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.

The first 1000 days of a child's life represent a critical window for brain development, during which nutrition exerts profound effects on the trajectories of neurodevelopment. Human Milk Oligosaccharides (HMOs), a major component of human milk, are largely indigestible by infants and are known to influence immunity, microbiome composition, and gut-brain signaling, but their direct role in neurodevelopment remains poorly understood. Here, we investigated the impact of HMOs on human cortical organoids, a physiologically relevant in vitro model of early brain development. We found that HMO treatment significantly enhanced neurite outgrowth and synaptogenesis in a dose-dependent manner. Global proteomic profiling further demonstrated the upregulation of proteins associated with neuronal differentiation, synaptic maturation, and cytoskeletal remodeling. Our findings suggest that HMOs can influence neurodevelopmental processes and highlight a potential role for maternal milk components in early brain development.

Abstract Background Although human milk (HM) confers important health benefits, how bioactive milk components (e.g., microbiota, oligosaccharides, and fatty acids) interact with infant genetics to influence childhood atopy remains poorly understood. Objective We investigated interactions between infant genomic susceptibility and exposure to maternal human milk components (HMCs) and assessed whether integrating these genetic and milk features improves prediction of childhood atopy. Methods Leveraging infant genomic and maternal HMC data from the CHILD Cohort Study, we conducted gene-milk interaction analysis using linear regression models that integrated polygenic risk scores (PRS) of nursing infants with multiple HMC types. Gradient-boosting machines (GBMs) were used to evaluate predictive performance of HMCs and infant PRS for childhood atopy. Results Childhood atopy was associated with interactions between infant genomics (e.g., PRS associated with atopy) and exposure to specific human milk microbes (e.g., Abiotrophia , P Bonf =0.005, β=0.29), as well as networks of co-occurring HMCs (e.g., a module containing Bifidobacterium longum , 2’-fucosyllactose, and eicosapentaenoic acid, P=0.009, β=-12.3). A GBM integrating HMCs and infant PRS achieved the highest predictive performance for childhood atopy with an area under the curve (AUC) of 0.78, outperforming models based on individual HMC types or PRS alone (AUC range: 0.54-0.63). Conclusion Integration of maternal HMC exposures with infant genomics reveals interaction effects that contribute to prediction of childhood atopy. Understanding how early-life exposures such as HMCs impact the health of children differently depending on their genomic profiles may facilitate the development of personalized intervention strategies to reduce the burden of these health outcomes during childhood. Key messages Interactions between infant polygenic risk and exposure to human milk components are associated with childhood atopy. Networks of co-occurring human milk microbiota, oligosaccharides, and fatty acids may influence childhood atopy, with effects varying by infant genomic susceptibility. Integration of human milk components with infant genomics improves prediction of childhood atopy compared with individual milk components or genomics alone. Capsule Summary This study demonstrates that interactions between infant polygenic risk and maternal milk components improve prediction of childhood atopy, highlighting opportunities for personalized early-life prevention strategies.

