Longitudinal change of selected human milk oligosaccharides and association to infants' growth, an observatory, single center, longitudinal cohort study.
ABSTRACT: BACKGROUND:Human milk is the recommended and sole nutrient source for newborns. One of the largest components of human milk is oligosaccharides (HMOs) with major constituents determined by the mother genotype for the fucosyltransferase 2 (FUT2, secretor) gene. HMO variation has been related with infant microbiota establishment, diarrhea incidence, morbidity and mortality, IgE associated eczema and body composition. OBJECTIVES:We investigated the (i) dependence of several major representative HMOs on the FUT2 status assessed through breast milk 2'Fucosyllactose (2'FL) and (ii) the relation of the 2'FL status with infant growth up to 4 months of life. DESIGN:From an open observatory, single center, longitudinal cohort study with quantitative human milk collection at 30, 60, and 120 days postpartum from 50 mothers, who gave birth to 25 female and 25 male singleton infants, we collected a representative sample of human milk. We quantified the following 5 representative HMOs: 2'FL, Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT), 3'Sialyllactose (3'SL) and 6'Sialyllactose (6'SL). We grouped the milk samples and corresponding infants according to the measured milk 2'FL concentrations at 30 days of lactation, which clustered around low concentrations (95% CI of mean 12-42 mg/L) and high concentrations (95% CI of mean 1880-2460 mg/L) with the former likely representing Secretor negative mothers. Infant anthropometric measures were recorded at birth, 1, 2 and 4 months of age. Relations among the quantified HMOs and the relation of the high and low 2'FL HMOs groups with infant growth parameters were investigated via linear mixed models. RESULTS:The milk samples with low 2'FL concentration had higher LNT and lower LNnT concentrations compared to the samples with high 2'FL. The milk 3'- and 6'SL concentrations were independent of 2'FL. Over lactation time we observed a drop in the concentration of 2'FL, LNT, LNnT and 6'SL, especially from 1 to 2 months, while 3'SL remained at relatively constant concentration from 1 month onwards. Up to 4 months of age, we did not observe significant differences in body weight, body length, body mass index and head circumference of the infants who consumed breast milk with low or high FUT2 associated HMO concentrations and composition. CONCLUSIONS:Our findings on HMO concentrations over time of lactation and clusters based on 2'FL concentrations confirm previous observations and suggest that LNnT and LNT are 'co-regulated' with the FUT2 dependent 2'FL concentration, with LNnT showing a positive and LNT a negative relation. Further, our findings also suggest that the relatively substantial variation in HMOs between the high and low 2'FL clusters do not impact infant growth of either sex up to 4 months of age. The study was registered in www.ClinicalTrial.gov (NCT01805011).
Project description: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.
Project description: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 SNP rs601338 was found to predominantly define the Secretor status Se-: 11.8% and it was highly correlated with 2'-fucosyllactose (2'FL, p < 0.001) and lacto-N-fucosylpentaose-I (LNFP-I, p < 0.001). 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.
Project description:This study tested the hypothesis that the fecal bacterial genera of breast-fed (BF) and formula-fed (FF) infants differ and that human milk oligosaccharides (HMOs) modulate the microbiota of BF infants.Fecal samples were obtained from BF (n = 16) or FF (n = 6) infants at 3-month postpartum. Human milk samples were collected on the same day when feces were collected. The microbiota was assessed by pyrosequencing of bacterial 16S ribosomal RNA genes. HMOs were measured by high-performance liquid chromatography-chip time-of-flight mass spectrometry.The overall microbiota of BF differed from that of FF (P = 0.005). Compared with FF, BF had higher relative abundances of Bacteroides, lower proportions of Clostridium XVIII, Lachnospiraceae incertae sedis, Streptococcus, Enterococcus, and Veillonella (P < 0.05). Bifidobacterium predominated in both BF and FF infants, with no difference in abundance between the 2 groups. The most abundant HMOs were lacto-N-tetraose + lacto-N-neotetraose (LNT + LNnT, 22.6%), followed by 2'-fucosyllactose (2'FL, 14.5%) and lacto-N-fucopentaose I (LNFP I, 9.5%). Partial least squares regression of HMO and microbiota showed several infant fecal bacterial genera could be predicted by their mothers' HMO profiles, and the important HMOs for the prediction of bacterial genera were identified by variable importance in the projection scores.These results strengthen the established relation between HMO and the infant microbiota and identify statistical means whereby infant bacterial genera can be predicted by milk HMO. Future studies are needed to validate these findings and determine whether the supplementation of formula with defined HMO could selectively modify the gut microbiota.
