Individual Human Milk Oligosaccharides Research Articles

A Cross-Sectional Assessment of Human Milk Oligosaccharide Composition of Vegan, Vegetarian, and Nonvegetarian Mothers.

Purpose: Recently, maternal nutrient intake has been associated with human milk oligosaccharides (HMOs) composition. The goal of this study was to assess HMO composition in breast milk samples from vegan, vegetarian, and nonvegetarian lactating women. Second, we assessed impact of maternal body mass index (BMI), age, parity, and lactation stage on HMO composition. Materials and Methods: A cross-sectional analysis of HMO composition from vegan (n = 26), vegetarian (n = 22), and nonvegetarian (n = 26) lactating women was carried out. The majority of participants took dietary supplements. Results: In an unadjusted bivariate model, there was no difference in individual HMO composition, total HMO-bound fucose and HMO-bound sialic acid, or diversity and evenness scores by diet group. When adjusting for factors that significantly differed between groups (maternal BMI and lactation stage), no differences in HMO composition were observed. Secretor status was significant for 13 of the outcome variables with the strongest positive relationship with total HMO (β = 0.922) and HMO-bound fucose (β = 0.910), and the strongest negative relationship with sialyl-lacto-N-tetraose b (LSTb) (β = -0.544). Lactation stage was significant for eight analytes, with the strongest positive impact on 3'sialyllactose (3'SL) (β = 0.433), and the strongest negative impact on 6'sialyllactose (6'SL) (β = -0.519). Maternal BMI had a significant positive relationship with total HMO composition (β = 0.113) and 3'SL (β = 0.325). Conclusions: Lactating women who consume plant-based diets do not produce different breast milk as it relates to HMO composition.

Infant-gut associated Bifidobacterium dentium strains utilize the galactose moiety and release lacto-N-triose from the human milk oligosaccharides lacto-N-tetraose and lacto-N-neotetraose

Much evidence suggests a role for human milk oligosaccharides (HMOs) in establishing the infant microbiota in the large intestine, but the response of particular bacteria to individual HMOs is not well known. Here twelve bacterial strains belonging to the genera Bifidobacterium, Enterococcus, Limosilactobacillus, Lactobacillus, Lacticaseibacillus, Staphylococcus and Streptococcus were isolated from infant faeces and their growth was analyzed in the presence of the major HMOs, 2′-fucosyllactose (2′FL), 3-fucosyllactose (3FL), 2′,3-difucosyllactose (DFL), lacto-N-tetraose (LNT) and lacto-N-neo-tetraose (LNnT), present in human milk. Only the isolated Bifidobacterium strains demonstrated the capability to utilize these HMOs as carbon sources. Bifidobacterium infantis Y538 efficiently consumed all tested HMOs. Contrarily, Bifidobacterium dentium strains Y510 and Y521 just metabolized LNT and LNnT. Both tetra-saccharides are hydrolyzed into galactose and lacto-N-triose (LNTII) by B. dentium. Interestingly, this species consumed only the galactose moiety during growth on LNT or LNnT, and excreted the LNTII moiety. Two β-galactosidases were characterized from B. dentium Y510, Bdg42A showed the highest activity towards LNT, hydrolyzing it into galactose and LNTII, and Bdg2A towards lactose, degrading efficiently also 6′-galactopyranosyl-N-acetylglucosamine, N-acetyl-lactosamine and LNnT. The work presented here supports the hypothesis that HMOs are mainly metabolized by Bifidobacterium species in the infant gut.

Associations of Human Milk Oligosaccharides With Otitis Media and Lower and Upper Respiratory Tract Infections up to 2 Years: The Ulm SPATZ Health Study.

