Balanced energy-protein (BEP) supplementation during pregnancy improves birth and growth outcomes, but its biological mechanisms remain unclear. In 309 mother-infant dyads from the MISAME-III trial in rural Burkina Faso, we integrated metabolomics, proteomics, metagenomics and milk composition profiling across maternal, milk, and infant compartment. Prenatal BEP was associated with sustained infant growth benefits through 12 months, whereas postnatal BEP increased milk B vitamins and altered lipid composition without measurable growth benefits. BEP supplementation was associated with coordinated metabolic and proteomic responses across maternal, milk and infant compartment, particularly involving glycan biosynthesis, lipid metabolism and protein turnover. Mediation analysis identified 432 molecular mediators, with milk IgA emerging as the most consistent mediator, although it accounted for only ~3% of the total effect on infant head circumference. Inter-omics networks revealed stronger maternal-infant metabolomic connectivity than milk-infant connectivity, supporting a prominent role for prenatal programming in driving BEP-associated growth benefits. ClinicalTrial.gov: NCT03533712.

BACKGROUND:The risk of vitamin B-3 (niacin) deficiency is increased if maize is consumed without improving bioavailability during food preparation and with limited consumption of niacin-rich foods. OBJECTIVES:We assessed human-milk vitamin B-3 concentrations and their relationship to child outcomes as a secondary analysis of a randomized controlled trial of nicotinamide supplementation to lactating mothers in rural Tanzania. METHODS:The Early Life Interventions for Childhood Growth and Development in Tanzania study, conducted in Haydom, Tanzania, randomly assigned lactating females to supplemental nicotinamide (250 mg/d compared with placebo) from study entry (infant age <2 wk) until 6 mo. Among 1173 mother/child dyads, human-milk nicotinamide and B3 vitamer concentrations at lactation months 1 and 5 were compared with international reference limits (fifth and 95th percentile) from the recent Mothers, Infants and Lactation Quality study, and child anthropometry and cognitive development [Malawi Developmental Assessment Tool (MDAT)] relationships were assessed. RESULTS:The proportion of human-milk total vitamin B-3 concentrations below the lower reference limit increased over time in both those without and with nicotinamide supplementation (2.9% compared with 0.5% ≤0.720 mg/L at 1 mo; 28.8% compared with 9.9% ≤0.549 mg/L at 5 mo). Maternal nicotinamide supplementation was associated with many participants having human-milk total vitamin B-3 concentrations exceeding the upper reference limit (72.9% ≥3.284 mg/L at 1 mo; 50.7% ≥2.760 mg/L at 5 mo), and a sustained higher median concentration with supplementation compared with placebo (by 291% at month 1 and 281% at month 5). No associations were observed between human-milk total vitamin B-3 or vitamer concentrations and child weight, height, or head circumference ≤18 mo or with MDAT scores at 18 mo. CONCLUSIONS:Without maternal nicotinamide supplementation, lactating females had a greater prevalence of human-milk total vitamin B-3 concentrations below the fifth percentile reference limit. Although supplementation was associated with greater total vitamin B-3 and vitamer concentrations, there was no association between human-milk vitamin B-3 and child growth or development outcomes ≤18 mo. This study was registered at clinicaltrials.gov as NCT03268902.

Vitamin B12 is acquired through the consumption of animal-source foods and supplements. In animal models, interventions with B12 and/or methionine influence fecal short-chain fatty acid (SCFA) concentration. Yet the relevance of dietary B12 to microbially produced SCFAs in humans is unknown. This study determined associations between dietary B12 and the gut microbiome in a deeply phenotyped cohort of healthy U.S. adults. Habitual diet and fecal shotgun metagenomes were integrated alongside measurements of fecal SCFAs, plasma SCFAs, and plasma B12 (n = 277). Vitamin B12 intake ranged from 2.4 to 1062 µg/day, and nearly all participants were B12 replete. Stratification of participants into adequate (2.4-8.51 µg/day) and high B12 intake (>8.51 µg/day) groups revealed the association of high intake with a reduction in bacteria capable of anaerobic B12 biosynthesis. High B12 intake was also associated with lower fecal SCFA concentrations even after controlling for fiber and methionine intake. Differences in microbial taxa between dietary groups were limited. However, machine learning models demonstrated the ability to predict fecal propionate and butyrate from microbial pathways in the adequate or no supplement groups, but not in the high intake or supplement groups. Our results indicate that dietary B12 greater than 8.51 µg/day may be associated with reduced microbial synthesis of B12 and lower fecal SCFA production.