Project description:Human milk contains a high concentration of indigestible oligosaccharides, which likely mediated the coevolution of the nursing infant with its gut microbiome. Specifically, Bifidobacterium longum subsp. infantis (B. infantis) often colonizes the infant gut and utilizes these human milk oligosaccharides (HMOs) to enrich their abundance. In this study, the physiology and mechanisms underlying B. infantis utilization of two HMO isomers lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) was investigated in addition to their carbohydrate constituents. Both LNT and LNnT utilization induced a significant shift in the ratio of secreted acetate to lactate (1.7-2.0) in contrast to the catabolism of their component carbohydrates (~1.5). Inefficient metabolism of LNnT prompts B. infantis to shunt carbon toward formic acid and ethanol secretion. The global transcriptome presents genomic features differentially expressed to catabolize these two HMO species that vary by a single glycosidic linkage. Furthermore, a measure of strain-level variation exists between B. infantis isolates. Regardless of strain, inefficient HMO metabolism induces the metabolic shift toward formic acid and ethanol production. Furthermore, bifidobacterial metabolites reduced LPS-induced inflammation in a cell culture model. Thus, differential metabolism of milk glycans potentially drives the emergent physiology of host-microbial interactions to impact infant health.
Project description:The development of infant gut microbiota is strongly influenced by nutrition. Human milk oligosaccharides (HMOSs) in breast milk selectively promote the growth of glycan-degrading microbes, which lays the basis of the microbial network. In this study, we investigated the trophic interaction between Bacteroides thetaiotaomicron and the butyrate-producing Anaerostipes caccae in the presence of early-life carbohydrates. Anaerobic bioreactors were set up to study the monocultures of B. thetaiotaomicron and the co-cultures of B. thetaiotaomicron with A. caccae in minimal media supplemented with lactose or a total human milk carbohydrate fraction. Bacterial growth (qPCR), metabolites (HPLC), and HMOS utilization (LC-ESI-MS2) were monitored. B. thetaiotaomicron displayed potent glycan catabolic capability with differential preference in degrading specific low molecular weight HMOSs, including the neutral trioses (2'-FL and 3-FL), neutral tetraoses (DFL, LNT, LNnT), neutral pentaoses (LNFP I, II, III, V), and acidic trioses (3'-SL and 6'-SL). In contrast, A. caccae was not able to utilize lactose and HMOSs. However, the signature metabolite of A. caccae, butyrate, was detected in co-culture with B. thetaiotaomicron. As such, A. caccae cross-fed on B. thetaiotaomicron-derived monosaccharides, acetate, and d-lactate for growth and concomitant butyrate production. This study provides a proof of concept that B. thetaiotaomicron could drive the butyrogenic metabolic network in the infant gut.
Project description:To study the variability in human milk oligosaccharide (HMO) composition of Chinese human milk over a 20-wk lactation period, HMO profiles of 30 mothers were analyzed using CE-LIF. This study showed that total HMO concentrations in Chinese human milk decreased significantly over a 20-wk lactation period, independent of the mother's SeLe status, although with individual variations. In addition, total acidic and neutral HMO concentrations in Chinese human milk decreased over lactation, and levels are driven by their mother's SeLe status. Analysis showed that total neutral fucosylated HMO concentrations in Chinese human milk were higher in the two secretor groups as compared to the nonsecretor group. On the basis of the total neutral fucosylated HMO concentrations in Chinese human milk, HMO profiles within the Se+Le+ group can be divided into two subgroups. HMOs that differed in level between Se+Le+ subgroups were 2'FL, DF-L, LNFP I, and F-LNO. HMO profiles in Dutch human milk also showed Se+Le+ subgroup division, with 2'FL, LNT, and F-LNO as the driving force.
