Diversification of a Fucosyllactose Transporter within the Genus Bifidobacterium.

Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose [FL]), are associated with the formation of a Bifidobacterium-rich gut microbial community. Phylogenetic analysis revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subsp. kashiwanohense and Bifidobacterium pseudocatenulatum, both of which possess FL-binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO consumption analyses showed that the FL transporter type from B. catenulatum subsp. kashiwanohense JCM 15439T conferred a novel HMO uptake pattern that includes complex fucosylated HMOs (lacto-N-fucopentaose II and lacto-N-difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that the H-antigen- or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria in HMO-rich environments. IMPORTANCE The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breast milk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated host-microbe symbiosis. While the coevolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within Bifidobacterium. We have shown that the diversification of the solute-binding proteins of the FL transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allow bifidobacteria to consume different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.

Human milk oligosaccharide profiles and child atopic dermatitis up to 2 years of age: The Ulm SPATZ Health Study.

BACKGROUND: Human milk oligosaccharides (HMOs) have several biological functions. Yet, very few studies have investigated the effect of HMOs on the development of allergies and even fewer on their specific associations with atopic dermatitis (AD) during early childhood. OBJECTIVE: This study investigated whether individual HMO concentrations, measured at two time points of lactation, were associated with reported diagnosis of AD in children up to two years of age. METHOD: Outcome data were available for HMOs measured in human milk samples collected at 6 weeks (n = 534) and 6 months (n = 356) of lactation. Associations of HMOs with AD, ascertained from parents and pediatricians at ages one and two years, were assessed in crude and adjusted logistic regression models. RESULTS: Few associations were statistically significant at the conventional level (p < .05), for example, 6-week Lacto-N-neotetraose with 2-year AD [OR 95%CI: 0.82 (0.66, 1.00)] and 6-month 3'-sialyllactose among non-secretor mothers with 1-year AD [2.59 (1.53, 6.81)]. Importantly, accounting for multiple testing, these and all further associations were not statistically significant (all p > .0031, which is the threshold for statistical significance after correction for multiple testing). CONCLUSION: Our findings suggest that the intake of different levels (or even absence) of the individual HMOs measured at 6 weeks and 6 months of lactation, in the current study, is not significantly associated with the development of AD in early childhood. Given the exploratory nature of our study and the limited sample size, these results should be interpreted with caution. The specific HMOs for which we show plausible associations at conventional level may warrant further research and investigation.

Gold standard for nutrition: a review of human milk oligosaccharide and its effects on infant gut microbiota.

Human milk is the gold standard for nutrition of infant growth, whose nutritional value is mainly attributed to human milk oligosaccharides (HMOs). HMOs, the third most abundant component of human milk after lactose and lipids, are complex sugars with unique structural diversity which are indigestible by the infant. Acting as prebiotics, multiple beneficial functions of HMO are believed to be exerted through interactions with the gut microbiota either directly or indirectly, such as supporting beneficial bacteria growth, anti-pathogenic effects, and modulation of intestinal epithelial cell response. Recent studies have highlighted that HMOs can boost infants health and reduce disease risk, revealing potential of HMOs in food additive and therapeutics. The present paper discusses recent research in respect to the impact of HMO on the infant gut microbiome, with emphasis on the molecular basis of mechanism underlying beneficial effects of HMOs.

New strategies for profiling and characterization of human milk oligosaccharides.

Human breast milk is an incredibly rich and complex biofluid composed of proteins, lipids and complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides (HMOs). Strikingly, HMOs are not digested by the infant but function as prebiotics for bacterial strains associated with numerous benefits. Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition, the analysis of HMO diversity and complexity is of utmost relevance. Using human milk samples from a cohort of Bangladeshi mothers participating in a study on malnutrition and stunting in children, we have characterized breast milk oligosaccharide composition by means of permethylation followed by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) analysis. This approach identified over 100 different glycoforms and showed a wide diversity of milk composition, with a predominance of fucosylated and sialylated HMOs over nonmodified HMOs. We observed that these samples contain on average 80 HMOs, with the highest permethylated masses detected being >5000 mass units. Here we report an easily implemented method developed for the separation, characterization and relative quantitation of large arrays of HMOs, including higher molecular weight sialylated HMOs. Our ultimate goal is to create a simple, high-throughput method, which can be used for full characterization of sialylated and/or fucosylated HMOs. These results demonstrate how current analytical techniques can be applied to characterize human milk composition, providing new tools to help the scientific community shed new light on the impact of HMOs during infant development.