BACKGROUND:Human milk (HM) contains essential components that support infant growth and development; however, evidence linking HM macronutrient intake to infant growth remains minimal. OBJECTIVES:This study aimed to evaluate the longitudinal association between HM macronutrient intake and infant growth. METHODS:This study includes 833 mother-infant pairs from the 4-site Mothers, Infants and Lactation Quality study (Bangladesh, Brazil, Denmark, and The Gambia) who were followed at 1-3.49 mo (M1), 3.5-5.99 mo (M2), and 6-8.5 mo (M3). HM macronutrients were measured by near-infrared spectroscopy at M1. HM volume intake was assessed at M1 on a subset of 533 participants using the stable isotope "dose-to-mother" method, except in Denmark, where test-weighing was used. HM macronutrient intake was estimated as the product of each macronutrient concentration and HM intake. Weight-for-age (WAZ), length-for-age (LAZ), weight-for-length (WLZ), and BMI-for-age (BAZ) z-scores were calculated at M1, M2, and M3, and analyzed according to WHO Growth Standards. Statistical analyses included linear mixed-effects models adjusted for site, maternal age, and BMI, infant birth weight, and sex. Interaction terms for site and Benjamini-Hochberg multiple testing comparisons were included. RESULTS:Positive longitudinal associations were observed between HM protein intake (g/d) at M1 and WAZ (β = 0.084), LAZ (β = 0.044), WLZ (β = 0.082), and BAZ (β = 0.092) between 1 and 8.5 mo. HM carbohydrate intake (g/d) at M1 was associated with higher WAZ (β = 0.034), LAZ (β = 0.016), WLZ (β = 0.036), and BAZ (β = 0.039) between 1 and 8.5 mo. Positive associations were also observed between HM lipid intake (g/d) and WAZ (β = 0.014), WLZ (β = 0.017), and BAZ (β = 0.018). Significant interactions with study site were observed for the associations between HM protein intake and LAZ in Brazil (β = 0.115) and between HM carbohydrate intake and WAZ in Bangladesh (β = 0.018), compared with The Gambia. CONCLUSIONS:These results reveal that HM macronutrient intake is positively associated with infant growth, and some associations may also be modified by site. This study was registered at clinicaltrials.gov as NCT03254329.

Background:The Multi-Omics for Mothers and Infants (MOMI) Consortium aims to define biological mechanisms associated with preterm birth, small for gestational age, preeclampsia, and stillbirth. Globally, the burden of adverse pregnancy outcomes (APOs) is highest in low- and middle-income countries (LMICs). The MOMI Consortium consists of six different LMIC sites established by The Alliance for Maternal and Newborn Health Improvement in Bangladesh, Pakistan, and Tanzania, the Global Alliance to Prevent Prematurity and Stillbirth in Bangladesh and Zambia, and the Interdisciplinary Group for Advanced Research on Birth Outcomes - DBT India Initiative. It also partners with five analytical partners and three bioinformatic teams. Methods:The combined MOMI cohort includes 24 321 pregnant women, a rich biorepository, and paired socioeconomic and clinical data. Considering the multifactorial aetiologies and molecular drivers of APOs, we applied an integrative multi-omics (genomics, metabolomics, proteomics, nutrient testing) approach to identify site-specific and cohort-wide signatures linked to distinct APOs. Conclusions:This protocol summarises the MOMI study design, sample and data collection methods, data harmonisation, and the various analytics platforms, and discusses potential outcomes for enhanced clinical care and novel diagnostic and therapeutic tools.

Protein and fat concentrations in donor human milk (DHM) can vary twofold to threefold and are influenced by the number of unique donors per pool. The aim of this study was to broadly characterize how the number of donors (2-10) randomly combined into a pool during milk bank processing influenced the variability of macronutrients, vitamins, minerals, and bioactive factors in DHM. The minimum number of donors required for 80% of the pools to meet pre-defined targets for true protein, fat, and disialyllacto-N-tetraose (DSLNT) was also evaluated. Monte Carlo simulation was used to create models that accounted for donor lifetime donation volume and milk bank production constraints. Variability in nutrients was quantified as a Nutrient Inequality Index (NII) which was computed as the ratio of the 90th percentile to the 10th percentile for each simulation. Random multi-donor pooling of 2-10 donors produced lower variability in DHM macronutrients than most vitamins and minerals. A priori targets of 0.9 g/dL of true protein, 3.5 g/dL of fat, and 210 µg/L of DSLNT could not be achieved with any random pooling scenario. The NII for lactose stabilized at less than 1.1 when there were 3+ donors per pool, while the NII for fat and true protein stabilized at less than 1.3 when there were 5+ donors per pool. The NII exceeded 1.5, even at 10 donors per pool, for several micronutrients including zinc, copper, sodium, iron, biotin, riboflavin, B6, B12, and pantothenic acid.

