Human milk proteins differentiate over the sex of newborns and across stages of lactation

Background & Aims Human milk (HM) is a complete food that meets the nutritional and energy demands of the newborns. It contains numerous bioactive components, including functional proteins. Variations in HM energy and lipid content have already been reported related to the newborn's sex, but differences between protein profiles are still scarce. This work aimed to identify differences between HM proteins produced by mothers of female and male newborns, in the lactation stages of colostrum and mature milk, and the metabolic pathways involved. Methods A total of 98 HM samples were collected from 39 lactating women and classified according to the newborn's sex, stages of lactation, and three mothers' age groups, and evaluated about protein concentration and one-dimensional electrophoretic profile. Next, to assess samples with the greatest differences, the HM proteins regarding the newborn's sex and the stages of lactation were compared using nano-LC-MS/MS, in 24 HM samples randomly rearranged into four groups: female and male infants, and colostrum and mature milk. Functional classification, metabolic pathways, and protein interaction networks were analyzed by Gene Ontology, KEGG, and STRING, respectively. Results The soluble protein content of HM decreased throughout lactation, with differences regarding isolated factors, such as mothers' age group, child's sex and stages of lactation, and also in terms of their interactions. A total of 146 proteins were identified, 42 of which showed different abundances over the sexes of newborns and 53 between the stages of lactation. In general, proteins related to metabolic processes were up-regulated for mothers of male infants and in the mature stage of lactation, while proteins related to defense were up-regulated in mothers of female infants and in the colostrum phase. Conclusion This study indicated that there are differentiated and specific nutritional and defense needs of newborns, by sex and by lactation phase, which is highly relevant for a more appropriate supply of food to infants receiving HM from donor mothers.