Background: Human milk oligosaccharides (HMOs) support and concurrently shape the neonatal immune system through various mechanisms. Thereby, they may contribute to lower incidence of infections in infants. However, there is limited evidence on the role of individual HMOs in the risk of otitis media (OM), as well as lower and upper respiratory tract infections (LRTI and URTI, respectively) in children up to 2 years.Objective: To investigate whether individual HMO concentrations measured at 6 weeks of lactation were associated with risk of OM, LRTI or URTI up to 2 years in breastfed infants. Associations with OM, LRTI and URTI were determined for the most prominent human milk oligosaccharides including 13 neutral, partly isomeric structures (trioses up to hexaoses), two acidic trioses, and lactose.Design: HMO measurements and physician reported data on infections were available from human milk samples collected at 6 weeks postpartum (n = 667). Associations of HMOs with infections were assessed in crude and adjusted models using modified Poisson regression.Results: Absolute concentrations (median [min, max], in g/L) of 2′-fucosyllactose (2′-FL) tended (p = 0.04) to be lower, while lacto-N-tetraose (LNT) was higher in the milk for infants with OM in the 1st year of life (p = 0.0046). In the milk of secretor mothers, LNT was significantly higher in the milk for infants with OM (RR [95% CI]: 0.98 [0.15, 2.60]) compared to infants without OM (RR [95% CI]: 0.76 [0.14, 2.90]) at 1 year (p = 0.0019). No statistically significant milk group differences and associations were observed for OM, LRTI, and URTI (p > 0.0031).Conclusion: Our findings suggest that neither prominent neutral individual HMOs (ranging from 2′-FL to LNDFHs) nor acidic human milk sialyllactoses or lactose are significantly associated with a reduced or increased risk of infections in infants up to 2 years of age. Further research is needed to determine whether specific HMOs could potentially reduce the incidence or alleviate the course of distinct infections in early life.

Utilization Efficiency of Human Milk Oligosaccharides by Human-Associated Akkermansia Is Strain Dependent.

ABSTRACTAkkermansia muciniphila is a mucin-degrading bacterium found in the human gut and is often associated with positive human health. However, despite being detected by as early as 1 month of age, little is known about the role of Akkermansia in the infant gut. Human milk oligosaccharides (HMOs) are abundant components of human milk and are structurally similar to the oligosaccharides that comprise mucin, the preferred growth substrate of human-associated Akkermansia. A limited subset of intestinal bacteria has been shown to grow well on HMOs and mucin. We therefore examined the ability of genomically diverse strains of Akkermansia to grow on HMOs. First, we screened 85 genomes representing the four known Akkermansia phylogroups to examine their metabolic potential to degrade HMOs. Furthermore, we examined the ability of representative isolates to grow on individual HMOs in a mucin background and analyzed the resulting metabolites. All Akkermansia genomes were equipped with an array of glycoside hydrolases associated with HMO deconstruction. Representative strains were all able to grow on HMOs with various efficiencies and growth yields. Strain CSUN-19, belonging to the AmIV phylogroup, grew to the highest level in the presence of fucosylated and sialylated HMOs. This activity may be partially related to the increased copy numbers and/or the enzyme activities of the α-fucosidases, α-sialidases, and β-galactosidases. This study examines the utilization of individual purified HMOs by Akkermansia strains representing all known phylogroups. Further studies are required to examine how HMO ingestion influences gut microbial ecology in infants harboring different Akkermansia phylogroups.IMPORTANCE Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk and provide several benefits to developing infants, including the recruitment of beneficial bacteria to the human gut. Akkermansia strains are largely considered beneficial bacteria and have been detected in colostrum, breast milk, and young infants. A. muciniphila MucT, belonging to the AmI phylogroup, contributes to the HMO deconstruction capacity of the infant. Here, using phylogenomics, we examined the genomic capacities of four Akkermansia phylogroups to deconstruct HMOs. Indeed, each phylogroup contained differences in their genomic capacities to deconstruct HMOs, and representative strains of each phylogroup were able to grow using HMOs. These Akkermansia-HMO interactions potentially influence gut microbial ecology in early life, a critical time for the development of the gut microbiome and infant health.

Levels of human milk oligosaccharides in breast milk of mothers delivering preterm infants of different gestational ages and their effects on early growth and development