Background/Objectives: Human milk (HM) is recognized as the optimal source of infant nutrition, particularly during the first six months of life. While its nutritional aspects and bioactive components are well studied, the HM metabolome remains less understood, particularly in low- and middle-income countries. This study utilized targeted metabolomics for HM profiling and investigated associations of the HM metabolome with maternal and infant characteristics. Methods: In total, 267 HM samples and demographic data from mothers participating in the Maternal and environmental Impact assessment on Neurodevelopment in Early childhood years (MINE) study were collected during enrolment (up to 6-months postpartum) and analyzed using the MxP® Quant 500 targeted metabolomics kit from Biocrates. Results: A total of 440 metabolites were quantified, mostly lipids such as triglycerides (59.73%), phosphatidylcholines (14.25%), and diglycerides (8.49%), and small molecules including amino acids (26.67%), amino acid-related compounds (21.33%), hexosylceramides (17.33%), and fatty acids (14.67%). Maternal age was positively correlated with a wide range of metabolites, mainly cholesteryl esters, sphingomyelins, triglycerides, and acylcarnitines, while child age was associated with metabolites belonging to acylcarnitine, phosphatidyl-choline, ceramide, diacylglycerol, sphingomyelin, and triglyceride classes. Child’s gender was associated with metabolites, including ceramides, phosphatidylcholines, and sphingomyelins. Pathway enrichment analysis revealed that the metabolites were significantly enriched in valine, leucine, and isoleucine biosynthesis; arginine biosynthesis; phenylalanine, tyrosine, and tryptophan biosynthesis; and glutathione metabolism; however, these reflect annotation-based clustering rather than evidence of active metabolic processes in HM. Conclusions: The HM metabolome varies with maternal and infant characteristics, particularly infant age, reflecting cross-sectional differences in milk composition among mother–infant dyads. Enrichment of metabolites annotated to amino acid and antioxidant-related pathways highlights coordinated representation of nutritionally relevant compounds. These findings provide new insight into the factors shaping HM composition in a low- and middle-income populations.

BACKGROUND:Currently, clinical care of preterm infants is limited by a dearth of information on the nutritional and bioactive composition of donor human milk (DHM). OBJECTIVES:The primary objective is to assess and compare the macronutrients, vitamins, minerals, human milk oligosaccharides (HMOs), and antimicrobial proteins in human milk from approved milk banks in a variety of low-, middle-, and high-income settings. The secondary objective is to explore factors that influence composition, including gestational stage (preterm compared with term), lactation stage, maternal age, lifetime donation volume, and storage duration. METHODS:We conducted a cross-sectional study of human milk collected systematically from unique, approved milk bank donors (n = 600) in Chile, Kenya, Poland, the United States, and Vietnam. True protein, total fat, lactose, HMOs, vitamins, minerals, and antimicrobial proteins were assessed. Analysis of variance and Tukey's tests were used to evaluate normally distributed data; Kruskal-Wallis and Dunn's tests were used for nonparametric data. Multiple regression was used to explore factors associated with milk composition. RESULTS:The only site with predominantly preterm donors was Kenya (26/50). Kenya also had an early donation model (mean lactation stage 1.6 ± 1.1 wk), whereas the remaining sites had a mature donation model (mean lactation stage ranged 14.2-21.4 wk). Most nutrients differed significantly (P < 0.05) between geographies. For instance, true protein in Kenya was 1.3 ± 0.3 g/dL compared with 0.8 ± 0.2 at all United States banks. Many minerals were higher in the early donation model whereas lactose and several water-soluble vitamins were higher in the mature donation model. Although the lactation stage typically had the most predictive value, most nutrient variation (>60%) remained unexplained. CONCLUSIONS:Clinical protocols for feeding DHM to preterm infants, including setting-specific fortifier products, may need to take into consideration the local donor milk banking model. Multidonor pooling warrants further investigation as a tool for milk banks to ensure minimum nutrient standards for DHM.

Human milk (HM) contains a multitude of nutritive and nonnutritive bioactive compounds that support infant growth, immunity and development, yet its complex composition remains poorly understood. Integrating diverse scientific disciplines from nutrition and global health to data science, the International Milk Composition (IMiC) Consortium was established to undertake a comprehensive harmonized analysis of HM from low, middle and high-resource settings to inform novel strategies for supporting maternal-child nutrition and health. IMiC is a collaboration of HM experts, data scientists and four mother-infant health studies, each contributing a subset of participants: Canada (CHILD Cohort, n = 400), Tanzania (ELICIT Trial, n = 200), Pakistan (VITAL-LW Trial, n = 150), and Burkina Faso (MISAME-3 Trial, n = 290). Altogether IMiC includes 1,946 HM samples across time-points ranging from birth to 5 months. Using HM-validated assays, we are measuring macronutrients, minerals, B-vitamins, fat-soluble vitamins, HM oligosaccharides, selected bioactive proteins, and untargeted metabolites, proteins, and bacteria. Multi-modal machine learning methods (extreme gradient boosting with late fusion and two-layered cross-validation) will be applied to predict infant growth and identify determinants of HM variation. Feature selection and pathway enrichment analyses will identify key HM components and biological pathways, respectively. While participant data (e.g., maternal characteristics, health, household characteristics) will be harmonized across studies to the extent possible, we will also employ a meta-analytic structure approach where HM effects will be estimated separately within each study, and then meta-analyzed across studies. IMiC was approved by the human research ethics board at the University of Manitoba. Contributing studies were approved by their respective primary institutions and local study centers, with all participants providing informed consent. Aiming to inform maternal, newborn, and infant nutritional recommendations and interventions, results will be disseminated through Open Access platforms, and data will be available for secondary analysis. ClinicalTrials.gov, identifier, NCT05119166.