Targeted LC-ESI-MS2 characterization of human milk oligosaccharide diversity at 6 to 16 weeks post-partum reveals clear staging effects and distinctive milk groups.

Many molecular components in human milk (HM), such as human milk oligosaccharides (HMOs), assist in the healthy development of infants. It has been hypothesized that the functional benefits of HM may be highly dependent on the abundance and individual fine structures of contained HMOs and that distinctive HM groups can be defined by their HMO profiles. However, the structural diversity and abundances of individual HMOs may also vary between milk donors and at different stages of lactations. Improvements in efficiency and selectivity of quantitative HMO analysis are essential to further expand our understanding about the impact of HMO variations on healthy early life development. Hence, we applied here a targeted, highly selective, and semi-quantitative LC-ESI-MS2 approach by analyzing 2 × 30 mature human milk samples collected at 6 and 16 weeks post-partum. The analytical approach covered the most abundant HMOs up to hexasaccharides and, for the first time, also assigned blood group A and B tetrasaccharides. Principal component analysis (PCA) was employed and allowed for automatic grouping and assignment of human milk samples to four human milk groups which are related to the maternal Secretor (Se) and Lewis (Le) genotypes. We found that HMO diversity varied significantly between these four HM groups. Variations were driven by HMOs being either dependent or independent of maternal genetic Se and Le status. We found preliminary evidence for an additional HM subgroup within the Se- and Le-positive HM group I. Furthermore, the abundances of 6 distinct HMO structures (including 6'-SL and 3-FL) changed significantly with progression of lactation. Graphical abstract.

Label-free targeted LC-ESI-MS2 analysis of human milk oligosaccharides (HMOS) and related human milk groups with enhanced structural selectivity.

Human milk (HM) supports the healthy development of neonates and exerts many of its beneficial effects via contained free human milk oligosaccharides (HMOS). These HMOS exhibit a complexity and structural diversity that pose a significant analytical challenge. A detailed characterization of HMOS is essential as every individual structure may have a different function/activity. Certain HMOS isomers may even fundamentally differ in their biological function, and especially their characterization by LC or LC-MS is often impaired by co-elution phenomena. Thus, more efficient analytical methodologies with enhanced structural selectivity are required. Therefore, we developed a negative ion mode LC-ESI-MS2 approach featuring straightforward sample preparation, environmentally friendly EtOH gradient elution, and enhanced, semiquantitative characterization of distinct native HMOS by multiple reaction monitoring (MRM). Our MRM-LC-MS setup takes advantage of highly selective, glycan configuration-dependent collision-induced dissociation (CID) fragments to identify individual neutral and acidic HMOS. Notably, many human milk oligosaccharide isomers could be distinguished in a retention time-independent manner. This contrasts with other contemporary MRM approaches relying on rather unspecific MRM transitions. Our method was used to determine the most abundant human milk tri-, tetra-, penta-, and hexaoses semiquantitatively in a single LC-MS assay. Detected HMO structures included fucosyllactoses (e.g., 2'-FL), lacto-N-difucotetraose (LDFT), lacto-N-tetraoses (LNTs), lacto-N-fucopentaoses (e.g., LNFP I, LNFP II and III), lacto-N-difucohexaoses (LNDFHs) as well as sialyllactoses (SLs) and tentatively assigned blood group A and B tetrasaccharides from which correct human milk type assignment could be also demonstrated. Correctness of milk typing was validated for milk groups I-IV by high pressure anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (HPAEC-PAD). Graphical Abstract ᅟ.

Prenatal Human Milk Oligosaccharides (HMOs) in the Context of BMI, Gestational Weight Gain, and Lipid Profile-An Association Study in Pregnant Women with Overweight or Obesity.

BACKGROUND: Human milk oligosaccharides (HMOs) are bioactive glycans first detected in human milk. Their presence in maternal blood during pregnancy suggests systemic functions. Dynamics and associations of the most abundant prenatal HMOs in relation to maternal BMI and serum lipids in a cohort of 87 pregnant women with either overweight or obesity are studied. METHODS: Serum HMOs (2'FL, 3'SL, 3'SLN, LDFT), serum lipids (total cholesterol, HDL, LDL, triglycerides), and BMI are measured at 15, 24, and 32 weeks of gestation. RESULTS: 2'FL and LDFT are negatively correlated to pre-pregnancy BMI and increase significantly slower between 15 and 24 weeks in highly obese women. Women without detectable increase of serum 2'FL (non-secretors) show a less pronounced gestational weight gain and lower BMI in the third trimester as compared to women phenotype as secretors. Higher early-pregnancy 2'FL is associated with high HDL and low triglycerides in pregnancy. On the other hand, higher 3'SL at 15 weeks is associated with higher triglycerides, LDL, and total cholesterol. CONCLUSIONS: Higher early-pregnancy 2'FL is associated with a cardioprotective lipid profile, whereas higher 3'SL is associated with an atherogenic lipid profile. Serum trajectories of 2'FL and LDFT in obese women suggest an obesity mediated delay of α-1,2-fucosylation.

