quantitative reverse transcription polymerase chain reaction (RT-qPCR), immunohistochemistry (IHC), and immunofluorescence to validate the gene-expression results.
Candidate urea transporters were assessed through gene-expression analyses, while bacterial communities in maternal stool, human milk, and infant stool were characterized using 16S rRNA sequencing and shotgun metagenomics. The metagenomic data revealed bacterial genes linked to urea metabolism. Laboratory experiments examined how varying urea levels affected bacterial growth and urease activity, while separate analyses compared urea concentrations across feeding sources and accounted for formula exposure in infant microbiome analyses.
Results
Breast milk urea increased during early lactation in the two North American cohorts, with comparable changes observed in mice. Age-matched concentrations were also similar across the three human cohorts.
In the NJKS cohort, concentrations increased by 0.21 mg/dL per week, similar to the 0.23 mg/dL weekly rise observed in USA-1 during the first 20 weeks of lactation. In USA-1, the rise was followed by a plateau. Urea levels declined between early and late lactation in 8 of 11 NJKS mothers (72.7%), but this decline was not statistically significant.
The amount of urea detected in human milk showed no significant association with the number of animal-protein food categories mothers reported consuming in the previous 24 hours or their blood urea concentrations. This points to a possible regulatory role for the mammary gland. Among the transporters examined, aquaporin 3 (AQP3) emerged as the strongest candidate for moving urea into milk. Its expression was concentrated in mammary epithelial cells, and mouse mammary tissue showed increased Aqp3 expression during lactation, whereas UT-B expression declined.
Gene-expression profiles did not support a complete mammary urea cycle. AQP3-mediated urea transfer was not directly demonstrated. At peak lactation, germ-free and conventional mice had comparable milk urea concentrations, suggesting that an intact maternal gut microbiota is not required for urea incorporation into milk.
The researchers next examined which bacteria might use the urea present in maternal milk. Breast milk and maternal fecal samples both harbored bacteria with potential urea-metabolizing activity. Genetic analysis also revealed genes encoding urease and urea transport machinery in milk and infant stool samples. Bifidobacterium, Streptococcus, and Klebsiella were among the bacterial groups carrying these genes in infant stool. Cesarean delivery was also associated with higher Klebsiella abundance at the genus level.
Urea exposure differed between feeding sources. Five of eight commercial infant formulas contained more urea than human milk at all three lactation stages assessed. In bacterial growth experiments, a tested strain of Bifidobacterium longum showed the greatest growth at urea levels comparable to those in early-lactation milk, while Klebsiella pneumoniae continued to grow more as urea availability increased.
These findings suggest that differences in available urea could give certain gut bacteria an advantage over others, including potentially harmful species, but changes in infant gut colonization were not directly tested.
Conclusion and Future Directions
The findings suggest that urea in human milk may influence early microbial development by serving as a nitrogen source for bacteria colonizing the infant intestine. Candidate urea-metabolizing bacteria were detected in complementary maternal reservoirs and the infant gut, but direct mother-to-infant transmission was not established.
The results also raise questions about how infant nutrition, particularly formula composition, could better support the bacterial communities associated with breastfeeding. Additional research should consider non-protein nitrogen alongside other milk-derived nutrients and determine how these resources influence microbiome development. The laboratory findings do not establish whether altering milk or formula urea concentrations changes infant gut communities or health outcomes.
Research into when the infant gut microbiome is most adaptable may help guide nutritional strategies, particularly for infants exposed to early-life factors such as Cesarean delivery or perinatal antibiotics.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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