Identification of early interventions to reduce/eliminate asthma - the most common chronic disease among children - could significantly reduce burden on the healthcare system. Large-scale asthma Exposome-Wide Association Studies (ExWAS) could identify potential interventions, however integration of diverse data is required to address association confounders. The CHILD Cohort Study has followed 3,454 healthy Canadian children and their families from early pregnancy, collecting exceptionally diverse data including 27,006 variables from participant questionnaires, clinical data, household and neighbourhood-level exposures, and sample-derived chemical analytic/omic datasets. Here, we report integration of these datasets into the CHILDdb database platform, and use these data to perform ExWAS and machine learning analyses, identifying and further characterizing associations between childhood asthma and 2,954 diverse early exposures (pregnancy-age 5). Significant asthma associations include antibiotic use, human milk components, DEHP-phthalate, and mother's prenatal cleaning product/disinfectant exposure. Subsequent analysis revealed epigenetic changes in the cord blood at birth, after prenatal cleaner exposure, and different microbiome and/or inflammatory cytokine changes associated with different asthma-associated exposures in the child. Collective results support asthma as a heterogeneous condition involving multiple etiologies, with associated endotypes, including significant prenatal exposures with potential transgenerational effects, and suggest targets for early interventions. ### Competing Interest Statement TE reports to have acted or acts as local PI for company-sponsored trials by DBV Therapeutics, Greer Stallergens, Novartis, and sub-investigator ALK-Abelló. He is Co-Investigator or scientific lead in three investigator-initiated oral immunotherapy trials supported by the SickKids Food Allergy and Anaphylaxis Program and serves as an associate editor for Allergy. He/his lab received unconditional/in-kind contributions from Macro Array Diagnostics and an unrestricted grant from ALK-Abelló. He holds scientific advisory board roles for ALK-Abelló, VAMED, Nutricia/Danone, Hipp, Sanofi, Greer-Stallergens, Allergy Therapeutics and Aimmune. TE reports lecture fees from Novartis, ThermoFisher, Nutricia/Danone, Aimmune, Sanofi, Schwalbe, MADX, ALK-Abelló. ### Funding Statement Key support for this work was provided by the Schroeder Allergy and Immunology Research Institute, Genome Canada, Genome BC, and CIHR, with additional support by Simon Fraser University and the Digital Research Alliance of Canada. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval for the CHILD Cohort Study, including the oversight of the CHILD biological samples and the CHILD database (CHILDdb), was obtained from the local Research Ethics Board of each study site: the University of British Columbia, the University of Alberta, the University of Manitoba, the Hospital for Sick Children, and McMaster University. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data described in the manuscript are available by registration to the CHILD database https://childstudy.ca/childdb/ and the submission of a formal request. More information about data access for the CHILD Cohort Study can be found at https://childstudy.ca/for-researchers/dataaccess/. Researchers interested in accessing CHILD Cohort Study data for their research should contact child{at}mcmaster.ca.

IntroductionHuman milk oligosaccharides (HMOs) are structurally diverse carbohydrates found in high concentrations in human milk, supporting infant immune development and gut microbiota colonization. Their composition is influenced by maternal Secretor (Se) status, determined by the FUT2 gene. While immunological HMO effects are increasingly recognized, the influence of an infant’s own Se status on immune responses remains understudied. This study explores how peripheral blood mononuclear cells (PBMCs) from Se-positive (Se+) and Se-negative (Se−) individuals respond to Se+ and Se− HMOs under bacterial and viral stimulation.MethodsPBMCs from 14 healthy adult donors, classified based on FUT2 expression, were exposed to 0.1% pooled Se+ or Se− HMOs, individual HMOs (2′-fucosyllactose and 3-fucosyllactose), and immune triggers (αCD3/CD28, LPS, and/or Poly I:C) for 24–48 h. Cytokine secretion (IFNγ, IL10, IL13, TNFα) was measured.ResultsBasal cytokine secretion did not differ between Se+ and Se− PBMCs. Upon stimulation, Se+ PBMCs secreted more IL10, particularly in response to Se+ or Se− HMOs. Individual HMOs did not replicate effects seen with pooled mixtures, highlighting the importance of HMO complexity. Under LPS stimulation, TNFα secretion was significantly reduced only with genotype-matched HMOs, suggesting Secretor-specific immune modulation.ConclusionThis is the first study showing that PBMC cytokine responses are shaped mainly by host Secretor status than HMO composition. Both genotype and HMO profile influence immune reactivity and should be considered in HMO research and personalized infant nutrition strategies.