Enzymatic modular synthesis of asymmetrically branched human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are intricate glycans that promote healthy growth of infants and have been incorporated into infant formula as food additives. Despite their importance, the limited availability of asymmetrically branched HMOs hinders the exploration of their structure and function relationships. Herein, we report an enzymatic modular strategy for the efficient synthesis of these HMOs. The key branching enzyme for the assembly of branched HMOs, human ß1,6-N-acetylglucosaminyltransferase 2 (GCNT2), was successfully expressed in Pichia pastoris for the first time. Then, it was integrated with six other bacterial glycosyltransferases to establish seven glycosylation modules. Each module comprises a one-pot multi-enzyme (OPME) system for in-situ generation of costly sugar nucleotide donors, combined with a glycosyltransferase for specific glycosylation. This approach enabled the synthesis of 31 branched HMOs and 13 linear HMOs in a stepwise manner with well-programmed synthetic routes. The binding details of these HMOs with related glycan-binding proteins were subsequently elucidated using glycan microarray assays to provide insights into their biological functions. This comprehensive collection of synthetic HMOs not only serves as standards for HMOs structure identification in complex biological samples but also significantly enhances the fields of HMOs glycomics, opening new avenues for biomedical applications.

Innovative direct introduction-ion mobility-mass spectrometry (DI-IM-MS) approach for fast and robust isomer-specific quantification in a complex matrix: Application to 2'-fucosyllactose (2'-FL) in breast milk.

Identification and specific quantification of isomers in a complex biological matrix by mass spectrometry alone is not an easy task due to their identical chemical formula and therefore their same mass-to-charge ratio (m/z). Here, the potential of direct introduction combined with ion mobility-mass spectrometry (DI-IM-MS) for rapid quantification of isomers as human milk oligosaccharides (HMOs) was investigated. Differences in HMO profiles between various analyzed breast milk samples were highlighted using the single ion mobility monitoring (SIM2) acquisition for high ion mobility resolution detection. Furthermore, the Se+ (secretor) or Se- (non-secretor) phenotype could be assigned to breast milk samples studied based on their HMO contents, especially on the response of 2'-fucosyllactose (2'-FL) and lacto-N-fucopentaose I (LNFP I). The possibility of quantifying a specific isomer in breast milk by DI-IM-MS was also investigated. The standard addition method allowed the determination of the 2'-FL despite the presence of other oligosaccharides, including 3-fucosyllactose (3-FL) isomer in breast milk. This proof-of-concept study demonstrated the high potential of such an approach for the rapid and convenient quantification of isomers in complex mixtures.

Probiotics in the New Era of Human Milk Oligosaccharides (HMOs): HMO Utilization and Beneficial Effects of Bifidobacterium longum subsp. infantis M-63 on Infant Health.

A healthy gut microbiome is crucial for the immune system and overall development of infants. Bifidobacterium has been known to be a predominant species in the infant gut; however, an emerging concern is the apparent loss of this genus, in particular, Bifidobacterium longum subsp. infantis (B. infantis) in the gut microbiome of infants in industrialized nations, underscoring the importance of restoring this beneficial bacterium. With the growing understanding of the gut microbiome, probiotics, especially infant-type human-residential bifidobacteria (HRB) strains like B. infantis, are gaining prominence for their unique ability to utilize HMOs and positively influence infant health. This article delves into the physiology of a probiotic strain, B. infantis M-63, its symbiotic relationship with HMOs, and its potential in improving gastrointestinal and allergic conditions in infants and children. Moreover, this article critically assesses the role of HMOs and the emerging trend of supplementing infant formulas with the prebiotic HMOs, which serve as fuel for beneficial gut bacteria, thereby emulating the protective effects of breastfeeding. The review highlights the potential of combining B. infantis M-63 with HMOs as a feasible strategy to improve health outcomes in infants and children, acknowledging the complexities and requirements for further research in this area.

