TABLE 1.

Multifunctional effects of human milk on infant and child outcomes.

Functional System		Findings [reference]
Neurocognitive Development	Observations	•A meta-analysis of studies involving >12,000 children reported that those breastfed (BF) for ≤6 mos versus >6 mos had 1.04- and 1.06-fold higher scores on intelligence tests than those never BF, respectively [7]. •Associations between variable concentrations of LC-PUFA (8–11) and choline and lutein [12] in human milk and IQ have been shown. •Synergistic associations of higher levels of both choline and DHA in human milk with better recognition memory in infants [12]. •Meta-analyses of RCT of LC-PUFA supplementation have not shown an impact of consumption of fish oil or DHA/EPA supplements in breastfeeding females on cognitive performance of their children [13, 14]
	Proposed Mechanisms or Associations with Human Milk Components	•Improved myelination by 2 y of age in BF children, including networks associated with a broad array of cognitive and behavioral skills [15] •Sialylated molecules, including gangliosides and sialoproteins, are present in the frontal cortex of infants [16] •In BF, but not in FF infants, ganglioside-bound sialic acid was correlated with ganglioside ceramide DHA and total n-3 fatty acid, suggesting potential interactions between human milk LC-PUFA and HMOs. [16]
	Limitations	•Small sample size studies, short-term studies with short follow-up. lack of diversity, lack of information on maternal and child genetic polymorphisms in LC-PUFA synthesis, lack of report of sociodemographic factors [13,14,17]. •Within human milk component interactions have not been explored (HMO to other components, HMO-HMO interactions).
Endocrine Development	Observations	•Type 2 Diabetes: BF reduced risk by 33% [18]. • Type 1 Diabetes: • Exclusive BF for >2 wk was associated with a 14% lower risk than in a shorter duration and/or a lack of BF [[19], [20], [21]].
	Proposed Mechanisms or Associations with Human Milk Components	•Breastfeeding is associated with lower preprandial serum glucose and insulin concentrations than in formula-feeding [22]. •Type 2 Diabetes: Postulated effects on appetite regulation, reduced weight gain during infancy, and/or nutrients in human milk that promote energy balance, independently of child or adult BMI [23]. • Type 1 Diabetes: • Lower gut permeability in BF than FF infants [24] • Immunomodulatory substances, such as lactoferrin, lysozyme, and secretory immunoglobin A (sIgA), as well as macrophages that affect the function of T- and B cells [25] • Other human milk components have been suggested, but mechanistic evidence in humans is lacking: SCFA [26,27] human milk-derived opioid peptides β-casomorphins [28], HMOs [29], fucosyltransferase-2 nonsecretor status [30], miRNA content of human milk exosomes [31]
	Limitations	•Type 1 Diabetes: Unknown whether human milk offers protection in high-risk populations with genetic predisposition [32] •Lack of well controlled studies with longitudinal outcomes and comprehensive information on human milk ecology and composition
Intestinal Development	Observations	•Endoscopic biopsies from healthy infants demonstrated 30% greater crypt length in FF than BF infants [33]. •Intestinal permeability in vivo was higher in FF than BF infants [24,34].
	Proposed Mechanisms or Associations with Human Milk Components	•Pathways regulating stem cell proliferation, differentiation, and migration, as well as barrier function and immune response were differentially expressed in exfoliated epithelial cells of BF vs. FF infants [35]. •Diet can affect, via gut colonization and cross talk with host epithelial cell, expression of genes associated with the innate immune system in infants [36].