Objective: To investigate the human milk oligosaccharides (HMOs) levels in breast milk of mothers delivering preterm infants and their effects on the early growth and development of infants. Methods: In this prospective cohort study, full-term and preterm newborns whose parents decided to breastfeed were recruited from Peking University Third Hospital between December 1, 2017 and November 30, 2018. The preterm infants were divided based on their gestational ages into extremely preterm (<28 weeks), very preterm (28-31+6 weeks) and moderate to late preterm (32-36+6 weeks) groups. Breast milk was collected from mothers at 7, 14, 28 and 120d postpartum. 368 breast milk samples were collected from 125 mothers in this study, including 54 mothers of full-term infants, 23 mothers of moderate to late preterm infants, 39 mothers of very preterm infants, and 9 mothers of extremely preterm infants. Ultra-performance liquid chromatography-mass spectrometer (UPLC-MS/MS) was used to determine the concentration of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 3'-sialyllactose (3'SL), A-tetrasaccharide (P1), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose Ⅱ (LNFP-Ⅱ) and lacto-N-fucopentaose Ⅴ (LNFP-Ⅴ). Secretor status of mothers was defined as 2'-fucosyllactose (2'FL) concentration in colostrum and transitional milk greater than 200 μg/mL. Weight gain and the occurrence of allergic diseases of infants were collected at 120 d(4 months) postpartum. The chi-square test or the Fisher's exact test was used for the comparison of categorical data between groups; Kruskal-Wallis test and Wilcoxon rank sum test were used for comparison of continuous data between groups. Nemenyi test was used for multiple comparison. Results: 79.2% (99/125) of the mothers were secretor. There were no statistical differences between groups in the secretor status of mothers (χ²=1.31,P>0.05). The total concentration of HMOs peaked at 1-2 weeks postpartum. Compared to the preterm milk, the HMOs from the term milk was trending downwards at an earlier time. In the breast milk of secretor mothers on 28 d, total concentration of HMOs significant differed among the three groups of preterm milk and the term milk, with the median value of 4 587.09,4 615.25,5 277.44,5 476.03 μg/mL, respectively (Kruskal-Wallis χ²=8.1234,P=0.044). When analyzed by the median weight gain of the infants (low vs high weight gain) at 4 months postpartum, 2'FL was significantly lower in the high weight gain group at 7 d (1 818.04 μg/mL vs 2 181.67 μg/mL, W=1 386,P=0.018), while LNT & LNnT were significantly higher (1 182.36 μg/mL vs 1 053.62 μg/mL, W=816,P=0.044). The level of 3FL at 120 d was significantly affected by presence of allergic disease in infants, breast milk from mothers of infants with allergic disease had lower 3FL than those from mothers of infants without allergic disease (256.17 μg/mL vs 286.18 μg/mL, W=564,P=0.026). Conclusions: The overall profiles of HMOs in breast milk of mothers delivering preterm infants was basically the same as that of mothers delivering term infants; individual HMOs play a role in weight gain and the development of allergic diseases in preterm infants, but the mechanism is unclear and needs further study.

The Mean of Milk: A Review of Human Milk Oligosaccharide Concentrations throughout Lactation.

Human milk oligosaccharides (HMOs) are non-digestible and structurally diverse complex carbohydrates that are highly abundant in human milk. To date, more than 200 different HMO structures have been identified. Their concentrations in human milk vary according to various factors such as lactation period, mother’s genetic secretor status, and length of gestation (term or preterm). The objective of this review is to assess and rank HMO concentrations from healthy mothers throughout lactation at a global level. To this aim, published data from pooled (secretor and non-secretor) human milk samples were used. When samples were reported as secretor or non-secretor, means were converted to a pooled level, using the reported mean of approximately 80/20% secretor/non-secretor frequency in the global population. This approach provides an estimate of HMO concentrations in the milk of an average, healthy mother independent of secretor status. Mean concentrations of HMOs were extracted and categorized by pre-defined lactation periods of colostrum (0–5 days), transitional milk (6–14 days), mature milk (15–90 days), and late milk (>90 days). Further categorizations were made by gestational length at birth, mother’s ethnicity, and analytical methodology. Data were excluded if they were from preterm milk, unknown sample size and mothers with any known disease status. A total of 57 peer-reviewed articles reporting individual HMO concentrations published between 1996 and 2020 were included in the review. Pooled HMO means reported from 31 countries were analyzed. In addition to individual HMO concentrations, 12 articles reporting total HMO concentrations were also analyzed as a basis for relative HMO abundance. Total HMOs were found as 17.7 g/L in colostrum, 13.3 g/L in transitional milk, and 11.3 g/L in mature milk. The results show that HMO concentrations differ largely for each individual HMO and vary with lactation stages. For instance, while 2′-FL significantly decreased from colostrum (3.18 g/L ± 0.9) to late milk (1.64 g/L ± 0.67), 3-FL showed a significant increase from colostrum (0.37 g/L ± 0.1) to late milk (0.92 g/L ± 0.5). Although pooled human milk contains a diverse HMO profile with more than 200 structures identified, the top 10 individual HMOs make up over 70% of total HMO concentration. In mature pooled human milk, the top 15 HMOs in decreasing order of magnitude are 2′-FL, LNDFH-I (DFLNT), LNFP-I, LNFP-II, LNT, 3-FL, 6′-SL, DSLNT, LNnT, DFL (LDFT), FDS-LNH, LNFP-III, 3′-SL, LST c, and TF-LNH.

