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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.

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., Competing Interests: TS, JH, and NS are employees of Société des Produits Nestlé, SA, Switzerland. This study was supported in part by a grant from Nestlé Product Technology Center-Nutrition, Société des Produits Nestlé S.A., Switzerland. WL was a consultant of and received travel support from Nestlé SA, Switzerland. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cho, Samuel, Li, Howell, Baluyot, Hazlett, Elison, Zhu, Hauser, Sprenger and Lin.)

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., (© 2023 S. Karger AG, Basel.)

Motivation: 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 Toolbox. We implemented several methods for analysis and exploration to provide interactive visualizations for easy comprehension and reporting of longitudinal microbiome data., Results: Based on the abundance of microbiome features such as taxa as well as functional capacity modules, and with the corresponding metadata per sample, the Microbiome Toolbox includes methods for (i) data analysis and exploration, (ii) data preparation including dataset-specific preprocessing and transformation, (iii) best feature selection for log-ratio denominators, (iv) two-group analysis, (v) microbiome trajectory prediction with feature importance over time, (vi) spline and linear regression statistical analysis for testing universality across different groups and differentiation of two trajectories, (vii) longitudinal anomaly detection on the microbiome trajectory and (viii) simulated intervention to return anomaly back to a reference trajectory., Availability and Implementation: The software tools are open source and implemented in Python. For developers interested in additional functionality of the Microbiome Toolbox, it is modular allowing for further extension with custom methods and analysis. The code, python package and the link to the interactive dashboard of Microbiome Toolbox are available on GitHub https://github.com/JelenaBanjac/microbiome-toolbox., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press.)

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., (© 2022. The Author(s).)

Early dietary exposure via human milk nutrients offers a window of opportunity to support cognitive and temperament development. While several studies have focused on associations of few pre-selected human milk nutrients with cognition and temperament, it is highly plausible that human milk nutrients synergistically and jointly support cognitive and behavioral development in early life. We aimed to discern the combined associations of three major classes of human milk nutrients with cognition and temperament during the first 6 months of life when human milk is the primary source of an infant's nutrition and explore whether there were persistent effects up to 18 months old. The Mullen Scales of Early Learning and Infant Behavior Questionnaires-Revised were used to assess cognition and temperament, respectively, of 54 exclusively/predominantly breastfed infants in the first 6 months of life, whose follow-ups were conducted at 6-9, 9-12, and 12-18 months old. Human milk samples were obtained from the mothers of the participants at less than 6 months of age and analyzed for fatty acids [total monounsaturated fatty acids, polyunsaturated fatty acid, total saturated fatty acid (TSFA), arachidonic acid (ARA), docosahexaenoic acid (DHA), ARA/DHA, omega-6/omega-3 polyunsaturated fatty acids ratio (n-6/n-3)], phospholipids [phosphatidylcholine, phosphatidylethanolamine (PE), phosphatidylinositol (PI), sphingomyelin], and choline [free choline, phosphocholine (PCho), glycerophosphocholine]. Feature selection was performed to select nutrients associated with cognition and temperament. The combined effects of selected nutrients were analyzed using multiple regression. A positive association between the arachidonic acid (ARA) and surgency was observed ( p = 0.024). A significant effect of DHA, n-6/n-3, PE, and TSFA concentrations on receptive language ( R 2 = 0.39, p = 0.025) and the elevated ARA, PCho, and PI with increased surgency ( R 2 = 0.43, p = 0.003) was identified, suggesting that DHA and ARA may have distinct roles for temperament and language functions. Furthermore, the exploratory association analyses suggest that the effects of human milk nutrients on R.L. and surgency may persist beyond the first 6 months of life, particularly surgency at 12-18 months ( p = 0.002). Our study highlighted that various human milk nutrients work together to support the development of cognition and temperament traits during early infancy., Competing Interests: TS, JH, and NS were the employees of Société des Produits Nestlé SA, Switzerland. WL was a consultant of and received travel support from Nestlé SA, Switzerland. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Samuel, Zhu, Howell, Cho, Baluyot, Hazlett, Elison, Wu, Hauser, Sprenger, Zhu and Lin.)

Human milk oligosaccharides (HMOs) are structurally diverse oligosaccharides present in breast milk, supporting the development of the gut microbiota and immune system. Previously, 2-HMO (2'fucosyllactose, lacto- N -neotetraose) compared to control formula feeding was associated with reduced risk of lower respiratory tract infections (LRTIs), in part linked to lower acetate and higher bifidobacteria proportions. Here, our objective was to gain further insight into additional molecular pathways linking the 2-HMO formula feeding and LRTI mitigation. From the same trial, we measured the microbiota composition and 743 known biochemical species in infant stool at 3 months of age using shotgun metagenomic sequencing and untargeted mass spectrometry metabolomics. We used multivariate analysis to identify biochemicals associated to 2-HMO formula feeding and LRTI and integrated those findings with the microbiota compositional data. Three molecular pathways stood out: increased gamma-glutamylation and N -acetylation of amino acids and decreased inflammatory signaling lipids. Integration of stool metagenomic data revealed some Bifidobacterium and Bacteroides species to be implicated. These findings deepen our understanding of the infant gut/microbiome co-metabolism in early life and provide evidence for how such metabolic changes may influence immune competence at distant mucosal sites such as the airways., (Copyright © 2022 Martin, Tytgat, Krogh Pedersen, Moine, Eklund, Berger and Sprenger.)