The Role of Human Milk Oligosaccharides in Myelination, Socio-Emotional and Language Development: Observational Data from Breast-Fed Infants in the United States of America.

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.

Human milk oligosaccharides in milk of mothers with term and preterm delivery at different lactation stage.

Human milk oligosaccharides (HMOs) are structurally diverse unconjugated glycans, and play crucial roles in protecting infants from infections. Preterm birth is one of the leading causes of neonatal mortality, and preterm infants are particularly vulnerable and are in need of improved outcomes from breast-feeding due to the presence of bioactive HMOs. However, studies on specific difference in HMOs as a function of gestation time have been very limited. We established an approach to extract and analyze HMOs based on 96-well plate extraction and mass spectrometry, and determined maternal phenotypes through distinctive fragments in product-ion spectra. We enrolled 85 women delivering at different gestation times (25-41 weeks), and observed different HMOs correlating with gestation time based on 233 samples from the 85 donors. With the increase of postpartum age, we observed a regular changing trajectory of HMOs in composition and relative abundance, and found significant differences in HMOs secreted at different postpartum times. Preterm delivery induced more variations between participants with different phenotypes compared with term delivery, and more HMOs varied with postpartum age in the population of secretors. The sialylation level in mature milk decreased for women delivering preterm while such decrease was not observed for women delivering on term.

Alterations of milk oligosaccharides in mothers with gestational diabetes mellitus impede colonization of beneficial bacteria and development of RORγt+ Treg cell-mediated immune tolerance in neonates.

Gestational diabetes mellitus (GDM) is an increasing public health concern that significantly increases the risk of early childhood allergic diseases. Altered maternal milk glycobiome may strongly affect gut microbiota and enteric-specific Treg cell-mediated development of immune tolerance in GDM infants. In this study, we found that, compared with healthy Chinese mothers, mothers with GDM had significantly lower levels of total and specific human milk oligosaccharides (HMOs) in their colostrum that subsequently increased with extension of lactation. This alteration in HMO profiles significantly delayed colonization of Lactobacillus and Bifidobacterium spp. in their breast-fed infants, resulting in a distinct gut microbial structure and metabolome. Further experiments in GDM mouse models indicated that decreased contents of milk oligosaccharides, mainly 3'-sialyllactose (3'-SL), in GDM maternal mice reduced colonization of bacteria, such as L. reuteri and L. johnsonii, in the neonatal gut, which impeded development of RORγt+ regulatory T (Treg) cell-mediated immune tolerance. Treatment of GDM neonates with 3'-SL, Lactobacillus reuteri (L. reuteri) and L. johnsonii promoted the proliferation of enteric Treg cells and expression of transcription factor RORγt, which may have contributed to compromising ovalbumin (OVA)-induced allergic responses. In vitro experiments showed that 3'-SL, metabolites of L. johnsonii, and lysates of L. reuteri stimulated differentiation of mouse RORγt+ Treg cells through multiple regulatory effects on Toll-like receptor, MAPK, p53, and NOD-like receptor signaling pathways. This study provides new ideas for the development of gut microbiota and immune tolerance in GDM newborns.

The First 1000 Days of Life: How Changes in the Microbiota Can Influence Food Allergy Onset in Children.

BACKGROUND: Allergic disease, including food allergies (FA)s, has been identified as a major global disease. The first 1000 days of life can be a "window of opportunity" or a "window of susceptibility", during which several factors can predispose children to FA development. Changes in the composition of the gut microbiota from pregnancy to infancy may play a pivotal role in this regard: some bacterial genera, such as Lactobacillus and Bifidobacterium, seem to be protective against FA development. On the contrary, Clostridium and Staphylococcus appear to be unprotective. METHODS: We conducted research on the most recent literature (2013-2023) using the PubMed and Scopus databases. We included original papers, clinical trials, meta-analyses, and reviews in English. Case reports, series, and letters were excluded. RESULTS: During pregnancy, the maternal diet can play a fundamental role in influencing the gut microbiota composition of newborns. After birth, human milk can promote the development of protective microbial species via human milk oligosaccharides (HMOs), which play a prebiotic role. Moreover, complementary feeding can modify the gut microbiota's composition. CONCLUSIONS: The first two years of life are a critical period, during which several factors can increase the risk of FA development in genetically predisposed children.

Human milk nutritional composition across lactational stages in Central Africa.