Project description:The purpose of this project was to determine the whole transcriptome response of Bifidobacterium bifidum SC555 to pooled and individual human milk oligosaccharides (HMO) relative to lactose Bacterial isolates grown on lactose, pooled human milk oligosaccharides (HMO), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 2’fucosyllactose (2’FL), 3-fucosyllactose (3FL), 6’sialyllactose (6’SL) and porcine mucin (MUC). RNA was extracted and sequenced, in duplicate, on an Illumina HiSeq. Early, mid, and late timepoints in response to pooled HMO were additionally sequenced in duplicate.
Project description:The purpose of this project was to determine the whole transcriptome response of Bifidobacterium longum subsp. Infantis to pooled and individual human milk oligosaccharides (HMO) relative to lactose Bacterial isolates grown on lactose, pooled human milk oligosaccharides (HMO), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 2’fucosyllactose (2’FL), 3-fucosyllactose (3FL), and 6’sialyllactose (6’SL). RNA was extracted and sequenced, in duplicate, on an Illumina HiSeq. Early, mid, and late timepoints in response to pooled HMO were additionally sequenced in duplicate.
Project description:Human milk oligosaccharides (HMOs) are chief maternal milk constituents that feed the intestinal microbiota and drive maturation of the infant gut. Our objective was to determine whether supplementing individual HMOs to a weanling diet alters growth and gut health in rats. Healthy three-week-old Sprague Dawley rat pups were randomized to control, 2'-O-fucosyllactose (2'FL)- and 3'sialyllactose (3'SL)-fortified diets alone or in combination at physiological doses for eight weeks. Body composition, intestinal permeability, serum cytokines, fecal microbiota composition, and messenger RNA (mRNA) expression in the gastrointestinal tract were assessed. Males fed a control diet were 10% heavier and displayed elevated interleukin (IL-18) (p = 0.01) in serum compared to all HMO-fortified groups at week 11. No differences in body composition were detected between groups. In females, HMOs did not affect body weight but 2'FL + 3'SL significantly increased cecum weight. All female HMO-fortified groups displayed significant reductions in intestinal permeability compared to controls (p = 0.02). All HMO-fortified diets altered gut microbiota composition and mRNA expression in the gastrointestinal tract, albeit differently according to sex. Supplementation with a fraction of the HMOs found in breast milk has a complex sex-dependent risk/benefit profile. Further long-term investigation of gut microbial profiles and supplementation with other HMOs during early development is warranted.
Project description:Human milk oligosaccharide (HMO) composition varies throughout lactation and can be influenced by maternal characteristics. This study describes HMO variation up to three months postpartum and explores the influences of maternal sociodemographic and anthropometric characteristics in a Brazilian prospective cohort. We followed 101 subjects from 28-35 gestational weeks (baseline) and throughout lactation at 2-8 (visit 1), 28-50 (visit 2) and 88-119 days postpartum (visit 3). Milk samples were collected at visits 1, 2 and 3, and 19 HMOs were quantified usinghigh-performance liquid chromatography with fluorescence detection (HPLC-FL). Friedman post-hoc test, Spearman rank correlation for maternal characteristics and HMOs and non-negative matrix factorization (NMF) were used to define the HMO profile. Most women were secretors (89.1%) and presented high proportion of 2'-fucosyllactose (2?FL) at all three sample times, while lacto-N-tetraose (LNT, 2-8 days) and lacto-N-fucopentaose II (LNFPII, 28-50 and 88-119 days) were the most abundant HMOs in non-secretor women. Over the course of lactation, total HMO weight concentrations (g/L) decreased, but total HMO molar concentrations (mmol/L) increased, highlighting differential changes in HMO composition over time. In addition, maternal pre-pregnancy body mass index (BMI) and parity influence the HMO composition in healthy women in this Brazilian cohort.