A Mediterranean Meal Plan Modulates Human Milk Composition in Women With Obesity

ObjectivesHuman milk (HM) is impacted by both maternal obesity and diet. HM from women with obesity has higher fat, leptin and insulin concentrations. Additionally, maternal dietary fat intake is associated with increased HM fat content. The objective of this study was to determine the impact of a Mediterranean meal plan on the composition of HM from women with obesity. MethodsIn this pilot study, thirteen women with obesity (body mass index (BMI) ≥ 30 kg/m2) enrolled at five months post-partum and followed a provided Mediterranean meal plan focused on reducing fat intake and increasing unsaturated fat and fiber intake for four weeks. Participants’ diet was assessed using Nutrition Data System for Research. HM was collected at baseline and after the 4-week intervention and the concentrations of metabolites, hormones and cytokines were assayed. Wilcoxon signed-rank tests and analysis of variance were used to assess changes in participant diet and HM composition. Summary statistics are presented as mean ± standard deviation. ResultsThe participants’ Healthy Eating Index Score, a measure of diet quality, significantly improved (38.3 ± 5.13 vs. 78.2 ± 6.75, p < 0.001) and total fat intake was significantly lower (99.6 ± 23.3 g vs. 66.9 ± 12.0 g/day, p < 0.001) after the intervention. Human milk leptin (694 ± 464 pg/ml vs. 437 ± 324 pg/ml, p = 0.007) and tumor necrosis factor α (0.515 ± 0.267 pg/ml vs. 0.310 ± 0.127 pg/ml, p = 0.010) concentrations were lower post intervention, whereas HM macronutrient composition was unchanged. The concentrations of several individual human milk oligosaccharides (HMO) and total HMOs (7720 ± 797 nmol/ml vs. 6986 ± 940 nmol/ml, p = 0.049), as well as HM tyrosine concentration (18.2 ± 6.16 nmol/ml vs. 14.5 ± 4.69 nmol/ml, p = 0.005) were significantly lower post intervention. ConclusionsThe composition of HM from women with obesity can be modulated by short-term adherence to a Mediterranean meal pattern. These findings merit further studies that use longer interventions and examine the impact of any changes in HM composition on infant growth and development. Modifying HM composition via a dietary intervention may provide a novel strategy to promote child development and health. Funding SourcesUSDA ARS #6026-51,000-010-05S and #6026-51,000-012-06S, NIH/NIDDK R01DK107516, Arkansas Children’s Research Institute/Arkansas Biosciences Institute GR037121.

Longitudinal Changes in Human Milk Oligosaccharefides (HMOs) Over the Course of 24 Months of Lactation

Human milk oligosaccharides (HMOs) are complex glycans that are highly abundant in human milk. While over 150 HMOs have been identified, it is unknown how individual HMOs change in concentration over 24 months of lactation. To understand how HMO concentrations change over 24 months of lactation. Breast milk samples were collected from participants in a longitudinal cohort study of Hispanic mother-infant pairs at 1, 6, 12, 18, and 24 months postpartum. Concentrations of 19 of the most abundant HMOs were measured using HPLC. Because the parent study is ongoing and not all participants have finished all time points yet, the sample sizes ranged per time point (n=207 at 1 month; n=109 at 6 months; n=83 at 12 months; n=59 at 18 months; and n=28 at 24 months). Approximately 88% of participants were classified as HMO secretors-a genetic factor that affects concentrations of HMOs such as 2'fucosyllactose (2'FL) and lacto-N-fucopentaose I-while the remaining 12% were classified as nonsecretors. Mixed models were used to examine changes in HMO concentrations and relative abundances over the course of lactation. The majority of HMOs significantly decreased in concentration over the course of lactation. The exceptions were 2'FL, sialyl-lacto-N-tetraose b, and disialyl-lacto-N-tetraose, which did not change with time, and 3-fucosyllactose (3FL) and 3'-sialyllactose (3'SL), which significantly increased. The concentration of 3FL increased 10-fold, from 195 (IQR 138-415) μg/mL at 1 month to 1930 (1100-2630) μg/mL at 24 months, while 3'SL increased 2-fold, from 277 (198-377) μg/mL to 568 (448-708) μg/mL over the same time period. These results indicate that HMOs do not decrease in concentration uniformly across lactation. In particular, 3FL and 3'SL increased over the course of lactation in this cohort. Future studies are required to fully understand the functions of these HMOs.