The African region encompasses the highest undernutrition burden with the highest neonatal and infant mortality rates globally. Under these circumstances, breastfeeding is one of the most effective ways to ensure child health and development. However, evidence on human milk (HM) composition from African women is scarce. This is of special concern, as we have no reference data from HM composition in the context of food insecurity in Africa. Furthermore, data on the evolution of HM across lactational stages in this setting lack as well. In the MITICA study, we conducted a cohort study among 48 Central-African women and their 50 infants to analyze the emergence of gut dysbiosis in infants and describe the mother-infant transmission of microbiota between birth and 6 months of age. In this context, we assessed nutritional components in HM of 48 lactating women in Central Africa through five sampling times from week 1 after birth until week 25. Unexpectedly, HM-type III (Secretor + and Lewis genes -) was predominant in HM from Central African women, and some nutrients differed significantly among HM-types. While lactose concentration increased across lactation periods, fatty acid concentration did not vary significantly. The overall median level of 16 detected individual human milk oligosaccharides (HMOs; core structures as well as fucosylated and sialylated ones) decreased from 7.3 g/l at week 1 to 3.5 g/l at week 25. The median levels of total amino acids in HM dropped from 12.8 mg/ml at week 1 to 7.4 mg/ml at week 25. In contrast, specific free amino acids increased between months 1 and 3 of lactation, e.g., free glutamic acid, glutamine, aspartic acid, and serine. In conclusion, HM-type distribution and certain nutrients differed from Western mother HM.

Food Insecurity and Maternal Diet Influence Human Milk Composition between the Infant's Birth and 6 Months after Birth in Central-Africa.

Although the World Health Organization (WHO) and UNICEF recommend that infants should be exclusively breastfed for the first 6 months of life, evidence is scarce on how the mother's undernourishment status at delivery and maternal dietary factors influence human milk (HM) composition during the first 6 months of life in regions with high food insecurity. The maternal undernourishment status at delivery, maternal diet, and HM nutrients were assessed among 46 women and their 48 vaginally born infants in Bangui at 1, 4, 11, 18, and 25 weeks after birth through 24-h recalls and food consumption questionnaires from December 2017 to June 2019 in the context of the "Mother-to-Infant TransmIssion of microbiota in Central-Africa" (MITICA) study. High food insecurity indexes during the follow-up were significantly associated with them having lower levels of many of the human milk oligosaccharides (HMOs) that were measured and with lower levels of retinol (aß-coef = −0.2, p value = 0.04), fatty acids (aß-coef = −7.2, p value = 0.03), and amino acids (aß-coef = −2121.0, p value < 0.001). On the contrary, women from food-insecure households displayed significantly higher levels of lactose in their HM (aß-coef = 3.3, p value = 0.02). In parallel, the consumption of meat, poultry, and fish was associated with higher HM levels of many of the HMOs that were measured, total amino acids (aß-coef = 5484.4, p value < 0.001), and with lower HM levels of lactose (aß-coef = −15.6, p value = 0.01). Food insecurity and maternal diet had a meaningful effect on HM composition with a possible impact being an infant undernourishment risk. Our results plead for consistent actions on food security as an effective manner to influence the nutritional content of HM and thereby, potentially improve infant survival and healthy growth.

The analysis of 2'-fucosyllactose concentration in Korean maternal milk using LC-MS/MS.

Despite health benefits reported recently, 2'-fucosyllactose (2'-FL) concentration in maternal milk was not conclusively reported because it varies between countries and mothers. Particularly, its distribution among Korean mothers was not obtained from a reliable sample group yet. Thus, a dynamic range for 2'-FL concentration in Korean mothers' milk was investigated from 102 samples. A quantitative method using multiple reaction monitoring (MRM) by triple-quadrupole-mass spectrometry has been evaluated by a standard procedure of method validation. The 2'-FL concentration was in the range of 0.4 to 2.6 g/L overall. While the samples from secretor mothers (n = 80) contained 1.0 to 2.8 g/L of 2'-FL, the maternal milk from non-secretor mothers (n = 22) had 0.01 to 0.06 g/L of 2'-FL only. In addition to the genetic variation of mothers, the lactation period impacted the 2'-FL concentration. The average 2'-FL concentration of the late-stage group (> 60 days) was 78% of that obtained from the first month of postpartum mothers. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01154-4.

Use of a Liquid Supplement Containing 2 Human Milk Oligosaccharides: The First Double-Blind, Randomized, Controlled Trial in Pre-term Infants.