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]., Competing Interests: This study received funding from Nestlé Nutrition, Société des Produits Nestlé S.A., Switzerland. DG, NS, BB, and CC were current employees of the funder. The funder had the following involvement with the study: study design, data analysis, decision to publish, and preparation of the manuscript. HP, OL, and AE were employees of Clinical Microbiomics, Denmark, which was involved in the sample and data analysis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bosheva, Tokodi, Krasnow, Pedersen, Lukjancenko, Eklund, Grathwohl, Sprenger, Berger, Cercamondi and 5 HMO Study Investigator Consortium.)

Human milk oligosaccharides (HMOs) have been researched by scientists for over 100 years, driven by the substantial evidence for the nutritional and health benefits of mother's milk. Yet research has truly bloomed during the last decade, thanks to progress in biotechnology, which has allowed the production of large amounts of bona fide HMOs. The availability of HMOs has been particularly crucial for the renewed interest in HMO research because of the low abundance or even absence of HMOs in farmed animal milk. This interest is reflected in the increasing number of original research publications and reviews on HMOs. Here, we provide an overview and critical discussion on structure-function relations of HMOs that highlight why they are such interesting and important components of human milk. Clinical observations in breastfed infants backed by basic research from animal models provide guidance as to what physiological roles for HMOs are to be expected. From an evidence-based nutrition viewpoint, we discuss the current data supporting the clinical relevance of specific HMOs based on randomised placebo-controlled clinical intervention trials in formula-fed infants., (© 2022 The Authors. Journal of Human Nutrition and Dietetics published by John Wiley & Sons Ltd on behalf of British Dietetic Association.)

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 × 10 7  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., (© 2022. The Author(s).)

The Apple Watch is capable of recording single-lead electrocardiograms (ECGs). To incorporate such devices in routine medical care, the reliability of such devices to obtain precordial leads needs to be validated. The purpose of this study was to assess the feasibility and reliability of a smartwatch (SW) to obtain precordial leads compared to standard ECGs. We included 100 participants (62 male, aged 62.8 ± 13.1 years) with sinus rhythm and recorded a standard 12-lead ECG and the precordial leads with the Apple Watch. The ECGs were quantitively compared. A total of 98 patients were able to record precordial leads without assistance. A strong correlation was observed between the amplitude of the standard and SW-ECGs' waves, in terms of P waves, QRS-complexes, and T waves (all p -values < 0.01). A significant correlation was observed between the two methods regarding the duration of the ECG waves (all p -values < 0.01). Assessment of polarity showed a significant and a strong concordance between the ECGs' waves in all six leads (91-100%, all p -values < 0.001). In conclusion, 98% of patients were able to record precordial leads using a SW without assistance. The SW is feasible and reliable for obtaining valid precordial-lead ECG recordings as a validated alternative to a standard ECG.