	Limitations	•Limited sample sizes (4-10 infants per group) •Lack of longitudinal data on diet and infant intestinal development •Need to delineate developmental from human milk effects (e.g., term vs. preterm) •Need to correlate gene expression with function and developmental outcomes
Immune Development	Observations	Immune cells: •CD4+ T cells: No consensus for an effect of BF [[37], [38], [39]] •CD8+ T cells: Longer BF was associated with increased CD8 T cell memory, but not memory B cell numbers, in the first 6 mo [39]. •Natural killer cells: Higher in BF than formula-fed infants at 6 mo of age [37]. •Tregs: Proportion of Tregs increased nearly two-fold between birth and 3 wk of age in BF then FF infants [40]. •IgA and IgG secreting B cells: Higher in FF than BF infants [41]. Cytokines •Serum proinflammatory TNF-α and IL-2 were higher, while TGF-β2 was lower, in FF than BF infants in the first year [42]. •BF neonates showed a specific and Treg-dependent reduction in proliferative T-cell responses to noninherited maternal antigens, associated with a reduction in inflammatory cytokine production [40]. IgA: •Earlier and greater IgA production in nasal and saliva samples from BF compared with FF infants in the first few days of life [43], but not at 7 wks [44]. •Inconsistent finding on salivary IgA, IgM, and IgG between 3 and 6 mos [45,46]. Thymic size and GALT: •Breastfeeding increases thymic size compared with formula-feeding [47]. There are no human studies on the effect of breastfeeding or breast milk on the development of GALT due to lack of access to human tissues.
	Proposed Mechanisms or Associations with Human Milk Components	•Higher IgA and IgG secreting B cells in FF possibly due to higher antigenic exposure [41]. •Human milk TGFβ may induce IgA production in infants [48]. •IgA production may be induced by Bifidobacterium species (enriched in BF) [19,49]. •Human milk IL-7 content, an important factor for lymphocyte development, was correlated with thymic size [50,51]. •Thymic size was also correlated with the number of CD8+ T cells, which are increased in BF infants [39].
	Limitations	•Only observational studies. •Small sample sizes and limitations in the detection of memory T cells. •Unknown if mechanisms underlying higher proinflammatory cytokines in formula-fed infants are direct effects of human milk components or are mediated through gut microbiota. •Discrepancies in salivary IgA levels may depend on antigenic exposure and infant gut microbiome composition. •There are no human infant studies on the effect of BF or human milk on the development of GALT, due to lack of access to such human tissues.
Clinical Immune Outcomes	Observations	Food Allergy: •Protective effect of exclusive breastfeeding against cow’s milk allergy in early childhood among high-risk infants is inconsistent [52,53]. Other Atopic Diseases: •Moderate evidence for a protective effect of human milk consumption against asthma in childhood, limited evidence to indicate no association between human milk consumption and atopic dermatitis in childhood, and inconclusive evidence to suggest a relationship between human milk consumption and atopic dermatitis from 0-24 mo of age [20]. •No association between duration of human milk consumption and allergic rhinitis in childhood [20]. •Evidence is insufficient to suggest any relationships between human milk consumption and asthma, atopic dermatitis, or allergic rhinitis during adolescence or adulthood, or between human milk consumption and food allergy at any life stage [53]. Gut Inflammatory Diseases: •Inconclusive evidence of human milk consumption on celiac disease [54]. •Limited, but consistent, case-control evidence suggests that shorter versus longer durations of any human milk feeding are associated with higher risk of IBD [20].
	Proposed Mechanisms or Associations with Human Milk Components	•Higher concentrations of total IgA and casein-specific IgA in human milk have been associated with protection against cow’s milk allergy [55,56]. •TGFβ is the most well-studied human milk cytokine in connection with infant atopic outcomes; however, a meta-analysis found no association between TGFβ in human milk and allergic outcomes [57]. •High levels of some human milk cytokines (e.g., IL-1β, IL-6, IL-10, and TGFβ) are associated with protection against food allergic disease [58], and human milk IL-6 and IGF-I may play a role in oral tolerance [59]. •Effects of HMO are inconsistent with regard to atopic eczema, cow’s milk allergy, asthma and eczema [[60], [61], [62]].
	Limitations	•High heterogeneity and low-quality evidence, lack of adequate statistical power to assess the impact of BF [53].