Early-life gut microbiome development is shaped by complex maternal and nutritional influences, yet the temporal and directional structure of these interactions remains unclear. In a longitudinal study of 152 mother-infant dyads in rural Burkina Faso, we examine how maternal gut and milk microbiomes, alongside milk components, influence infant gut microbiome development during the first 6 months. At 1-2 months, the infant gut microbiome clusters into three types: Escherichia-dominated, Bifidobacterium-dominated, and a diverse, pathogen-prevalent profile, which become less distinct by 5-6 months. Early infant gut microbiomes associate with maternal prenatal gut microbiota and early milk microbiome and oligosaccharides, while later variation links to other milk nutrients. Furthermore, early infant gut profiles predict subsequent milk composition, suggesting potential bidirectional communication between infant needs and maternal lactational physiology. These findings offer insights into early-life microbial development and inform future mechanistic studies and microbiome-targeted interventions, particularly in low-resource settings.

Background/Objectives: Metabolic profiling of human milk (HM) is indispensable for elucidating mother-milk-infant relationships. Methods: We evaluated the Biocrates MxP® Quant 500 assay for HM-targeted metabolomics (106 small molecules, 524 lipids) and analyzed in a feasibility test HM from apparently healthy Brazilian mothers (A: 2–8, B: 28–50, C: 88–119 days postpartum, ntotal = 25). Results: Of the 630 possible signatures detectable with this assay, 506 were above the limits of detection in an HM-pool (10 µL) used for assay evaluation, 12 of them above the upper limit of quantitation. Analyzing five different HM-pool volumes (2–20 µL) revealed acceptable linearity for 458 metabolites. Intraday accuracy of 80–120% was attained by 469 metabolites after spiking and for 342 after a 1:2 dilution. Analyzing HM from Brazilian mothers revealed significantly lower concentrations in colostrum vs. mature milk for many flow-injection analyses (FIA) and only a few LC-MS metabolites, including triglycerides, sphingomyelins, and phosphatidylcholines. Higher concentrations at the later lactation stages were found predominantly for amino acids and related compounds. Conclusions: The MxP Quant® 500 assay is a useful tool for HM metabolic profiling, minimizing analytical bias between matrices, and enhancing our ability to study milk as a biological system.

The human milk content of some micronutrients including thiamine depends on maternal status, and if low, breastfed infants are at risk of deficiency. Thiamine deficiency remains an important cause of morbidity and mortality among infants in Asia. We aimed to explore correlations between maternal thiamine diphosphate (ThDP) or erythrocyte transketolase activation coefficient (ETKac) and human milk thiamine concentration (MTh) and between MTh and infant ThDP or ETKac among breastfed infants < 6 months of age in northern Lao PDR. Hospitalized infants (aged ≥ 21 days) with symptoms suggestive of thiamine deficiency were eligible. Infants in a community comparison group were matched by age, sex and residence. Venous whole blood ThDP and MTh were determined by HPLC-FLD, and ETKac in washed erythrocytes by UV spectrophotometry. Associations between biomarkers were assessed using Spearman's ρ correlations and linear regression. Among all women combined (n = 489), the prevalence of ThDP < 95 nmol/L was 78.5%, elevated ETKac (> 1.25) 52.6%, and low MTh (< 90 µg/L) 45.4%. Maternal ThDP was moderately correlated with MTh (ρ = 0.50) and ETKac was strongly correlated with MTh (ρ = -0.71). Among all infants combined (n = 359), the prevalence of ThDP < 95 nmol/L was 79.2% and elevated ETKac (> 1.25) 50.2%. MTh was moderately correlated with infant ThDP concentration (ρ = 0.39) and with infant ETKac (ρ = -0.52). Maternal thiamine status predicts the thiamine concentration in human milk, and thiamine status of breastfed infants < 6 months of age depends on the thiamine provided through this milk. Effective interventions are needed to improve maternal and infant thiamine status and wellbeing. TRIAL REGISTRATION: Clinicaltrials.gov NCT03626337.