Background: Bovine milk-derived oligosaccharides (MOS) containing primarily galacto-oligosaccharides with inherent concentrations of sialylated oligosaccharides can be added to infant formula to enhance the oligosaccharide profile., Objective: To investigate the effects of an MOS-supplemented infant formula on gut microbiota and intestinal immunity., Methods: In a double-blind, randomized, controlled trial, healthy term formula-fed infants aged 21-26 d either received an intact protein cow milk-based formula (control group, CG, n = 112) or the same formula containing 7.2 g MOS/L (experimental group, EG, n = 114) until the age of 6 mo. Exclusively human milk-fed infants (HFI, n = 70) from an observational study served as the reference. Fecal samples collected at baseline, and the ages of 2.5 and 4 mo were assessed for microbiota (16S ribosomal RNA-based approaches), metabolites, and biomarkers of gut health and immune response., Results: Aged 2.5 and 4 mo, redundancy analysis (P = 0.002) and average phylogenetic distance (P < 0.05) showed that the overall microbiota composition in EG was different from CG and closer to that of HFI. Similarly, EG caesarean-born infants were different from CG caesarean- or vaginally born infants and approaching HFI vaginally born infants. Relative bifidobacteria abundance was higher in EG compared with CG (P < 0.05) approaching HFI. At the age of 4 mo, counts of Clostridioides difficile and Clostridium perfringens were ∼90% (P < 0.001) and ∼65% (P < 0.01) lower in EG compared with CG, respectively. Geometric LS mean (95% CI) fecal secretory IgA in EG was twice that of CG [70 (57, 85) compared with 34 (28, 42) mg/g, P < 0.001] and closer to HFI. Fecal oral polio vaccine-specific IgA was ∼50% higher in EG compared with CG (P = 0.065). Compared with CG, EG and HFI had lower fecal calcium excretion (by ∼30%, P < 0.005) and fecal pH (P < 0.001), and higher lactate concentration (P < 0.001)., Conclusions: Infant formula with MOS shifts the gut microbiota and metabolic signature closer to that of HFI, has a strong bifidogenic effect, reduces fecal pathogens, and improves the intestinal immune response., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Background & Aims: High hydrostatic pressure (HHP) processing is a non-thermal method proposed as an alternative to Holder pasteurization (HoP) for the treatment of human milk. HHP preserves numerous milk bioactive components that are degraded by HoP, but no data are available for milk oligosaccharides (HMOs) or the formation of Maillard reaction products, which may be deleterious for preterm newborns., Methods: We evaluated the impact of HHP processing of human milk on 22 HMOs measured by liquid chromatography with fluorescence detection and on furosine, lactuloselysine, carboxymethyllysine (CML) and carboxyethyllysine (CEL) measured by liquid chromatography with tandem mass spectrometric detection (LC-MS/MS), four established indicators of the Maillard reaction. Human raw milk was sterilized by HoP (62.5 °C for 30 min) or processed by HHP (350 MPa at 38 °C)., Results: Neither HHP nor HoP processing affected the concentration of HMOs, but HoP significantly increased furosine, lactuloselysine, CML and CEL levels in milk., Conclusions: Our findings demonstrate that HPP treatment preserves HMOs and avoids formation of Maillard reaction products. Our study confirms and extends previous findings that HHP treatment of human milk provides safe milk, with fewer detrimental effects on the biochemically active milk components than HoP., Competing Interests: Conflict of interest The authors declare no conflict of interest. NS is an employee of Société des Produits Nestlé S.A. Switzerland., (Copyright © 2021 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Acute respiratory infections (ARIs) are one of the most common causes of morbidity and mortality in young children. The aim of our study was to examine whether variation in maternal FUT2 (α1,2-fucosyltransferase 2) and FUT3 (α1,3/4-fucosyltransferase 3) genes, which shape fucosylated human milk oligosaccharides (HMOs) in breast milk, are associated with the occurrence of ARIs in breastfed infants as well as the influence of the nasopharyngeal microbiome on ARI risk. Occurrences of ARIs were prospectively recorded in a cohort of 240 breastfed Bangladeshi infants from birth to 2 years. Secretor and Lewis status was established by sequencing of FUT2/3 genes. The nasopharyngeal microbiome was characterized by shotgun metagenomics, complemented by specific detection of respiratory pathogens; 88.6% of mothers and 91% of infants were identified as secretors. Maternal secretor status was associated with reduced ARI incidence among these infants in the period from birth to 6 months (incidence rate ratio [IRR], 0.66; 95% confidence interval [CI], 0.47 to 0.94; P =  0.020), but not at later time periods. The nasopharyngeal microbiome, despite precise characterization to the species level, was not predictive of subsequent ARIs. The observed risk reduction of ARIs among infants of secretor mothers during the predominant breastfeeding period is consistent with the hypothesis that fucosylated oligosaccharides in human milk contribute to protection against respiratory infections. However, we found no evidence that modulation of the nasopharyngeal microbiome influenced ARI risk. IMPORTANCE The observed risk reduction of acute respiratory infections (ARIs) among infants of secretor mothers during the predominant breastfeeding period is consistent with the hypothesis that fucosylated oligosaccharides in human milk contribute to protection against respiratory infections. Respiratory pathogens were only weak modulators of risk, and the nasopharyngeal microbiome did not influence ARI risk, suggesting that the associated protective effects of human milk oligosaccharides (HMOs) are not conveyed via changes in the nasopharyngeal microbiome. Our observations add to the evidence for a role of fucosylated HMOs in protection against respiratory infections in exclusively or predominantly breastfed infants in low-resource settings. There is no indication that the nasopharyngeal microbiome substantially modulates the risk of subsequent mild ARIs. Larger studies are needed to provide mechanistic insights on links between secretor status, HMOs, and risk of respiratory infections.