Microbial production of human milk oligosaccharide lactodifucotetraose

Human milk oligosaccharides (HMOs) are potent bioactive compounds that modulate neonatal health and are of interest for development as potential drug treatments for adult diseases. The potential of these molecules, their limited access from natural sources, and difficulty in large-scale isolation of individual HMOs for studies and applications have motivated the development of chemical syntheses and in vitro enzymatic catalysis strategies. Whole cell biocatalysts are emerging as alternative self-regulating production platforms that have the potential to reduce the cost for enzymatic synthesis of HMOs. Whole cell biocatalysts for the production of short-chained, linear and small monofucosylated HMOs have been reported but those for fucosylated structures with higher complexity have not been explored. In this study, we established a strategy for producing a difucosylated HMO, lactodifucotetraose (LDFT), from lactose and L-fucose in Escherichia coli. We used two bacterial fucosyltransferases with narrow acceptor selectivity to drive the sequential fucosylation of lactose and intermediate 2′-fucosyllactose (2′-FL) to produce LDFT. Deletion of substrate degradation pathways that decoupled cellular growth from LDFT production, enhanced expression of native substrate transporters and modular induction of the genes in the LDFT biosynthetic pathway allowed complete conversion of lactose into LDFT and minor quantities of the side product 3-fucosyllactose (3-FL). Overall, 5.1 g/L of LDFT was produced from 3 g/L lactose and 3 g/L L-fucose in 24 h. Our results demonstrate promising applications of engineered microbial biosystems for the production of multi-fucosylated HMOs for biochemical studies.

A47 SYNTHETIC HUMAN MILK OLIGOSACCHARIDES PREVENT EXPERIMENTAL NECROTIZING ENTEROCOLITIS VIA DIVERGENT TRANSCRIPTOMIC RESPONSES.

Abstract Background Breastmilk reduces the risk of necrotizing enterocolitis (NEC) in preterm infants, but the bioactive components mediating this effect are not well understood. Human milk oligosaccharides (HMOs) reduce NEC both in humans and in relevant animal models. However, it is unclear if there are functional differences between individual oligosaccharides. Aims The objective of this study was to compare the intestinal transcriptome responses of individual HMOs using complementary in vitro and in vivo models of NEC. Methods RNA sequencing was performed on Caco-2Bbe1 gut epithelial cells after exposure to commercially-purified 2’-fucosyllactose (2’FL), 3-fucosyllactose, 6’-siallyllactose, lacto-N-tetraose (LNT) or lacto-N-neotetraose for 24hr at 37°C for 24 h (n=3). Signaling pathways were analyzed in murine- and human-derived NEC enteroids by qPCR. To validate these findings, five-day-old mouse pups were orally gavaged formula with or without individual HMOs, followed by NEC induction with hypoxia (5% O2, 95% N2) and lipopolysaccharide (4 mg/kg/day). Coded ileal sections (n=6–7/group) were analyzed for mucosal injury by histology, immune fluorescence, immunohistochemistry, and gene expression via qPCR. Results The HMO transcriptome clustered into divergent functional categories including metabolic process, protein processing and responses to external stimuli. Each synthetic HMO induced a unique transcriptome and exhibited varying effects on the intestinal epithelial functions and biological pathways. This was confirmed in the murine model of NEC, as both LNT and 2FL mitigated NEC injury with comparable recovery of intestinal cell proliferation (Ki67) and expression of stem cells (Lgr5+). Both qPCR and immunofluorescence staining showed differences between 2FL- and LNT-fed pups in host inflammatory and immune responses. Conclusions This study demonstrates that synthetic HMOs ameliorate intestinal injury in experimental NEC. However, the mechanisms by which individual oligosaccharides act on the intestine differ, suggesting that single synthetic HMOs may not fully recapitulate the benefits of pooled HMOs. Future studies will further delineate structure-function relationships of synthetic HMOs on host intestinal innate and adaptive immune responses. Funding Agencies CIHRFerring Canada Medical Student Research grant

Human milk oligosaccharides, infant growth, and adiposity over the first 4 months of lactation.

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. 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. 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. 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. 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.