INTRODUCTION:Human milk oligosaccharides (HMOs) arise from tightly regulated glycosylation pathways and exert pleiotropic effects on infant immune, microbial, and neurodevelopmental trajectories. Vitamin D influences gene transcription and cellular metabolism, yet its potential role in mammary glycosylation remains unexplored. We examined whether maternal vitamin D status during lactation is associated with HMO composition and whether effects vary by maternal secretor phenotype. METHODS:We performed a post hoc analysis of two clinical studies: the NICHD vitamin D lactation randomized controlled trial (2005-2012) and the Lactation Immune Trial pilot (n = 88). Serum 25-hydroxyvitamin D [25(OH)D, vitamin D status] and 19 HMOs concentrations were measured at 1 and 4 months postpartum. Secretor status was defined by presence of 2'-fucosyllactose. Statistical analyses were conducted using SAS 9.4 with p < 0.05. RESULTS:The cohort comprised 41% White, 7% Black, and 52% Hispanic women. Black and Hispanic mothers exhibited lower 25(OH)D concentrations than White mothers (p < 0.001), and Hispanic mothers were more likely to be secretors (p = 0.006). In secretors, higher 25(OH)D was significantly associated with increased fucosylated and complex HMOs, including lacto-N-fucopentaose II, lacto-N-hexaose, and fucosyl-disialyl-lacto-N-hexaose I at 1 month, with sustained association for the latter at 4 months. In non-secretors, higher 25(OH)D was associated with lower 6'-sialyllactose at 4 months. Distinct HMO patterns were observed by secretor phenotype. CONCLUSION:Vitamin D status is associated with differential HMO profiles during early lactation, supporting a potential role for vitamin D-mediated regulation of mammary glycosylation that is modified by secretor status.

The infant gut resistome is established early in life and is shaped by perinatal exposures, yet the mechanisms underlying its modulation remain unclear. We combined shotgun metagenomics of fecal samples from 57 one-month-old infants and paired milk samples from 50 mothers in the MAMI cohort to investigate the influence of maternal secretor status on early-life resistome development. Longitudinal follow-up at 6 and 12 months, and also further validation in the independent Lifelines NEXT (LLNEXT) cohort, support our findings. Cesarean section (C-section) was associated with increased antibiotic resistance gene (ARG) diversity, whereas exclusive breastfeeding reduced ARG abundance and diversity. Maternal secretor status further modified resistome composition among exclusively breastfed infants. Human milk oligosaccharide profiling identified specific glycans underlying these associations, with 2'-fucosyllactose and 6'-sialyllactose showing negative correlations with distinct ARG classes. These findings identify human milk composition as a key determinant of early-life resistome assembly and a potential target for modulating antimicrobial resistance.

Human milk oligosaccharides (HMOs) are bioactive components in human milk that influence infant health, yet their role in growth trajectories remains unclear. This study examined associations between concentrations of individual HMOs and child growth outcomes: length-for-age (LAZ), weight-for-age (WAZ), and weight-for-length (WLZ) z-scores—at 24 months of age in a population-based cohort from León, Nicaragua. Data were analyzed from 295 mother–infant dyads. HMO concentrations in human milk collected one month postpartum were measured using high-performance liquid chromatography with fluorescence detection. Infant growth was assessed at 10–14 days, 6, and 24 months, with z-scores calculated per WHO standards. Associations between HMOs and growth outcomes were estimated using generalized linear regression models: one unweighted model and two weighted by inverse probability of censoring to address potential bias from differences in breastfeeding duration. Models were adjusted for birth weight, infant sex, maternal secretor status, maternal age, maternal education, and household poverty. Growth associations differed by specific HMOs. 3′-sialyllactose (3′SL; β = 0.11, 95% CI: 0.02, 0.21) and difucosyllactose (DFLac; β = 0.04, 95% CI: 0.00, 0.09) were positively associated with LAZ, suggesting a role in promoting linear growth. In contrast, fucosyllacto-N-hexaose (FLNH; β = −0.07, 95% CI: −0.11, −0.01) was negatively associated with WAZ, and disialyllacto-N-hexaose (DSLNH; β = −0.13, 95% CI: −0.21, −0.05) was negatively associated with WLZ, indicating a more complex relationship with weight gain. These associations remained consistent after IPCW adjustment. Distinct HMOs were differentially associated with child growth patterns. The positive associations of 3′SL and DFLac with LAZ and the negative associations of FLNH and DSLNH with WAZ and WLZ underscore the complexity of HMO–growth relationships and highlight the need for future studies to determine whether targeted HMO modulation or supplementation could optimize early growth outcomes. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The author(s) received no specific funding for this work. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The protocol used in this study was reviewed and approved by the Ethical Committee for Biomedical Research of the Universidad Nacional Autonoma de Nicaragua UNAN-León (Acta No.2 2017) and the University of North Carolina at Chapel Hill Institutional Review Board (Study #: 16-2079, approved 9/01/2016). Written informed consent was collected from mothers and the parents of all children and minor mothers in this study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data underlying the results presented in the study contain de-identified human participant information. De-identified data may be made available upon reasonable request to the corresponding author, subject to approval by the relevant Institutional Review Board and data use agreements.