Background: The relationship between human milk oligosaccharides (HMO) and child growth has been investigated only insufficiently with ambiguous results. Therefore, this study examines potential influencing factors of HMO concentrations and how HMO are associated with child growth parameters., Methods: Milk samples from the German LIFE Child cohort of healthy children were analyzed for 9 HMO. Putative associations with maternal and child cofactors and child height, head circumference and BMI between 3 months and 7 years of age were examined. Secretor status, defined as the presence of 2'-fucosyllactose, was investigated for associations with infant outcomes., Results: Our population consisted of 21 (14.7%) non-secretor and 122 (85.3%) secretor mothers. Maternal age was significantly associated with higher 3'SL concentrations; gestational age was associated with LNT, 6'SL and LNFP-I. Pre-pregnancy BMI was negatively associated with LNnT only in non-secretors. The growth velocity of non-secretors' children was inversely associated with LNnT at 3 months to 1 year (R = 0.95 [0.90, 0.99], p = 0.014), 1 to 2 years (R = 0.80 [0.72, 0.88], p < 0.001) and 5 to 6 years (R = 0.71 [0.57, 0.87], p = 0.002). 2'FL was negatively associated with BMI consistently, reaching statistical significance at 3 months and 4 and 5 years. Children of non-secretors showed higher BMI at 3 months, 6 months, and 3, 6, and 7 years of age., Conclusion: We found that some associations between HMO and infant growth may extend beyond the infancy and breastfeeding periods. They highlight the importance of both maternal and infant parameters in the understanding of the underlying associations., Trial Registration: The study is registered with ClinicalTrial.gov: NCT02550236 ., (© 2021. The Author(s).)

Early life is characterized by developmental milestones such as holding up the head, turning over, sitting up and walking that are typically achieved sequentially in specific time windows. Similarly, the early gut microbiome maturation can be characterized by specific temporal microorganism acquisition, colonization and selection with differential functional features over time. This orchestrated microbial sequence occurs from birth during the first years of age before the microbiome reaches an adult-like composition and function between 3 and 5 years of age. Increasingly, these different steps of microbiome development are recognized as crucial windows of opportunity for long term health, primarily linked to appropriate immune and metabolic development. For instance, microbiome disruptors such as preterm and Cesarean-section birth, malnutrition and antibiotic use are associated with increased risk to negatively affect long-term immune and metabolic health. Different age discriminant microbiome taxa and functionalities are used to describe age-appropriate microbiome development, and advanced modelling techniques enable an understanding and visualization of an optimal microbiome maturation trajectory. Specific microbiome features can be related to later health conditions, however, whether such features have a causal relationship is the topic of intense research. Early life nutrition is an important microbiome modulator, and 'Mother Nature' provides the model with breast milk as the sole source of nutrition for the early postnatal period, while dietary choices during the prenatal and weaning period are to a large extent guided by tradition and culture. Increasing evidence suggests prenatal maternal diet and infant and child nutrition impact the infant microbiome trajectory and immune competence development. The lack of a universal feeding reference for such phases represents a knowledge gap, but also a great opportunity to provide adequate nutritional guidance to maintain an age-appropriate microbiome for long term health. Here, we provide a narrative review and perspective on our current understanding of age-appropriate microbiome maturation, its relation to long term health and how nutrition shapes and influences this relationship.

(1) Background: Human milk oligosaccharides (HMOs) may support immune protection, partly via their action on the early-life gut microbiota. Exploratory findings of a randomized placebo-controlled trial associated 2'fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) formula feeding with reduced risk for reported bronchitis and lower respiratory tract illnesses (LRTI), as well as changes in gut microbiota composition. We sought to identify putative gut microbial mechanisms linked with these clinical observations. (2) Methods: We used stool microbiota composition, metabolites including organic acids and gut health markers in several machine-learning-based classification tools related prospectively to experiencing reported bronchitis or LRTI, as compared to no reported respiratory illness. We performed preclinical epithelial barrier function modelling to add mechanistic insight to these clinical observations. (3) Results: Among the main features discriminant for infants who did not experience any reported bronchitis ( n = 80/106) or LRTI ( n = 70/103) were the 2-HMO formula containing 2'FL and LNnT, higher acetate, fucosylated glycans and Bifidobacterium , as well as lower succinate, butyrate, propionate and 5-aminovalerate, along with Carnobacteriaceae members and Escherichia . Acetate correlated with several Bifidobacterium species. By univariate analysis, infants experiencing no bronchitis or LRTI, compared with those who did, showed higher acetate ( p < 0.007) and B. longum subsp. infantis ( p ≤ 0.03). In vitro experiments demonstrate that 2'FL, LNnT and lacto-N-tetraose (LNT) stimulated B. longum subsp. infantis (ATCC15697) metabolic activity. Metabolites in spent culture media, primarily due to acetate, supported epithelial barrier protection. (4) Conclusions: An early-life gut ecology characterized by Bifidobacterium -species-driven metabolic changes partly explains the observed clinical outcomes of reduced risk for bronchitis and LRTI in infants fed a formula with HMOs. (Trial registry number NCT01715246.).