Time of Lactation and Maternal Fucosyltransferase Genetic Polymorphisms Determine the Variability in Human Milk Oligosaccharides.

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. R2 = 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.

Targeted LC-ESI-MS2 characterization of human milk oligosaccharide diversity at 6 to 16\xa0weeks 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

Human Milk Oligosaccharides and Hispanic Infant Weight Gain in the First 6 Months.

The aim of this study was to determine whether human milk oligosaccharides (HMOs) at 1 month predicted infant weight gain at 6 months and whether associations varied by HMO secretor status. Participants were 157 Hispanic mother-infant pairs. Human milk samples were collected at 1 month. Nineteen individual HMOs were analyzed using high-performance liquid chromatography, and secretor status was determined by the presence of 2'-fucosyllactose or lacto-N-fucopentaose (LNFP) I. Infant weight was measured at 1 and 6 months. Path analysis was used to test effects of HMO composition on infant weight gain, adjusting for maternal age, prepregnancy BMI, and infant age, sex, and birth weight. In the total sample, higher LNFPII predicted lower infant weight gain (g1 = -4.1, P = 0.004); this was observed in both nonsecretor (g1 = -3.0, P = 0.006) and secretor groups (g1 = -4.7, P = 0.014). In the nonsecretor group, higher lacto-N-neotetraose (g1 = 7.6, P = 0.011) and disialyllacto-N-tetraose (g1 = 14.3, P = 0.002) predicted higher infant weight gain. There were no other associations in the secretor group. Our data suggest that higher LNFPII in human milk may decrease obesity risk across all infants, whereas higher lacto-N-neotetraose and disialyllacto-N-tetraose may increase obesity risk in infants of nonsecretors only.

Key genes involved in utilization of highly abundant HMO in breastmilk are present in B. infantis EVC001 allowing for superior colonization of the breastfed infant gut compared to other commercial probiotic strains

Purpose: Breast milk contains a high concentration of complex glycans known as human milk oligosaccharides (HMO). HMO influence the composition of the intestinal microbiome of breastfed infants by serving as growth substrates to select strains of bifidobacteria. More than 200 individual HMO structures have been identified in breastmilk. However, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and 2’-Fucosyllactose (2’FL) in FUT2+ mothers, are disproportionally abundant. The biological role for their abundance in the context of bacterial colonization of the infant gut has not been determined. In a previous study, Bifidobacterium …

Human Milk Oligosaccharide Supplementation Affects Intestinal Barrier Function and Microbial Composition in the Gastrointestinal Tract of Young Sprague Dawley Rats.

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.

Neutral Human Milk Oligosaccharides Are Associated with Multiple Fixed and Modifiable Maternal and Infant Characteristics.

We aimed to identify if maternal and infant factors were associated with neutral human milk oligosaccharides (HMOs) variability and examined the associations between HMOs concentration and infant growth and disease status in healthy Chinese mothers over a 6-month lactation period. We recruited mothers and their full-term infants as our subjects. At 1–5 days, 8–14 days, 4 weeks, and 6 months postpartum, all participants were interviewed to collect breast milk samples, obtain follow-up data and measure infant length and weight at their local hospital. A total of 23 neutral HMOs were analyzed by high performance liquid chromatography (HPLC)- mass spectrometer (MS). Secretor and Lewis phenotype were determined by the concentration of 2′-fucosyllactose (2′-FL) and Lacto-N-fucopentaose (LNFP)-II. The associations between maternal and infant factors with HMOs concentrations were investigated. A total of 464 human breast milk samples were collected from 116 mothers at four different time points. In total, 76.7% mothers were found to be Secretor and Lewis positive phenotype (Se+Le+), 17.2% were Se-Le+, 4.3% were Se+Le-, and 1.7% were Se-Le-. Several individual HMOs, including 2′-FL, Lactodifucotetraose (LDFT), LNFP-I were determined by Secretor phenotype. Most individual HMOs decreased at the later stage of lactation, except 3′-FL. We suggest that Secretor phenotype and lactation stage could influence most of the neutral HMOs. Concentrations of specific HMOs may be associated with maternal age, allergic history, pre-pregnancy body mass index (BMI), parity, delivery mode, infant gestational age and gender.

