Human Milk Oligosaccharides Increase Mucin Expression in Experimental Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease that is highly lethal in premature infants. Human milk oligosaccharides (HMOs) efficiently reduce the incidence of NEC. However, the protective mechanism of HMO treatment is unknown. It is hypothesized that HMOs protect against NEC by inhibiting the damage to intestinal epithelial cells. C57BL/6 pups are challenged with hypoxia and cold stress to induce NEC. All pups are sacrificed after 72 h. It is found that HMO administration reduces the concentrations of IL-8 in the serum and ileum of all NEC mice. Ileum toll-like receptor 4 (TLR4) protein expression and nuclear factor kappa-B (NFκB) pathway activation are inhibited. The proliferative ability of enterocytes in the ileum is restored as determined by labeling with proliferation markers (Ki67, SOX9). In a 3D culture intestinal crypt organoids study, HMO treatment improves the maturation of organoid cells and increases the ratio of proliferative cells under lipopolysaccharides (LPS) treatment. HMO treatment downregulates TLR4 expression in the organoid cells, thus reducing the effect of LPS. HMOs protect intestinal epithelial cells from injury by accelerating the turnover of crypt cells by reducing the expression of TLR4 on intestinal epithelial cells.

Objective:Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2’-Fucosyllactose (2’-FL) and 6’-Sialyllactose (6’-SL), can reduce NEC through inhibition of TLR4 signaling.Design:NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2’-FL, 6’-SL or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, human tissue explants, and via in silico modeling.Results:Supplementation of infant formula with either 2’-FL and/or 6’-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2’-FL and 6’-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2’-FL and 6’-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling.Conclusion:2’-FL and 6’-SL, but not lactose, prevent NEC in mice and piglet models, and attenuate NEC-inflammation in human ileum, in part through TLR4 inhibition.

Necrotizing enterocolitis (NEC) is a life-threatening gastrointestinal emergency in preterm infants, particularly those born before 32 weeks of gestation or weighing less than 1500 g. This review explores the central role of nutrition in NEC prevention, emphasizing the importance of human milk in shaping gut microbiota, modulating immune responses, and enhancing intestinal maturation. The immature gut barrier and dysregulated immune system in preterm neonates make them susceptible to NEC, especially in the context of formula feeding and inappropriate feeding practices. Human milk components such as human milk oligosaccharides, secretory immunoglobulin A, lactoferrin, and growth factors provide immunological and epithelial protection. Strategies including early enteral feeding, trophic feeding, moderate volume advancement, and standardized feeding protocols have shown promise in reducing NEC risk. The review also highlights the role of human milk fortification and the emerging benefits of targeted fortification. Probiotic supplementation, particularly multistrain preparations, has demonstrated efficacy in lowering NEC incidence, although safety concerns remain, especially in extremely low birth weight infants. Donor human milk, while facing supply and logistical challenges, offers a safer alternative to formula and supports better clinical outcomes. Emerging interventions, such as amniotic fluid-derived therapies, microbiome-based treatments, and exosomes, show potential in experimental models. Quality improvement initiatives, multidisciplinary care, and patient-centred outcomes, including parental well-being and neurodevelopment, are also discussed. Overall, evidence-based feeding strategies centered around human milk are likely to reduce NEC risk, enhance growth, and improve long-term outcomes in this vulnerable population.

Gastrointestinal perforation and peritonitis in infants and children: Experience with 179 cases over ten years

Preterm infants who receive human milk instead of formula are 6- to 10-times less likely to develop necrotizing enterocolitis (NEC), one of the most common and devastating intestinal disorders that affects 5–10% of all very-low-birth-weight infants. Combined data from in vitro tissue culture models, in vivo preclinical studies in animal models, as well human mother-infant cohort studies support the hypothesis that human milk oligosaccharides (HMOs), complex sugars that are highly abundant in human milk but not in infant formula, contribute to the beneficial effects of human milk feeding in reducing NEC. The almost 20-year long journey of testing this hypothesis took an interesting turn during HMO in vivo efficacy testing and structure elucidation, suggesting that the original hypothesis may indeed be correct and specific HMO reduce NEC risk, however, the underlying mechanisms are likely different than originally postulated.

Digestion of human milk oligosaccharides by healthy infants evaluated by the lactulose hydrogen breath test.