Background: The relationship between human milk oligosaccharides (HMOs) and infant growth and adiposity is not fully understood and comprehensive studies are missing from the current literature., Methods: We screened and recruited 370 healthy, pregnant women and their infants from seven European countries. Breastmilk samples were collected using standardized procedures at six time points over 4 months, as were infant parameters. Correlations and associations between HMO area under the curve, anthropometric data, and fat mass at 4 months were tested., Results: Lacto-N-neotetraose had a negative correlation with the change in length (rs = -0.18, P = 0.02). Sialyllacto-N-tetraose c (LSTc) had a positive correlation with weight for length (rs = 0.19, P = 0.015). Infants at the 25th upper percentile were fed milk higher in 3'-sialyllactose and LSTc (P = 0.017 and P = 0.006, respectively) compared to the lower 25th percentile of the weight-for-length z-score gain over 4 months of lactation. No significant associations between growth and body composition and Lewis or secretor-dependent HMOs like 2'-fucosyllactose were identified., Conclusions: Changes in the HMO composition of breastmilk during the first 4 months appear to have little influence on infant growth and body composition in this cohort of healthy mothers and infants., Impact: Modest associations exist between individual HMO and infant growth outcomes at least in healthy growing populations. Our study provides a comprehensive investigation of associations between all major HMO and infant growth and adiposity including several time points. Certain groups of HMOs, like the sialylated, may be associated with adiposity during the first months of lactation. HMO may modulate the risk of future metabolic disease. Future population studies need to address the role of specific groups of HMOs in the context of health and disease to understand the long-term impact., (© 2021. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Background: Genetic polymorphisms leading to variations in human milk oligosaccharide (HMO) composition have been reported. Alpha-Tetrasaccharide (A-tetra), an HMO, has been shown to only be present (>limit of detection; A-tetra+) in the human milk (HM) of women with blood type A, suggesting genetic origins determining the presence or absence (A-tetra-) of A-tetra in HM., Objectives: This study aimed to determine whether associations exist between HMO concentrations and cognitive development, and whether the associations vary between A-tetra+ and A-tetra- groups in children (<25 months old)., Methods: We enrolled typically developing children (2-25 months old; mean, 10 months old) who were at least partially breastfed at the study visit. The Mullen Scales of Early Learning (MSEL) were used as the primary outcome measure to assess early cognitive development. Linear mixed effects models were employed by stratifying children based on A-tetra levels (A-tetra+ or A-tetra-) to assess associations between age-removed HMO concentrations and both MSEL composite scores and the 5 subdomain scores., Results: A total of 99 mother-child dyads and 183 HM samples were included (A-tetra+: 57 samples, 33 dyads; A-tetra-: 126 samples, 66 dyads). No significant association was observed between HMOs and MSEL when all samples were analyzed together. The composite score and 3'-sialyllactose (3'-SL) levels were positively associated [P = 0.002; effect size (EF), 13.12; 95% CI, 5.36-20.80] in the A-tetra + group. This association was driven by the receptive (adjusted P = 0.015; EF, 9.95; 95% CI, 3.91-15.99) and expressive (adjusted P = 0.048; EF, 7.53; 95% CI, 2.51-13.79) language subdomain scores. Furthermore, there was an interaction between 3'-SL and age for receptive language (adjusted P = 0.03; EF, -14.93; 95% CI, -25.29 to -4.24)., Conclusions: Our study reports the association of 3'-SL and cognition, particularly language functions, in typically developing children who received HM containing detectable A-tetra during infancy., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Breastmilk contains bioactive molecules essential for brain and cognitive development. While sialylated human milk oligosaccharides (HMOs) have been implicated in phenotypic programming, their selective role and underlying mechanisms remained elusive. Here, we investigated the long-term consequences of a selective lactational deprivation of a specific sialylated HMO in mice. We capitalized on a knock-out (KO) mouse model (B6.129-St6gal1 tm2Jxm /J) lacking the gene responsible for the synthesis of sialyl(alpha2,6)lactose (6'SL), one of the two sources of sialic acid (Neu5Ac) to the lactating offspring. Neu5Ac is involved in the formation of brain structures sustaining cognition. To deprive lactating offspring of 6'SL, we cross-fostered newborn wild-type (WT) pups to KO dams, which provide 6'SL-deficient milk. To test whether lactational 6'SL deprivation affects cognitive capabilities in adulthood, we assessed attention, perseveration, and memory. To detail the associated endophenotypes, we investigated hippocampal electrophysiology, plasma metabolomics, and gut microbiota composition. To investigate the underlying molecular mechanisms, we assessed gene expression (at eye-opening and in adulthood) in two brain regions mediating executive functions and memory (hippocampus and prefrontal cortex, PFC). Compared to control mice, WT offspring deprived of 6'SL during lactation exhibited consistent alterations in all cognitive functions addressed, hippocampal electrophysiology, and in pathways regulating the serotonergic system (identified through gut microbiota and plasma metabolomics). These were associated with a site- (PFC) and time-specific (eye-opening) reduced expression of genes involved in central nervous system development. Our data suggest that 6'SL in maternal milk adjusts cognitive development through a short-term upregulation of genes modulating neuronal patterning in the PFC., (© 2021. The Author(s).)