A safety evaluation of mixed human milk oligosaccharides in rats

Human milk oligosaccharides (HMOs) are indigestible carbohydrates representing the third largest fraction of solutes in human breastmilk. They provide valuable prebiotic and anti-pathogenic functions in breastfed infants, but are not yet included in most infant formula products. Recent biotechnological advances now facilitate large-scale production of HMOs, providing infant formula manufacturers with the ability to supplement their products with HMOs to mimic human breastmilk. Although the safety of individual HMOs has been confirmed in preclinical toxicological studies, the safety of HMO mixtures has not been tested. We therefore performed bacterial reverse mutation and in vitro micronucleus tests and conducted a repeated-dose oral toxicity study in rats with a mixture of five HMOs (HMO MIX I), containing 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL). HMO MIX I was not genotoxic and did not induce adverse effects in the repeated dose study. The no-observed-adverse-effect-level (NOAEL) for HMO MIX I in this study is 10% in the diet (equivalent to 5.67 g HMO MIX I/kg bw/day for males and 6.97 g HMO MIX I/kg bw/day for females). Our results provide strong evidence for the safety of HMO MIX I in infant products and general foods.

Human Milk Oligosaccharide, Phospholipid, and Ganglioside Concentrations in Breast Milk from United Arab Emirates Mothers: Results from the MISC Cohort.

Human milk oligosaccharides (HMOs), phospholipids (PLs), and gangliosides (GAs) are components of human breast milk that play important roles in the development of the rapidly growing infant. The differences in these components in human milk from the United Arab Emirates (UAE) were studied in a cross-sectional trial. High-performance liquid chromatography‒mass spectrometry was used to determine HMO, PL, and GA concentrations in transitional (5–15 days) and mature (at 6 months post-partum) breast milk of mothers of the United Arab Emirates (UAE). The results showed that the average HMO (12 species), PL (7 species), and GA (2 species) concentrations quantified in the UAE mothers’ transitional milk samples were (in mg/L) 8204 ± 2389, 269 ± 89, and 21.18 ± 11.46, respectively, while in mature milk, the respective concentrations were (in mg/L) 3905 ± 1466, 220 ± 85, and 20.18 ± 9.75. The individual HMO concentrations measured in this study were all significantly higher in transitional milk than in mature milk, except for 3 fucosyllactose, which was higher in mature milk. In this study, secretor and non-secretor phenotype mothers showed no significant difference in the total HMO concentration. For the PL and GA components, changes in the individual PL and GA species distribution was observed between transitional milk and mature milk. However, the changes were within the ranges found in human milk from other regions.

Human Milk Oligosaccharides and Infant Weight in the First 6 Months of Life (P11-053-19)

ObjectivesThe effects of breastfeeding on reduced risk of obesity are inconclusive, although smaller studies suggest that specific human milk components may be relevant to infant weight gain. The primary aim of this study was to determine whether individual human milk oligosaccharides (HMOs) in the first month of life are associated with infant weight in the first 6 months of life. A secondary aim was to assess associations separately by HMO secretor status. MethodsParticipants were 156 Hispanic mother-infant pairs. Breast milk was collected at 1 month. HMO composition was analyzed using high-pressure liquid chromatography, and HMO secretor status was determined by the presence or near absence of 2’-fucosyllactose or lacto-N-fucopentaose (LNFP) I. Infant weight was measured at 1 and 6 months, and infant weight-for-age z scores (WAZ) and WAZ changes were calculated. Multiple linear regressions were used to examine the effect of HMO composition on WAZ, adjusting for maternal age, pre-pregnancy BMI, infant age, sex, and birth weight. ResultsIn the total sample, HMO composition at 1 month was not significantly associated with WAZ at 1 month. In the total sample, higher LNFPII at 1 month predicted lower WAZ gain (β = –0.27, P = 0.01) and lower WAZ status (β = –0.26, P = 0.01) at 6 months. In infants of secretor mothers, higher LNFPII at 1 month predicted lower WAZ gain at 6 months (β = –0.23, P = 0.02); in infants of non-secretor mothers, LNFPII at 1 month predicted lower WAZ status at 6 months (β = –0.40, P = 0.03). Similar significant associations were observed when infant weight replaced WAZ in the models. No other HMOs were significantly related to infant WAZ or weight. ConclusionsOur data suggest that greater exposure to LNFPII in the first month of life may be protective against rapid infant weight gain in the first 6 months, regardless of HMO secretor status. These findings provide additional evidence that specific human milk components may diminish infant obesity risk. Funding SourcesSupported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIH R01 DK110793) and the Gerber Foundation.