ObjectiveThis study investigated how HMO profiles in Chinese mothers’ milk are affected by lactation stage and delivery mode (C-section vs. vaginal).Study designThe study was based on a cross-sectional multi-center human milk study in China. Twenty-four HMOs were quantified using UPLC/MS/MS, and statistical tools were used to identify profiles and clusters of HMOs. The relationship between HMO concentrations, delivery mode and infant eczema was explored at each lactation stage.ResultsThe study included 635 mother-infant pairs. The concentrations of HMOs varied according to lactation stage and five distinct HMO clusters were observed based on significant associations between HMOs. Milk from mothers who delivered by C-section had significantly lower concentrations of 2’FL, LNnT, 3’SL, LNFP-III, DFLNHa, DSLNT, LNDFH-II, LNnDFH-II, and DFpLNnH compared to vaginal delivery. The odds ratio for eczema was reduced in infants whose mothers had higher concentrations of LSTb in mature milk (>90 days).ConclusionHMOs in Chinese human milk vary based on lactation stage and cluster based on their structure. C-section delivery lowers the concentrations of several HMOs in the milk compared to vaginal delivery. Higher LSTb inversely associated with parent-reported infant eczema. Further research is required to confirm these findings and to understand the underlying mechanisms.

During weaning period, the intake of dietary fibres changes and increases dramatically. Given the considerable structural differences, we hypothesized that different fibres may vary in their function. The objective of the study was to explore the impact of specific dietary fibres (arabinoxylan, cellulose, pectin and xyloglucan) on the gut microbiome of children below 3 years. By using in vitro fecal fermentation experiments, cellular models and cohort data analysis, we assessed how these fibres and their combinations influence infants’ gut microbiota composition, diversity, metabolite production and possible actions on the gut epithelial barrier function. We found that the fermentation with arabinoxylan, xyloglucan and pectin resulted in an increased production of short-chain fatty acids. These fibres also promoted the generation of metabolites with potential health benefits, such as indole-3-lactic acid. By combining the in vitro fermentation and cellular co-culture experiments, arabinoxylan and xyloglucan were found to maintain gut epithelial barrier integrity upon lipopolysaccharide (LPS) challenge, and a blend of cellulose, pectin, and xyloglucan dampened different LPS induced cytokines. Moreover, pectin was found to supportthe growth of a wide range of microbial species in vitro and correlated positively with α-diversity in young children in an observational cohort. Our findings provide insights into the potential benefits of diverse fibre intakes during early life. Further studies are needed to investigate the causal relationship, mechanisms and the effectiveness of specific fibres on the gut microbiome development in young children.

Early microbial colonization influences immune development, with lower abundance of human milk oligosaccharide (HMO)-utilizing bifidobacteria linked to immune-related disorder risk. Here, we demonstrate that synbiotic supplementation with Bifidobacterium infantis (LMG 11588) and a blend of six structurally diverse HMOs plus Bifidobacterium lactis (CNCM I-3446) altered gut microbiome composition, changed systemic immune cell-networks, and reduced persistence of a T helper 2 (Th2) bias in formula-fed infants, following a pattern observed in breastfed infants. In preclinical models, synbiotic reduces lower respiratory tract infection (LRI) severity and aberrant type-2 immune responses and confers lasting immune benefits into adulthood. These effects are associated with changes in the circulating metabolome, including increased 12(S)-hydroxyheptadecatrienoic acid (12-HHT), which correlates with improved infection outcomes and phenocopies synbiotic-mediated protection when administered orally. Together, these findings indicate that infant-type synbiotic supplementation during a critical early-life window can imprint immune function and promote disease tolerance.