There is growing evidence supporting the benefit of human milk oligosaccharides (HMOs) on reducing risk of illnesses and improving immune function in newborn infants, but evidence in pre-term infants is lacking. This randomized, double-blind, placebo-controlled trial (NCT03607942) of pre-term infants evaluated the effects of HMO supplementation on feeding tolerance, growth, and safety in 7 neonatal units in France. Pre-term infants (27-33 weeks' gestation, birth weight <1,700 g) were randomized early after birth to receive HMO supplement (n = 43) [2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) in a 10:1 ratio (0.374 g/kg body weight/day)] or an isocaloric placebo (n = 43) consisting of only glucose (0.140 g/kg/day) until discharge from the neonatal unit. Anthropometric z-scores were calculated using Fenton growth standards. Primary outcome was feeding tolerance, measured by non-inferiority (NI) in days to reach full enteral feeding (FEF) from birth in HMO vs. placebo group (NI margin = 4+ days). Mean number of days on intervention prior to FEF was 8.9 and 10.3 days in HMO and placebo, respectively. Non-inferiority in time to reach FEF in HMO (vs. placebo) was achieved [LS mean difference (95% CI) = -2.16 (-5.33, 1.00); upper bound of 95% CI < NI margin] in full analysis set and similar for per protocol. Adjusted mean time to reach FEF from birth was 2 days shorter in HMO (12.2) vs. placebo (14.3), although not statistically significant (p = 0.177). There was no difference in weight-for-age z-scores between groups throughout the FEF period until discharge. Length-for-age z-scores were higher in HMO at FEF day 14 [0.29 (0.02, 0.56), p = 0.037] and 21 [0.31 (0.02, 0.61), p = 0.037]. Head circumference-for-age z-score was higher in HMO vs. placebo at discharge [0.42 (0.12, 0.71), p = 0.007]. Occurrence of adverse events (AEs) was similar in both groups and relatively common in this population, whereas 2.3 and 14.3%, respectively, experienced investigator-confirmed, related AEs. HMO supplementation is safe and well-tolerated in pre-term infants. After 9 days of supplementation, the HMO group reached FEF 2 days earlier vs. placebo, although the difference was not statistically significant. In addition, HMO supplementation supports early postnatal growth, which may have a positive impact on long-term growth and developmental outcomes.

Human Milk Oligosaccharides Are Present in Amniotic Fluid and Show Specific Patterns Dependent on Gestational Age.

(1) Background: Human milk oligosaccharides (HMOs) are already found in maternal circulation in early pregnancy, changing with gestational age. HMOs are also present in cord blood and amniotic fluid (AF). We aimed to assess HMO profiles in AF over the course of gestation. (2) Methods: AF was collected during diagnostic amniocentesis, fetal surgery, or C-section from 77 women with a gestational age of ranging from 14.3 to 40.9 weeks. Samples were analysed using high performance liquid chromatography with fluorescence detection. (3) Results: We found lactose and up to 16 HMO structures in all AF samples investigated, starting at 14 weeks of gestation. Overall, 3'-sialyllactose (3'SL) and 2'-fucosyllactose (2'FL) were the most abundant HMOs. Individual and total HMO concentrations were significantly positively correlated with gestational age. HMO composition also changed between early, mid- and late pregnancy, with relative concentrations of 3'SL significantly decreasing (44%, 25%, 24%) and 2'FL increasing (7%, 13%, 21%), respectively. (4) Conclusion: Our study shows that HMOs are already present in AF early in pregnancy. This demonstrates extensive contact of the fetus with a broad variety of HMOs, suggesting roles for HMOs in fetal tissue development during the time course of pregnancy.

Determining the metabolic fate of human milk oligosaccharides: it may just be more complex than you think?

Human milk oligosaccharides (HMOs) are a class of structurally diverse and complex unconjugated glycans present in breast milk, which act as selective substrates for several genera of select microbes and inhibit the colonisation of pathogenic bacteria. Yet, not all infants are breastfed, instead being fed with formula milks which may or may not contain HMOs. Currently, formula milks only possess two HMOs: 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT), which have been suggested to be similarly effective as human breast milk in supporting age-related growth. However, the in vivo evidence regarding their ability to beneficially reduce respiratory infections along with altering the composition of an infant's microbiota is limited at best. Thus, this review will explore the concept of HMOs and their metabolic fate, and summarise previous in vitro and in vivo clinical data regarding HMOs, with specific regard to 2'FL and LNnT.