Importance:Infant milk feeding type (eg, human milk vs formula) and infant gut microbes have each been associated with differences in microbial metabolites and childhood blood pressure; however, evidence remains limited regarding how specific infant gut microbes, at a species or strain level, in combination with milk feeding type, shape microbial metabolites and blood pressure. Objective:To investigate whether human milk feeding and infant gut microbes, including Bifidobacterium longum subsp infantis (B infantis) and other milk-degrading microbes, are associated with infant fecal metabolites and childhood systolic blood pressure (SBP). Design, Setting, and Participants:This cohort study was part of the Canadian Healthy Infant Longitudinal Development (CHILD) cohort study, a prospective multicenter, contemporary, population-based cohort of pregnant mothers and their offspring recruited between 2009 and 2012. Data were collected from 2009 to 2018 and analyzed from January to December 2024. Participants included a subset of children born at 35 weeks of gestation or later without congenital abnormalities or respiratory distress syndrome and with available data on gut microbiome, fecal metabolome, SBP, and covariates. Exposures:Gut microbiome, fecal metabolome, and human milk feeding status at ages 3 months and 1 year. Main Outcomes and Measures:Age-, sex-, and height-specific SBP percentile, measured at ages 3 and 5 years. Results:A total of children (610 [46.1%] girls; 982 children [74.2%] delivered vaginally; mean [SD] maternal age at delivery, 33.3 [4.5] years) were included. At age 3 months, but not at age 1 year, human milk feeding and presence of B infantis showed interactive associations with infant fecal metabolites at ages 3 months and 1 year and SBP at ages 3 and 5 years. Among infants harboring B infantis at age 3 months, mixed feeding (difference, -14.81 [95% CI, -27.05 to -2.56] percentile) and exclusive human milk feeding (difference, -17.16 [95% CI, -29.48 to -4.83] percentile) were associated with a lower childhood SBP, whereas no association was observed among infants without B infantis. Several additional infant gut microbes (eg, Eggerthella lenta, Veillonella dispar) and fecal metabolites (eg, creatinine, succinic acid) also demonstrated feeding- or B infantis-dependent associations with childhood SBP. Conclusions and Relevance:In this cohort study, early-life interactions between human milk feeding and B infantis, among other bacteria, were associated with the infant fecal metabolome and childhood SBP, underscoring the potential importance of early-life nutrition-microbe interplay in cardiometabolic health.

Human milk oligosaccharides (HMOs) are complex carbohydrates unique to human milk, and a wealth of observational and mechanistic studies indicate that HMOs are key to infant health by supporting gut microbiota and immune development. This review synthesizes evidence from randomized clinical trials evaluating whether supplementation with human-identical milk oligosaccharides (HiMOs), i.e., synthetic HMOs, in infants and young children improves health outcomes. We identified 12 randomized clinical trials: 8 in healthy infants, 3 in special populations of infants, and 1 in young children. We selected only trials with a randomized, parallel group design; most of the included trials also had an observational human milk-fed control group. The most widely evaluated HiMO was 2'-fucosyllactose used alone or in combination with other HiMOs. In some trials, other bioactive components were included in the control and/or intervention formula groups, complicating interpretation. All trials in healthy infants confirmed the noninferiority of HiMO-supplemented formula on growth and tolerability relative to control formula. Results were mixed with respect to reductions in morbidity, and all studies were underpowered for more severe morbidity outcomes. Stool microbiota and biomarkers of inflammation and gut function generally shifted in a direction closer to human milk-fed infants with HiMO intervention. Some growth improvements were noted in association with HiMO intervention in preterm infants and in infants with severe acute malnutrition. HiMO supplementation may be a promising intervention to improve child health, but due to the heterogeneity and limitations of the clinical trials that have been undertaken, many questions remain about the nature of the benefits and the specific populations who might benefit.