Human Milk Oligosaccharides Inhibit Candida albicans Invasion of Human Premature Intestinal Epithelial Cells ,

A safety evaluation of mixed human milk oligosaccharides in rats

Background: Infant formula has a similar composition to breast milk, especially on key components like proteins, fats, and functional oligosaccharides. However, significant differences exist between bovine milk-based formula and human breast milk, particularly in the abundance and diversity of human milk oligosaccharides (HMOs). HMOs are pivotal in shaping the infant gut microbiome and immune system, with potential implications for infant health and development.Objectives: The literature review aimed to explore the role of HMOs in shaping the gut microbiome and strengthening the intestinal immune system in infants.Methods: Descriptive research was carried out in May-August 2023 using the literature review method. The design and implementation of this study referred to the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) for Randomized Controlled Trials (RCTs). Literature sources were obtained from PubMed, Elsevier, Medline, Nature, PLOS, MPDI, JAMA, and Scopus publication databases with a limitation of the last ten years (2013-2023). One hundred twenty articles were obtained with these keywords and then selected in stages according to predetermined criteria. Six articles that met the criteria were retrieved from 2013-2023. The articles obtained were compiled, analyzed, and concluded by looking for similarities and dissimilarities, giving views, comparing, and summarizing.Results: The review revealed that HMOs significantly influenced the composition and function of the infant's intestinal microbiota, inhibiting harmful pathogens in the intestine. Therefore, HMOs as prebiotics were crucial in promoting intestinal immune system health in infants.Conclusion: Human Milk Oligosaccharides (HMOs) in infant formula substantially impacted the gut microbiome composition and the infant's intestinal immune system. Studies consistently showed that HMOs influenced the growth and diversity of gut bacteria, leading to outcomes similar to those seen in breastfed infants.

Structure-Function Relationships of Human Milk Oligosaccharides

Necrotizing enterocolitis (NEC) and late-onset sepsis (LOS) are two major contributors to death among preterm infants. Oropharyngeal administration of colostrum (OAC) has been proved as an easy, safe, and economically viable technique to help preterm neonates to build up their immunity. In this review, we assessed the effects of OAC on preterm infants. Several mainstream databases were searched including PubMed, EMBASE, Cochrane Central Register of Controlled Trials (CENTRAL), and a website of clinical trials. Randomized controlled trials (RCTs) comparing OAC vs. placebo or no intervention in preterm infants (gestation age <34 weeks or birth weight <1500 g) were eligible. Overall, nine RCTs (n = 689) were included in the review. Meta-analysis showed no statistical significance in terms of the incidence of NEC (RR = 0.59, 95% CI = 0.33–1.06, p = 0.08), LOS (RR = 0.78, 95% CI = 0.60–1.03, p = 0.08) and mortality rate (RR = 0.63, 95% CI = 0.38–1.05, p = 0.07). No significant difference was found in the subgroup analysis, apart from the group of the undeveloped region in NEC and mortality. In addition, time was significantly reduced in terms of achieving full enteral feeding (MD = −3.60, 95% CI = −6.55–0.64, p = 0.02) and hospital stay (MD = −10.38, 95% CI = −18.47–2.29, p = 0.01). The results show that OAC does not reduce the incidences of NEC, LOS, and death in preterm infants, but there is a trend toward a positive effect. It is therefore recommended as routine care for preterm infants in the NICU.