Maternal genetics is a key determinant of human milk oligosaccharide (HMO) composition in human milk. Beyond genetic status, other factors influencing the HMO profile are poorly defined. Thus, we aimed to review the existing evidence on the associations between nongenetic maternal and infant factors and HMO composition. A systematic search was performed on PubMed and Web of Science (without a time restriction) to identify any relevant studies published. In total, 1056 results were obtained, of which 29 articles were selected to be included in this review. The range of factors investigated include lactation stage, maternal pre-pregnancy BMI (ppBMI), maternal age, parity, maternal diet, mode of delivery, infant gestational age, and infant sex. The data suggest that, beyond maternal genetics, HMO composition seems to be influenced by all these factors, but the underlining mechanisms remain speculative. The published evidence is discussed in this review, along with potential implications for infant growth and development. For example, 2'-fucosyllactose, which was reportedly increased in mothers with higher ppBMIs, was also associated with increased infant weight and height. In addition, greater levels of sialylated HMOs after preterm birth may support brain development in these infants., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Because of the recognized health benefits of breast milk, it is recommended as the sole nutrition source during the first 6 months of life. Among the bioactive components are human milk oligosaccharides (HMOs) that exert part of their activity via the gut microbiota. Here, we investigated the gut microbiota fermentation of HMO 2'fucosyllactose (2'-FL), using two in vitro models (48 h fecal incubations and the long-term mucosal simulator of the human intestinal microbial ecosystem [M-SHIME ® ]) with fecal samples from 3-month-old breastfed (BF) infants as well as 2-3 year old toddlers. The short-term model allowed the screening of five donors for each group and provided supportive data for the M-SHIME ® study. A key finding was the strong and immediate increase in the relative abundance of Bifidobacteriaceae following 2'-FL fermentation by both the BF infant and toddler microbiota in the M-SHIME ® . At the metabolic level, while decreasing branched-chain fatty acids, 2'-FL strongly increased acetate production together with increases in the health-related propionate and butyrate whilst gas production only mildly increased. Notably, consistently lower gas production was observed with 2'-FL fermentation as compared to lactose, suggesting that reduced discomfort during the dynamic microbiome establishment in early life may be an advantage along with the bifidogenic effect observed.

Rationale: Human milk oligosaccharides (HMOs) vary among mothers and genetic factors contribute to this variability. We assessed changes in HMO concentrations during the first year of lactation and the relationship with FUT2 Secretor group and FUT3 Lewis group defining genetic polymorphisms. Methods: Milk samples were collected from lactating mothers participating in the LIFE Child cohort in Leipzig, Germany. The concentrations of 24 HMOs in milk samples collected at 3 months ( N = 156), 6 months ( N = 122), and 12 months ( N = 28) were measured using liquid chromatography. Concentrations of HMOs were compared at all time-points and were tested for their associations with FUT2 and FUT3 genetic variations by sPLS regression. Results: FUT2 < 0.001) and lacto-N-fucosylpentaose-I (LNFP-I, p < 0.001) and lacto-N-fucosylpentaose-I (LNFP-I, p SNPs rs28362459 and rs812936 were found to define Lewis status (Le-: 5.9%) and correlated with lacto-N-fucosylpentaose-II (LNFP-II, FUT3 SNPs rs28362459 and rs812936 were found to define Lewis status (Le-: 5.9%) and correlated with lacto-N-fucosylpentaose-II (LNFP-II, p < 0.001). A polygenic score predicted the abundance of 2'FL levels within Secretors' milk (adj. R 2 = 0.58, p < 0.001). Mean concentrations of most of the individual HMOs, as well as the sums of the measured HMOs, the fucosylated HMOs, and the neutral HMOs were lower at 6 and 12 months compared to 3 months ( p < 0.001). Conclusions: Secretor and Lewis status defined by specific FUT2 and FUT3 SNPs are confirmed to be good proxies for specific individual HMOs and milk group variabilities. The polygenic score developed here is an opportunity for clinicians to predict 2'FL levels in milk of future mothers. These results show opportunities to strengthen our understanding of factors controlling FUT2 and FUT3 functionality, the temporal changes and variability of HMO composition during lactation and eventually their significance for infant development., (Copyright © 2020 Lefebvre, Shevlyakova, Charpagne, Marquis, Vogel, Kirsten, Kiess, Austin, Sprenger and Binia.)

Numerous benefits of breastfeeding over infant formula are fully established. The superiority of human milk over bovine milk-based formula is partly due to human milk oligosaccharides (HMOs), a family of over 100 molecules present specifically and substantially in human milk that resemble mucosal glycans. To uncover novel physiological functions and pathways of HMOs, we screened a panel of 165 G-protein coupled receptors (GPCRs) using a blend of 6 HMOs (3'-O-sialyllactose (3'SL), 6'-O-sialyllactose (6'SL), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), 2-O-fucosyllactose (2'FL), and difucosyllactose (diFL)), and followed up positive hits with standard receptor assays. The HMO blend specifically activated GPR35. LNT and 6'SL individually activated GPR35, and they showed synergy when used together. In addition, in vitro fermentation of infant stool samples showed that 2'FL upregulates the production of the GPR35 agonist kynurenic acid (KYNA) by the microbiota. LNT + 6'SL and KYNA showed additive activation of GPR35. Activation by 6'SL and LNT of GPR35, a receptor mediating attenuation of pain and colitis, is to our knowledge the first demonstration of GPCR activation by any HMO. In addition, we demonstrated a remarkable cooperation between nutrition and microbiota towards activation of a host receptor highlighting the close interplay between environment and host-microbe interactions.