Serum and Fecal Metabolomes	Observations	•BF infants have higher fatty acid metabolism compared with formula-fed infants, as shown by higher levels of free fatty acids, lysophosphatidylcholines, and long-chain acylcarnitines, as well as increased markers of β-oxidation in serum [63]. •FF infants have higher levels of circulating amino acids and amino acid degradation products in serum than BF [[64], [65], [66], [67], [68], [69], [70], [71], [72]]. •Higher levels of microbial degradation products of protein in feces of FF than BF infants [63,66,70,73]. •BF infants introduced to complementary feeding before 6 mos had higher serum BCAA at 12 mo of age than to BF infants who were exclusively BF to 6 mo of age [63,64].
	Proposed Mechanisms or Associations with Human Milk Components	•Differences in dietary composition between human milk and formula •Nutrients in human milk are packaged differently within the human milk matrix (e.g., fats with the MFGM)
	Limitations	•Further studies on human milk composition, including the human milk matrix, and nutrient absorption could determine if changes in human milk composition over time, or even during the day, alter the degree to which human milk nutrients are metabolized and absorbed •Few studies have examined the timing of the introduction of complementary feeding on metabolism •Limited evidence on interactions between diet, microbiome and metabolome and outcomes, such as growth
Gut Microbiome	Observations	•Gut microbiota at 12 mos differed compared with those weaned from human milk before 6 mo of age [74]. •BF infants supplemented with formula where BF was associated with higher B. breve and B. bifidum; cessation of BF resulted in faster maturation of the gut microbiome (Firmicutes) [75]. •Richness and diversity of microbiota were highest in infants who were not BF, lower in partially BF infants, and lowest in exclusively BF infants. Increasing exclusivity of breastfeeding was associated with greater relative abundance of Bifidobacteriaceae and Enterobacteriaceae and lower relative abundance of Lachnospiraceae, Veillonellaceae, and Ruminococcacae [74].
	Proposed Mechanisms or Associations with Human Milk Components	•Glycans cleaved from human milk proteins by microbial glycohydrolases [76] and peptides produced by proteolytic digestion in vivo [77] are bifidogenic •HMOs act as prebiotics and shape the infant microbiome [78]. •B. longum subsp. infantis, directly internalize intact HMOs by specific transporters and degrade them intracellularly [79], which allows it to outcompete other Bifidobacterium species/strains (B. bifidum and some B. longum strains) that use extracellular glycosidase(s) •Bifidobacterium populations increase more rapidly and are more abundant in infants fed by secretor mothers than those fed by nonsecretor mothers and specific Bifidobacterium strains that can use 2′FL are enriched in the stools of the infants receiving human milk of secretors vs. nonsecretors [80,81,82]. •Fucosylated α1-2 oligosaccharides are degraded by some Bacteroides (particularly B. fragilis) and Akkermansia (A. muciniphila MucT) [83], which are commonly present in the infant gut [75]. •Secretor status interacts with route of delivery; infants born by CS who were fed secretor human milk had a less dysbiotic gut microbiota compared with vaginally-delivered infants than did CS infants who received nonsecretor human milk [84]. •Human milk contains a milk microbiota, which has been implicated in seeding the infant microbiota [85].
	Limitations	•Data on microbiome composition of body sites besides fecal are limited •Most studies are limited to 16S rRNA analysis, with small sample sizes •Data on Archaea, viruses and fungi in human milk and infant stool are limited •HMO diversity and human milk microbiota composition and metabolic function are influenced by milk composition environmental factors, genetics, geographical location, and other factors [86,87], including differences based on secretor status [85,88,89], which are often not documented. •Low biomass samples, such as human milk may yield spurious results due to environmental contamination •16s rRNA signatures do not reflect viable human milk microbes [90] •Few studies addressed microbiome function (e.g., metagenomics and metabolomics) and human milk matrix •Studies on BF and host-microbe interactions and long-term outcomes are limited

BF, breastfeeding; CS, Cesarean section; 2′FL, 2′-fucosyllactose; FF, formula-feeding; GALT, gut-associated lymphoid tissue; HMO, human milk oligosaccharide; IBD, inflammatory bowel disease; SCFA, short-chain fatty acids; Tregs, T-regulatory cells; TGFβ, transforming growth factor-beta.