The presence of gut microbiota-brain-axis has been widely reported. However, few studies have focused on uncovering the potential associations during a time-period that our brain and gut microbiota undergo rapid maturation. We evaluated the potential associations between characteristics of gut microbiota and cognition and temperament using an accelerated longitudinal design in typically developing children over 0-3 years of age. Specifically, we extracted gut microbiota characteristics at three scale levels: diversity measures, microbial networks, and subject-wise longitudinal trajectory features, shedding light on how associations between cognition/temperament and gut microbiota may differ at global (diversity), ecological (microbial networks) and subject-wise levels. Our findings illustrated that associations between gut microbiota and temperament/cognition varied with the analytical approaches and highlighted differential gut microbial features in association with cognition and temperament traits-diversity measures and microbial networks largely with cognition while subject-wise trajectories with temperament. In addition, Ruminococcus bromii exhibited significant associations with cognitions spanning over multiple subdomains. Finally, the associations of gut microbiota with temperament and cognition converge on the potential interplay of language ability and social behaviors and highlight the importance of age-appropriate gut microbiota on early cognition/temperament development.

Cow's milk protein allergy (CMPA) in infancy is associated with intestinal microbial dysbiosis, characterised by low Bifidobacteriaceae levels. The present study aimed to investigate the impact of two human milk oligosaccharides (HMO), lactose (L), and their combination on the faecal microbiome and metabolome of infants with CMPA. Stool samples of 12 term infants with probable CMPA (mean age 4.3 months) were analysed using a validated intestinal fermentation assay (SIFWtechnology). For each substrate (i.e. HMO (2'-fucosyllactose [2'-FL] and lacto-N-neotetraose [LNnT]), L and HMO + L), taxonomic microbiome characterisation and untargeted metabolite profiling were performed at multiple timepoints. At baseline, the tested faecal microbiota overall displayed low abundances of Bifidobacteriaceae. Fermentation with either HMO or lactose significantly enriched Bifidobacterium The increase in HMO-utilising bifidobacteria was associated with a significant rise in levels of short-chain fatty acids, aromatic lactic acids and N-acetylated amino acids, with additive effects being observed for HMO + L. The above data suggest that the combination of 2'-FL, LNnT and lactose helps to alleviate the previously reported CMPA-associated intestinal bacterial dysbiosis and induces the production of several beneficial metabolites. The clinical significance of these findings for infants with CMPA requires further investigation.

Background and aimsGut health and microbiome development are closely linked in early life, with human milk oligosaccharides (HMOs) playing a key role. This study reports results through 4 months of age from a trial evaluating an infant formula containing a synbiotic blend of HMOs and probiotics, focusing on growth, gastrointestinal (GI) tolerance, and gut health biomarkers from birth to 15 months.Materials and methodsHealthy infants aged ≤14 days were randomized to receive either the experimental formula (SYN; control formula supplemented with six HMOs and two probiotics [B. infantis, B. lactis]) or the control formula (CTRL; partially hydrolyzed 100% whey-based formula). A non-randomized breastfed (BF) group served as a reference. The primary endpoint was weight gain velocity in SYN vs. CTRL through 4 months of age. Secondary endpoints included fecal outcomes (abundance of bifidobacteria, immune and gut health markers), GI tolerance, and adverse events (AEs).ResultsThe full analysis set (FAS) included 313 infants (118 in SYN, 114 in CTRL, and 81 BF), while the per-protocol population (PP) included 227 infants (84 in SYN, 84 in CTRL, and 59 BF). Weight gain velocity through 4 months in the SYN group was non-inferior to that in the CTRL group in both FAS and PP analyses (both p < 0.0001). Parent-reported GI tolerance and stool patterns were similar between SYN and CTRL groups through 4 months. At 3 months, Bifidobacteria abundance was significantly higher in the SYN group compared to the CTRL group (p = 0.004). Fecal pH was lower in the SYN group than in the CTRL group (p = 0.018) and more closely resembled that of the BF group. Immune and gut health markers were similar between the SYN and BF groups. No significant differences in AEs were observed across groups.ConclusionThe synbiotic-supplemented infant formula supported healthy, age-appropriate growth, good GI tolerance, and increased the abundance of beneficial bifidobacteria through 4 months of age.Clinical trial registrationhttps://clinicaltrials.gov/study/NCT04962594.