BACKGROUND:Infant gut microbiota colonization is important for supporting normal development and long-term health of children. Human milk oligosaccharides (HMOs) influence the composition of the gut microbiota, but their specific effects, particularly after breastfeeding, remain poorly understood. OBJECTIVES:We aimed to deepen the understanding of how HMOs associate with the gut microbiota composition at 3 mo and at 13 mo of age. In addition, we assessed the role of HMOs as microbiome-rebalancing agents in cesarean-delivered infants. METHODS:We analyzed fecal samples from infants at 3 mo (n = 517) and 13 mo (n = 522), along with human milk samples at 3 mo, from a population-based cohort. Gut microbiota was profiled by 16S rRNA sequencing, and 19 HMOs were quantified by high-performance liquid chromatography with fluorescent detection. Dirichlet multinomial mixtures clustering was used to identify bacterial fecal community types (FCTs) and multinomial logistic regression models to study the association between HMOs and FCTs. Permutational multivariate analysis of variance and linear regression models were used to associate HMOs with gut microbiota diversity measures and Spearman correlation to bacterial genera. RESULTS:HMOs were associated with gut microbiota FCTs, diversity measures, and bacterial genera at 3 and 13 mo of age. At 3 mo, disialyllacto-N-tetraose and the structurally related lacto-N-sialyllactose b showed notable associations with the gut microbiota, whereas at 13 mo, fucodisialyllacto-N-hexaose was associated with multiple gut microbiota metrics. Maternal secretor status was associated with the gut microbiota beta diversity (R2 = 0.003, P < 0.05) and decreased Shannon diversity (b = -0.24, P < 0.05) at 3 mo, with diminishing associations at 13 mo (observed richness, b = -11, P < 0.05). Although no individual HMOs showed microbiome-rebalancing effects in cesarean-born infants, infants fed by nonsecretor mothers exhibited stronger cesarean-related microbiota patterns compared with those fed by secretors. CONCLUSIONS:HMOs exhibit age-dependent and structure-specific associations with infant gut microbiota, extending beyond breastfeeding.

PurposeTo investigate the human milk oligosaccharide (HMO) composition, longitudinal change and the influence of maternal factors in a multi-ethnic Asian cohort.MethodsIn the prospective GUSTO mother-offspring cohort, maternal sociodemographic, genetic, and obstetric characteristics were related to the concentrations of the 19 most-abundant HMOs (quantified by HPLC, n = 248 mothers) at 3-weeks (n = 205) and 3-months (n = 114) postpartum (71 matched cases, 28.6%).ResultsMothers providing samples were Chinese (73.8%), Malay (14.5%) and Indian (11.7%). Across ethnicities, individual HMO concentrations and proportions of secretors, were comparable. Approximately 70% were secretors, determined by distinctly higher 2′-fucosyllactose (2’-FL, 3-weeks: 16.0% of total HMO; 3-months: 10.4%) and lacto-N-fucopentaose-I (LNFP-I), compared with non-secretors (2’-FL, 3-weeks, 0.3%; 3-months: 0.2%). 3-fucosyllactose (3-FL) comprised 9.5% and 21.7% of total HMO concentration in secretors and non-secretors, respectively, at 3-weeks, and 30.4% and 42.9%, respectively, at 3-months. Compared with term, preterm cases had higher 3′-sialyllactose (3′SL) at 3-weeks [adjusted mean difference 1.23 SDs (95%CI 0.44–2.01); p = 0.002]. Longitudinal changes in individual HMO concentrations from 3-weeks to 3-months were generally consistent across secretor groups. HMO-secretor-phenotype showed 97% concordance with status predicted by the FUT2 single nucleotide polymorphism (SNP) rs1047781.ConclusionChinese, Malay and Indian women exhibited similar HMO compositions, with the FUT2 rs1047781 SNP being a strong determinant of secretor status. Preterm delivery may influence specific early postpartum HMO concentrations, including a higher 3′SL concentration. The concentration of 2’-FL was lower, while 3-FL was higher, compared with published reports on non-Asians, suggesting multi-ethnic studies of infant nutrition and health should consider Asian HMO biology.