Commensal gut microbiota and probiotics have numerous effects on the host's metabolic and protective systems, which occur primarily through the intestinal epithelial cell interface. Prebiotics, like galacto-oligosaccharides (GOS) are widely used to modulate their function and abundance. However, important structure-function relations may exist, requiring a detailed structural characterization. Here, we detailed the structural characterization of bovine whey derived oligosaccharide preparations enriched with GOS or not, dubbed GOS-enriched milk oligosaccharides (GMOS) or MOS, respectively. We explore GMOS's and MOS's potential to improve intestinal epithelial barrier function, assessed in a model based on barrier disruptive effects of the Clostridioides difficile toxin A. GMOS and MOS contain mainly GOS species composed of β1-6- and β1-3-linked galactoses, and 3'- and 6'-sialyllactose. Both GMOS and MOS, combined with lactobacilli, like Lactobacillus rhamnosus (LPR, NCC4007), gave synergistic epithelial barrier protection, while no such effect was observed with Bifidobacterium longum (BL NCC3001), Escherichia coli (Nissle) or fructo-oligosaccharides. Mechanistically, for barrier protection with MOS, (i) viable LPR was required, (ii) acidification of growth medium was not enough, (iii) LPR did not directly neutralize toxin A, and (iv) physical proximity of LPR with the intestinal epithelial cells was necessary. This is the first study, highlighting the importance of structure-function specificity and the necessity of the simultaneous presence of prebiotic, probiotic and host cell interactions required for a biological effect.

Background: Post-natal gut maturation in infants interrelates maturation of the morphology, digestive, and immunological functions and gut microbiota development. Here, we explored both microbiota development and markers of gut barrier and maturation in healthy term infants during their early life to assess the interconnection of gut functions during different infant formulae regimes., Methods: A total of 203 infants were enrolled in this randomized double-blind controlled trial including a breastfed reference group. Infants were fed starter formulae for the first four weeks of life, supplemented with different combination of nutrients (lactoferrin, probiotics ( Bifidobacterium animal subsp. Lactis ) and prebiotics (Bovine Milk-derived Oligosaccharides-BMOS)) and subsequently fed the control formula up to eight weeks of life. Stool microbiota profiles and biomarkers of early gut maturation, calprotectin (primary outcome), elastase, α-1 antitrypsin (AAT) and neopterin were measured in feces at one, two, four, and eight weeks., Results: Infants fed formula containing BMOS had lower mean calprotectin levels over the first two to four weeks compared to the other formula groups. Elastase and AAT levels were closer to levels observed in breastfed infants. No differences were observed for neopterin. Global differences between the bacterial communities of all groups were assessed by constrained multivariate analysis with hypothesis testing. The canonical correspondence analysis (CCA) at genus level showed overlap between microbiota profiles at one and four weeks of age in the BMOS supplemented formula group with the breastfed reference, dominated by bifidobacteria. Microbiota profiles of all groups at four weeks were significantly associated with the calprotectin levels at 4 (CCA, p = 0.018) and eight weeks of age (CCA, p = 0.026)., Conclusion: A meaningful correlation was observed between changes in microbiota composition and gut maturation marker calprotectin. The supplementation with BMOS seems to favor gut maturation closer to that of breastfed infants.