Human milk is the recommended sole source of nutrition for infants during the first 6 months of age, thanks to its composition rich in nutritious and bioactive components. Progress in analytics has allowed for a detailed description of its components and their variability within and among mothers. This is especially valid for the human milk oligosaccharides (HMOs) that represent one of the major human milk compound groups. The stages of lactation and maternal genotypes are the main contributors to the variability of HMOs, although other maternal and environmental factors also contribute to the variation, which may be important for adaptation in evolutionary terms. Today, mainly individual HMOs or structural groups of HMOs were associated with infant outcome measures, ranging from anthropometry to immunity and brain development (social and cognitive skills). Mechanistic insights can partly explain some findings, yet there is a lack of consistency between the different observational studies of breastfed infants. Gaining a better understanding of the reasons behind these disparate findings is the key element going forward. Furthermore, studying human milk components, like HMOs, and their expected benefits using a systems biology approach can reveal further important insights. Here, we discuss recent findings with the perspective to learn more about the link to health outcomes.

Human Milk Oligosaccharides (HMOs) have been proposed to be instrumental in building immune competence. To explore the role of HMOs in allergy prevention, twenty-one HMOs were quantified in breast milk samples and associated with sensitisation in infants. 2 '-fucosyllactose (2 '-FL) levels were positively associated with an increased risk of sensitisation, atopic dermatitis and recurrent skin rash. Interestingly, 2 '-FL levels, ranging from 1.35 to 1.95 g/L, were associated with a higher prevalence of non-allergic and non-sensitised infants. The role of 2 '-FL and lacto-N-neotetraose (LNnT) was further investigated in allergic sensitisation models in vivo. Oral administration of HMOs decreased allergic sensitisation. This was associated with gut microbiota and short-chain fatty acid (SCFA) production changes. Aligned with the clinical associations, the decreased sensitisation was not observed with lower and higher tested doses of the HMOs supporting a U-shape association between 2 '-FL and LNnT levels and allergic sensitisation risk reduction in humans and mice.Trial registration: ClinicalTrials.gov identifier: NCT02550236.

The community of microorganisms colonizing the gut changes during the first postnatal years of life. This ecosystem, henceforth described as the microbiome, modulates infant physiology and health, but uncertainty remains about the significance of variation in microbiome composition and function. Some may be tolerable, yet some microbiomes may be less healthy than others. Most efforts to identify parameters of microbiome health focus on adults, and derived concepts may not directly translate to early life that is characterized by dynamic and sequential changes. Data suggest that an orderly progression from an immature neonatal microbiome to a mature adult state is preferable to delayed or over-rapid development. This can be parameterized as a "microbiome development trajectory". Diet modifies early life microbiome development and is the principal modifiable factor to this end. Infants fed with infant formulas show different microbiome development trajectories from breastfed infants. Early data suggest that formulas containing a specific blend of human milk oligosaccharides partially mitigate this difference. Introduction of a complementary diet complexifies the identification of diet-microbiome development interactions. A better understanding will only be achievable through detailed, longitudinal characterization of large cohorts.

Human milk contains all nutritive and bioactive compounds to give infants the best possible start in life. Human milk bioactives cover a broad range of components, including immune cells, antimicrobial proteins, microbes, and human milk oligosaccharides (HMOs). Over the last decade, HMOs have gained special attention as their industrial production has allowed the study of their structure-function relation in reductionist experimental setups. This has shed light on how HMOs steer microbiome and immune system development in early life but also how HMOs affect infant health (e.g., antibiotic use, respiratory tract infections). We are on the verge of a new era where we can examine human milk as a complex biological system. This allows not only study of the mode of action and causality of individual human milk components but also investigation of synergistic effects that might exist between different bioactives. This new wave in human milk research is largely fueled by significant advances in analytical tools in the field of systems biology and network analysis. It will be exciting to explore how human milk composition is affected by different factors, how different human milk compounds work together, and how this influences healthy infant development.