BACKGROUND:Gestational diabetes mellitus (GDM) increases offspring obesity risk, but whether this occurs via changes in human milk composition, including alterations in human milk oligosaccharides (HMOs), is unknown. OBJECTIVES:This study aimed to identify differences in HMO concentrations in mothers with and without GDM and test whether GDM-associated HMOs are associated with infant growth, body composition, and fecal microbiome characteristics over the first 6-mo of life. METHODS:Human milk was collected at 1-mo postpartum from 337 females (49 with GDM) who fed their infants breastmilk exclusively. HMOs were quantified by high-performance liquid chromatography and multivariate regression models were used to test differences in HMO concentrations by GDM status (false discovery rate adjustment for multiple testing set at q < 0.05). HMOs associated with GDM were then tested for associations with infant growth, body composition, and 1 and 6-mo infant fecal microbial abundances measured by metagenomic whole-genome sequencing. RESULTS:Participants with GDM had ∼1 SD higher milk 6'sialyllactose (6'SL) {[β (95% confidence interval): 0.58 (0.20, 0.96)] and lacto-N-fucopentaose III (LNFP III) III [95% CI: 0.55 (0.16, 0.94)]} compared with those without GDM and 6'SL concentration was also positively associated with weight and length gain. Although infants of mothers with GDM had lower 1-mo fecal α-diversity and altered abundances of 6 of 56 microbial species detected compared with those without GDM, microbial features were not associated with the concentration of either 6'SL or LNFP III and evidence for mediation of GDM-growth and GDM-microbiome by HMOs was not found. CONCLUSIONS:Mothers with a GDM diagnosis had higher milk concentrations of LNFP III and 6'SL, and 6'SL was in turn associated with increased infant growth rate, but neither HMO was associated with differential infant gut microbial abundances. The results suggest that the link between 6'SL and faster infant growth, if causal, occurs via mechanisms independent of the infant gut microbiome. This study was registered at clinicaltrials.gov as NCT03301753.

Establishing the biological context of microbial metabolites remains a major challenge. We present microbiomeMASST, a metadata-driven network graph that maps metabolites across 467 available datasets with 144,424 mass spectrometry files from humans, animals, and microbial culture systems. MicrobiomeMASST integrates monocultures, synthetic communities, and host-associated samples across multiple body sites and plants. MS/MS spectra can be queried to trace occurrence across hosts, experimental conditions, and interventions, enabling cross-study integration. We demonstrate this framework by contextualizing microbial-conjugated bile acids and interrogating microbiome-mediated drug metabolism. Screening gut bacteria revealed deprolylation of the angiotensin-converting enzyme (ACE) inhibitor prodrug enalapril. Using microbiomeMASST, we traced this metabolite across human cohorts, microbial isolates, environmental samples, and in Gorilla gorilla . Structural modeling and enzymatic assays showed that microbial deprolylation abolishes ACE inhibition, thereby inactivating its therapeutic effect. Together, microbiomeMASST links MS/MS spectra to biological context, converting isolated observations into an interpretable microbiome map for cross-study analysis.

Insulin resistance (IR) has emerged as a risk factor for lactation insufficiency and delays the onset of milk secretion after childbirth, termed secretory activation (SA). This may cause inadequate infant weight gain and early breastfeeding cessation. However, the mechanisms underlying delayed SA in insulin resistant women are unknown. To investigate this, we characterized the mammary transcriptomes and IR-related hormones of 75 breastfeeding women with healthy term infants during postpartum days 1-5. Participants were divided into IR tertiles based on plasma leptin-to-adiponectin ratio measurements. Those in the highest tertile had later SA onset with greater neonatal weight loss during postpartum days 1-5. Transcriptomic analysis on postpartum day 2 (n=4 high IR vs. n=8 low IR participants) showed transient suppression of mammary insulin and prolactin signaling genes, increased pro-inflammatory gene expression and altered expression of >200 mammary mitochondrial genes. These alterations were absent on postpartum days 3-5. Cultured mammary epithelial cells (MECs) treated with insulin showed upregulation of prolactin signaling and oxidative phosphorylation (OXPHOS) genes, with imaging and bioenergetic studies demonstrating that insulin promotes mitochondrial biogenesis and OXPHOS. Thus, our findings delineate roles for insulin in mammary bioenergetics and highlight mitochondrial dysfunction as a mechanism for delayed SA in insulin resistant women.