Background: Numerous studies have shown that specific components of breast milk, considered separately, are associated with disease status in the mother or the child using univariate analyses. However, very few studies have considered multivariate analysis approaches to evaluate the relationship between multiple breast milk components simultaneously., Aim: Here we aimed at visualizing breast milk component complex interactions in the context of the allergy status of the mother or the child., Methods: Milk samples were collected from lactating mothers participating in the Leipziger Forschungszentrum für Zivilisationskrankheiten (LIFE) Child cohort in Leipzig, Germany. A total of 156 breast milk samples, collected at 3 months after birth from mother/infant pairs, were analyzed for 51 breast milk components. Correlation, principal component analysis (PCA) and graphical discovery analysis were used., Result: Correlations ranging from 0.40 to 0.96 were observed between breast milk fatty acid and breast milk phospholipids levels and correlations ranging from 0 to 0.76 between specific human milk oligosaccharides (HMO) were observed. No separation of the data based on the risk of allergy in the infants was identified using PCA. When graphical discovery analysis was used, dependencies between maternal plasma immunoglobulin E (IgE) level and the breast milk immune marker transforming growth factor-beta 2 (TGF-ß2), between TGF-ß2, breast milk immunoglobulin A (IgA) and TGF-ß1 as well as between breast milk total protein and birth weight were observed. Graphical discovery analysis also exemplifies a possible competition for the fucosyl group between 2'FL, LNFP-I and 3'FL in the HMO group. Additionally, dependencies between immune component IgA and specific HMO (6'SL and blood group A antigen tetraose type 5 or PI-HMO) were identified., Conclusion: Graphical discovery analysis applied to complex matrices such as breast milk composition can aid in understanding the complexity of interactions between breast milk components and possible relations to health parameters in the mother or the infant. This approach can lead to novel discoveries in the context of health and diseases such as allergy. Our study thus represents the first attempt to visualize the complexity and the inter-dependency of breast milk components., Competing Interests: WK received an unconditional grant by Societé des Produits Nestlé S.A. to carry out this analysis. MS, ENC, EE, SH, SN, NS, CN and CB were employed by Societé des Produits Nestlé S.A. during the conduct of the study. This does not alter the adherence to PLOS ONE policies on sharing data and materials. AK, LM, and MV have no conflict of interest to disclose for this work. Societé des Produits Nestlé S.A. and authors of this publication hold patents in the context of allergy management: EE, SH, SN, NS, CN, CB. Results of the current work are not used in currently filed or published patents.

Human milk oligosaccharides (HMOs) may provide health benefits to infants partly by shaping the development of the early-life intestinal microbiota. In a randomized double-blinded controlled multicentric clinical trial, healthy term infants received either infant formula (control) or the same formula with two HMOs (2'-fucosyllactose and lacto-N- neo tetraose; test) from enrollment (0 to 14 days) to 6 months. Then, all infants received the same follow-up formula without HMOs until 12 months of age. Breastfed infants (BF) served as a reference group. Stool microbiota at 3 and 12 months, analyzed by 16S rRNA gene sequencing, clustered into seven fecal community types (FCTs) with marked differences in total microbial abundances. Three of the four 12-month FCTs were likely precursors of the adult enterotypes. At 3 months, microbiota composition in the test group ( n  = 58) appeared closer to that of BF ( n  = 35) than control ( n  = 63) by microbiota alpha (within group) and beta (between groups) diversity analyses and distribution of FCTs. While bifidobacteriaceae dominated two FCTs, its abundance was significantly higher in one (FCT BiH for Bifidobacteriaceae at high abundance) than in the other (FCT Bi for Bifidobacteriaceae ). HMO supplementation increased the number of infants with FCT BiH (predominant in BF) at the expense of FCT Bi (predominant in control). We explored the association of the FCTs with reported morbidities and medication use up to 12 months. Formula-fed infants with FCT BiH at 3 months were significantly less likely to require antibiotics during the first year than those with FCT Bi. Previously reported lower rates of infection-related medication use with HMOs may therefore be linked to gut microbiota community types. (This study has been registered at ClinicalTrials.gov under registration number NCT01715246.) IMPORTANCE Human milk is the sole and recommended nutrition for the newborn infant and contains one of the largest constituents of diverse oligosaccharides, dubbed human milk oligosaccharides (HMOs). Preclinical and clinical association studies indicate that HMOs have multiple physiological functions largely mediated through the establishment of the gut microbiome. Until recently, HMOs were not available to investigate their role in randomized controlled intervention trials. To our knowledge, this is the first report on the effects of 2 HMOs on establishing microbiota in newborn infants. We provide a detailed description of the microbiota changes observed upon feeding a formula with 2 HMOs in comparison to breastfed reference infants' microbiota. Then, we associate the microbiota to long-term health as assessed by prescribed antibiotic use., (Copyright © 2020 Berger et al.)

Human milk oligosaccharide (HMO) composition varies among lactating mothers and changes during the course of lactation period. Interindividual variation is largely driven by fucosyltransferase (FUT2 and FUT3) polymorphisms resulting in 4 distinct milk groups. Little is known regarding whether maternal physiological status contributes to HMO variability. We characterized the trajectories of 20 major HMOs and explored whether maternal pre-pregnancy body mass index (ppBMI), mode of delivery, or parity may affect milk HMO composition. Using longitudinal breastmilk samples from healthy mothers (n = 290) across 7 European countries, we characterized HMO composion and employed mixed linear models to explore associations of maternal characteristics with individual HMOs. We observed HMO-specific temporal trajectories and milk group dependencies. We observed relatively small but significant differences in HMO concentrations based on maternal ppBMI, mode of delivery and parity. Our findings suggest that HMO composition to be regulated time-dependently by an enzyme as well as substrate availability and that ppBMI, mode of delivery, and parity may influence maternal physiology to affect glycosylation marginally within the initital period of lactation. Our observational study is the largest European standardized and longitudinal (up to 4 months) milk collection study assessing HMO concentrations and basic maternal characteristics. Time of lactation and milk groups had the biggest impact on HMO variation. Future studies need to elucidate these observations and assess the physiological significance for the breastfed infant.