Cow’s milk protein allergy (CMPA) is the most common food allergy in infancy. CMPA is associated with gut microbial dysbiosis, characterized by low Bifidobacteriaceae levels. This study investigated the impact of a human milk oligosaccharide (HMO) mixture of 2’-fucosyllactose (2’-FL) and lacto- N -neotetraose (LNnT), as well as lactose (L), on the fecal microbiome of infants with clinically diagnosed CMPA (n=12), using the ex vivo intestinal fermentation SIFR technology. The fecal microbiota displayed considerable heterogeneity, with low abundances of Bifidobacteriaceae found in most infants, except two breastfed infants. HMO and lactose both enriched Bifidobacteriaceae via stimulation of Operational Taxonomic Units (OTUs) related to B. breve , B. longum , B. pseudocatenulatum and, for HMO+L, also B. bifidum . All supplementations significantly boosted the production of acetate, propionate, aromatic lactic acids and N-acetylated amino acids, with additive effects observed for combining HMO+L. Additional contributors to HMO fermentation were OTUs related to Bacteroides fragilis, Blautia obeum/wexlerae and Ruminococcus gnavus, while OTUs related to Escherichia coli and Veillonella parvula were involved in lactose fermentation, suggesting involvement of substrate-specific fermentation pathways. In conclusion, the observed bifidogenic effect of HMO confirms the potential of 2’-FL and LNnT to ameliorate CMPA-associated dysbiosis, with additive effects found for the abundance of B. bifidum and bifidobacterial metabolite levels. The clinical significance of the partial reversal of fecal microbial dysbiosis and enhanced production of beneficial metabolites on the clinical course of CMPA requires further investigation.

Infancy is a critical period for neurodevelopment, which includes myelination, synaptogenesis, synaptic pruning, and the development of motor, social-emotional, and cognitive functions. Human milk provides essential nutrients to the infant’s developing brain, especially during the first postnatal months. Human milk oligosaccharides (HMOs) are a major component of human milk, and there is growing evidence of the association of individual HMOs with cognitive development in early life. However, to our knowledge, no study has explained these associations with a mechanism of action. Here, we investigated possible mediating associations between HMOs in human milk, brain myelination (measured via myelin water fraction), and measures of motor, language (collected via the Bayley Scales of Infant and Toddler Development (Bayley-III)), and socioemotional development (collected via the Ages and Stages Questionnaire: Social-Emotional Version (ASQ-SE)) in healthy term-born breast-fed infants. The results revealed an association between 6′Sialyllactose and social skills that was mediated by myelination. Furthermore, associations of fucosylated HMOs with language outcomes were observed that were not mediated by myelination. These observations indicate the roles of specific HMOs in neurodevelopment and associated functional outcomes, such as social-emotional function and language development.

Summary The gut microbiome changes rapidly under the influence of different factors such as age, dietary changes or medications to name just a few. To analyze and understand such changes we present a microbiome analysis toolbox. We implemented several methods for analysis and exploration to provide interactive visualizations for easy comprehension and reporting of longitudinal microbiome data. Based on abundance of microbiome features such as taxa as well as functional capacity modules, and with the corresponding metadata per sample, the toolbox includes methods for 1) data analysis and exploration, 2) data preparation including dataset-specific preprocessing and transformation, 3) best feature selection for log-ratio denominators, 4) two-group analysis, 5) microbiome trajectory prediction with feature importance over time, 6) spline and linear regression statistical analysis for testing universality across different groups and differentiation of two trajectories, 7) longitudinal anomaly detection on the microbiome trajectory, and 8) simulated intervention to return anomaly back to a reference trajectory. Availability and implementation The software tools are open source and implemented in Python. The link to the interactive dashboard is https://microbiome-toolbox.herokuapp.com/. For developers interested in additional functionality of the toolbox, the Python package can be downloaded from https://pypi.org/project/microbiome-toolbox/. The toolbox is modular allowing for further extension with custom methods and analysis. The code is available on Github https://github.com/JelenaBanjac/microbiome-toolbox. Contact ShaillayKumar.Dogra@rd.nestle.com Supplementary Information Supplementary data are available at Bioinformatics online.

Cow’s milk protein allergy (CMPA) is a prevalent food allergy among infants and young children. We conducted a randomized, multicenter intervention study involving 194 non-breastfed infants with CMPA until 12 months of age (clinical trial registration: NCT03085134). One exploratory objective was to assess the effects of a whey-based extensively hydrolyzed formula (EHF) supplemented with 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT) on the fecal microbiome and metabolome in this population. Thus, fecal samples were collected at baseline, 1 and 3 months from enrollment, as well as at 12 months of age. Human milk oligosaccharides (HMO) supplementation led to the enrichment of bifidobacteria in the gut microbiome and delayed the shift of the microbiome composition toward an adult-like pattern. We identified specific HMO-mediated changes in fecal amino acid degradation and bile acid conjugation, particularly in infants commencing the HMO-supplemented formula before the age of three months. Thus, HMO supplementation partially corrected the dysbiosis commonly observed in infants with CMPA. Further investigation is necessary to determine the clinical significance of these findings in terms of a reduced incidence of respiratory infections and other potential health benefits.