Highly pathogenic avian influenza A (H5N1) (H5N1 hereafter) is an emerging pathogen in mammals. The recent recognition of H5N1 in dairy cattle increases opportunities for human exposure and infection and may accelerate a trajectory toward sustained human-to-human transmission. Furthermore, the presence of virus at high concentration in unpasteurized milk raises new risks for humans, especially infants and children. Milk has been identified as a vehicle for viral transmission in and between mammalian species, including humans. Sialic acids (SAs) found on cell surfaces are important mediators of species susceptibility to specific influenza strains and play an important role in viral tropism. New data demonstrate that SA receptors with α2,3 linkages capable of binding avian influenza strains are present in human mammary tissue. The presence of SA receptors that can bind avian influenza and a comparative analysis of viral transmission risk of raw and pasteurized milk in several mammalian species have implications for human milk feeding. During this period of sporadic human infections with H5N1, further research and collaboration is warranted to address the potential risk of human milk contamination. Infants and children are particularly vulnerable to emerging infections during pandemics and have unique needs that may be overlooked. Pandemic preparedness must address the needs of all populations at all life stages, including pregnancy and infancy, and must include support for the safety of human milk.

Abstract Human milk contains a diverse array of metabolites that contribute to infant nutrition, immune development, and microbial colonization. The maternal factors shaping the milk metabolome, and the relative contribution of genetics or diet vs. other factors, remain poorly understood. Here, we profiled 458 milk metabolites in 349 one-month postpartum human milk samples and integrated metabolomic data with maternal diet, clinical, transcriptomic, and genomic measurements. Maternal diet was broadly associated with milk metabolite composition, with significant correlations identified between dietary features and 323 metabolites. Coffee consumption strongly predicted milk quinic acid and 1,3-dimethyluric acid abundance, while high-fiber dietary patterns were associated with metabolites including proline-betaine and N-acetylornithine. Integration of milk transcriptomic and metabolomic data via machine learning identified biologically plausible gene-metabolite pairs, including associations between QPRT expression and quinolinic acid, and DPEP1 and cysteine-glycine dipeptide. Genome-wide association analyses identified nine study-wide significant metabolite quantitative trait loci, including novel milk-specific associations near PDE6A affecting purine metabolites and near GNE affecting free sialic acid. Comparison with plasma metabolite studies demonstrated both shared and milk-specific genetic regulation of metabolites. Finally, we found that of all tested maternal features, diet explained the largest proportion of variation in the milk metabolome. Together, these findings demonstrate that the human milk metabolome reflects both maternal exposures and mammary gland-specific biology. This work establishes a framework for understanding how genetic and environmental factors shape milk composition.

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.

Lactoferrin is an iron-binding glycoprotein in human milk (HM) that reduces the risk of neonatal sepsis. Data from low- and middle-income countries regarding the determinants of HM lactoferrin concentration is limited. Our objectives were to assess how HM lactoferrin concentrations change over time and identify factors associated with lactoferrin concentration. From a pregnancy cohort in Sylhet, Bangladesh, we enrolled 99 women to join the lactation sub-cohort and provide HM samples at a median of 50 (T1) and 146 (T2) days postpartum. We measured HM lactoferrin concentrations with meso-scale discovery and examined associations with predictors including indices of maternal nutritional status [body mass index (BMI), mid-upper arm circumference (MUAC), hemoglobin], depression scores, infant gestational age, and birthweight-for-gestational age z-score. HM lactoferrin concentration increased by 21% from T1 to T2. Higher gestational age at birth was associated with lower HM lactoferrin concentration at T1. Higher maternal MUAC was associated with higher HM lactoferrin concentration at T2. In rural Bangladeshi women, HM lactoferrin concentration increased during the postpartum period. Higher lactoferrin was associated with earlier gestational age at delivery and better maternal nutritional status. Interventions to improve maternal nutritional status might also increase HM lactoferrin concentration and ultimately, benefit child outcomes.