While ample research on independent associations between infant cognition and gut microbiota composition and human milk (HM) oligosaccharides (HMOs) has been reported, studies on how the interactions between gut microbiota and HMOs may yield associations with cognitive development in infancy are lacking. We aimed to determine how HMOs and species of Bacteroides and Bifidobacterium genera interact with each other and their associations with cognitive development in typically developing infants. A total of 105 mother-infant dyads were included in this study. The enrolled infants [2.9–12 months old (8.09 ± 2.48)] were at least predominantly breastfed at 4 months old. A total of 170 HM samples from the mothers and fecal samples of the children were collected longitudinally. Using the Mullen Scales of Early Learning to assess cognition and the scores as the outcomes, linear mixed effects models including both the levels of eight HMOs and relative abundance of Bacteroides and Bifidobacterium species as main associations and their interactions were employed with adjusting covariates; infant sex, delivery mode, maternal education, site, and batch effects of HMOs. Additionally, regression models stratifying infants based on the A-tetrasaccharide (A-tetra) status of the HM they received were also employed to determine if the associations depend on the A-tetra status. With Bacteroides species, we observed significant associations with motor functions, while Bif. catenulatum showed a negative association with visual reception in the detectable A-tetra group both as main effect (value of p = 0.012) and in interaction with LNFP-I (value of p = 0.007). Additionally, 3-FL showed a positive association with gross motor (p = 0.027) and visual reception (p = 0.041). Furthermore, significant associations were observed with the interaction terms mainly in the undetectable A-tetra group. Specifically, we observed negative associations for Bifidobacterium species and LNT [breve (p = 0.011) and longum (p = 0.022)], and positive associations for expressive language with 3′-SL and Bif. bifidum (p = 0.01), 6′-SL and B. fragilis (p = 0.019), and LNFP-I and Bif. kashiwanohense (p = 0.048), respectively. Our findings suggest that gut microbiota and HMOs are both independently and interactively associated with early cognitive development. In particular, the diverse interactions between HMOs and Bacteroides and Bifidobacterium species reveal different candidate pathways through which HMOs, Bifidobacterium and Bacteroides species potentially interact to impact cognitive development in infancy.

Background Human milk oligosaccharides (HMOs) have important biological functions for a healthy development in early life. Objective This study aimed to investigate gut maturation effects of an infant formula containing five HMOs (2′-fucosyllactose, 2′,3-di-fucosyllactose, lacto-N-tetraose, 3′-sialyllactose, and 6′-sialyllactose). Methods In a multicenter study, healthy infants (7–21 days old) were randomly assigned to a standard cow’s milk-based infant formula (control group, CG); the same formula with 1.5 g/L HMOs (test group 1, TG1); or with 2.5 g/L HMOs (test group 2, TG2). A human milk-fed group (HMG) was enrolled as a reference. Fecal samples collected at baseline (n∼150/formula group; HMG n = 60), age 3 (n∼140/formula group; HMG n = 65) and 6 (n∼115/formula group; HMG n = 60) months were analyzed for microbiome (shotgun metagenomics), metabolism, and biomarkers. Results At both post-baseline visits, weighted UniFrac analysis indicated different microbiota compositions in the two test groups (TGs) compared to CG (P < 0.01) with coordinates closer to that of HMG. The relative abundance of Bifidobacterium longum subsp. infantis (B. infantis) was higher in TGs vs. CG (P < 0.05; except at 6 months: TG2 vs. CG P = 0.083). Bifidobacterium abundance was higher by ∼45% in TGs vs. CG at 6-month approaching HMG. At both post-baseline visits, toxigenic Clostridioides difficile abundance was 75–85% lower in TGs vs. CG (P < 0.05) and comparable with HMG. Fecal pH was significantly lower in TGs vs. CG, and the overall organic acid profile was different in TGs vs. CG, approaching HMG. At 3 months, TGs (vs. CG) had higher secretory immunoglobulin A (sIgA) and lower alpha-1-antitrypsin (P < 0.05). At 6 months, sIgA in TG2 vs. CG remained higher (P < 0.05), and calprotectin was lower in TG1 (P < 0.05) vs. CG. Conclusion Infant formula with a specific blend of five HMOs supports the development of the intestinal immune system and gut barrier function and shifts the gut microbiome closer to that of breastfed infants with higher bifidobacteria, particularly B. infantis, and lower toxigenic Clostridioides difficile. Clinical Trial Registration [https://clinicaltrials.gov/ct2/show/], identifier [NCT03722550].

Human milk oligosaccharides play a key role in the maturation of the infant gut microbiome and immune system and are hypothesized to affect growth. This study examined the temporal changes of 24 HMOs and their associations to infant growth and appetitive traits in an exploratory, prospective, observational, study of 41 Filipino mother-infant dyads. Exclusively breastfed, healthy, term infants were enrolled at 21–26 days of age (≈ 0.75 mo) and followed for 6 months. Infant growth measures and appetitive traits were collected at visit 1 (V1) (≈ 0.75 mo), V2 (≈ 1.5 mo), V3 (2.5 mo), V4 (2.75 mo), V5 (4 mo), and V6 (6 mo), while HMOs were measured at V1, V2, V3 and V5. Overall exposure to each HMO was summarized as area under the curve from baseline to 4 months of age and examined in association with each measure of growth at 6 months using linear regression adjusted for maternal age at birth, infant sex, birth weight, and mode of delivery. We saw modest associations between several HMOs and infant growth parameters. Our results suggest that specific HMOs, partly as proxy for milk groups (defined by Secretor and Lewis status), may be associated with head circumference and length, increasing their relevance especially in populations at the lower end of the WHO growth curve. We did not identify the same HMOs associated with infant appetitive traits, indicating that at least in our cohort, changes in appetite were not driving the observed associations between HMOs and growth. Clinical trial registration: NCT03387124.

Abstract Background Human milk oligosaccharides (HMOs) have important and diverse biological functions in early life. This study tested the safety and efficacy of a starter infant formula containing Limosilactobacillus (L.) reuteri DSM 17938 and supplemented with 2’-fucosyllactose (2’FL). Methods Healthy infants < 14 days old (n = 289) were randomly assigned to a bovine milk-based formula containing L. reuteri DSM 17938 at 1 × 107 CFU/g (control group; CG) or the same formula with added 1.0 g/L 2’FL (experimental group; EG) until 6 months of age. A non-randomized breastfed group served as reference (BF; n = 60). The primary endpoint was weight gain through 4 months of age in the formula-fed infants. Secondary endpoints included additional anthropometric measures, gastrointestinal tolerance, stooling characteristics, adverse events (AEs), fecal microbiota and metabolism, and gut immunity and health biomarkers in all feeding groups. Results Weight gain in EG was non-inferior to CG as shown by a mean difference [95% CI] of 0.26 [-1.26, 1.79] g/day with the lower bound of the 95% CI above the non-inferiority margin (-3 g/day). Anthropometric Z-scores, parent-reported stooling characteristics, gastrointestinal symptoms and associated behaviors, and AEs were comparable between formula groups. Redundancy analysis indicated that the microbiota composition in EG was different from CG at age 2 (p = 0.050) and 3 months (p = 0.052), approaching BF. Similarly, between sample phylogenetic distance (weighted UniFrac) for BF vs EG was smaller than for BF vs CG at 3-month age (p = 0.045). At age 1 month, Clostridioides difficile counts were significantly lower in EG than CG. Bifidobacterium relative abundance in EG tracked towards that in BF. Fecal biomarkers and metabolic profile were comparable between CG and EG. Conclusion L. reuteri-containing infant formula with 2’FL supports age-appropriate growth, is well-tolerated and may play a role in shifting the gut microbial pattern towards that of breastfed infants. Trial Registration The trial was registered on ClinicalTrials.gov ( NCT03090360 ) on 24/03/2017.