Necrotising enterocolitis (NEC) is an inflammatory intestinal disorder affecting premature infants. Despite the worldwide improvement of health care practices and facilities raising the survival rates of neonatal premature infants, there has not been any improvement in treatment options or mortality rates for NEC. There has been an extensive volume of research into NEC, though to date there has not been any evidence to directly associate a causal agent to this devastating disease, nor have there been any conclusive observations of NEC prior to birth. The only key prognostic signal for NEC is that onset and severity of the disease are significantly associated with the prematurity of the neonatal infant. During the process of birth, the infant transitions from the near sterile conditions of the womb to the outside environment teeming with bacteria. Only then does the infant develop a symbiotic relationship as a host to beneficial bacteria. Upon this transition the community of gut microbes develops and aids in a range of host functions, namely digestion and absorption of nutrients, as well as the immune response. It is also at this time that NEC can begin to develop in the lower gastrointestinal tract. This coinciding of factors implicates the colonisation of the gut with bacteria in the development of NEC, however studies to date have failed to provide consistent reports of a causative pathogen or a characteristic gut microbiome structure associated with NEC. The purpose of this study was to characterise the structure of the bacterial community in the gastrointestinal tract of infants with and without NEC in order to identify a community distinctive to infants with NEC. This was addressed using non-invasive faecal sampling of premature infants in a large, prospectively enrolled cohort from across England, sampled over a twenty-day window spanning ten days prior to and ten days following the onset of NEC. Using established V4 16S rRNA protocols, bacterial taxa within environmental samples could be characterised without relying on classic culture dependent methods. With this amplification technique it was possible to utilise small amounts of bacterial DNA isolated from infant faecal samples while at the same time mitigating the selection bias associated with culture-based techniques. Short read sequencing (illumina MiSeq) was performed on a total of 656 faecal samples from 132 infants spanning eight neonatal intensive care units across England. All infants had gestational durations of less than 35 weeks. 44 infants had NEC (225 samples) and 88 were assigned as controls (431 samples) according to key risk factors defined by medical practitioners. Taxonomic abundances assigned with the QIIME informatics pipeline were normalised using non-reductive negative binomial normalisation. Local contributions to beta-diversity (LCBD) scores were used to quantify taxonomic changes in the community structure through subset regression. Non-metric multidimensional scaling (NMDS) was used to establish risk factors that best described NEC and control samples. The Random Forest machine learning algorithm was used to establish taxa that best discriminated between NEC and control infants, as well as to identify any conserved pathogens. Subset regression identified feeding regime, mode of delivery and age at sampling as significant discriminating factors for the NEC status of infants based on sample LCBD values. However, NMDS plots of sample LCBD values showed no clear clustering of samples according to NEC status. Canonical correlation analysis (CCA) indicated that this variability was due to inter-individual differences. Of the risk factors that could be accounted for, feeding regime was the most effective in differentiating community structures of NEC and control infant samples. There was also evidence that initial communities were influenced by delivery method. Three subgroups of infants based on these influential risk factors and with sufficient sampling depth were established and analysed separately in addition to collective, population-scale analysis. Random Forest analysis demonstrated that reduced abundance of the genus Bifidobacterium was significantly associated with NEC across all sub-groups of infants. Additionally, this method of analysis indicated no clear pathogenic taxa that consistently spanned the population. For infants with NEC that were delivered by caesarean section and fed both formula and breast milk, there was increased abundance of the genus Dialister when sampled shortly after birth. Infants that did not develop NEC, who were delivered vaginally and fed both formula and breast milk were seen to have a significantly greater abundance of the genus Veillonella over time, relative to NEC subjects. There were no additional taxonomic differences that could be ascertained in the sub-group of infants who were vaginally delivered and fed breast milk exclusively. Overall, the unique nature of the microbiome and the high degree of inter-individual variation within the community made direct comparisons between NEC and non-NEC subjects difficult. However, by accounting for factors that were significantly associated with NEC status it was possible to observe consistent association of increased bifidobacterial abundance in infants that did not develop NEC. This highlights the importance of large scale studies and case-control assignment when analysing complex community structures such as that of the human gastrointestinal tract, as well as the powerful, deep analytical analysis provided by machine learning algorithms. Further work should look to establish the impact and role of Bifidobacteria in the human gut community to inform early interventions in healthcare settings for infants at risk of NEC, focussing specifically on encouraging the development of a community structure reflective of that observed in premature infants that do not develop NEC.

Probiotics for preterm infants on the NICU

Human milk oligosaccharides (HMOs) are bioactive factors that benefit neonatal health, but little is known about effects on growth in very preterm infants (<32 weeks' gestation). We aimed to quantify HMO concentrations in human milk fed to very preterm infants during the neonatal hospitalization and investigate associations of HMOs with infant size and body composition at term-equivalent age. In 82 human-milk-fed very preterm infants, we measured HMO concentrations at two time points. We measured anthropometrics and body composition with air displacement plethysmography at term-equivalent age. We calculated means of individual and total HMOs, constructed tertiles of mean HMO concentrations, and assessed differences in outcomes comparing infants in the highest and intermediate tertiles with the lowest tertile using linear mixed effects models, adjusted for potential confounders. The mean (SD) infant gestational age was 28.2 (2.2) weeks, and birthweight was 1063 (386) grams. Exposure to the highest (vs. lowest) tertile of HMO concentrations was not associated with anthropometric or body composition z-scores at term-corrected age. Exposure to the intermediate (vs. lowest) tertile of 3FL was associated with a greater head circumference z-score (0.61, 95% CI 0.15, 1.07). Overall, the results do not support that higher HMO intakes influence growth outcomes in this very preterm cohort.

Human milk oligosaccharides (HMOs) represent the thirdmost abundantfraction of biomolecules in human milk (HM) and play a crucial rolein infant health and development. The unique contributions of HMOsto healthy development of breast-fed infants are assumed to rely onthe extraordinary complexity and diversity of HMO isomeric structures,which in turn still cause a huge analytical challenge. Many contemporaryanalytical methods aiming for more detailed HMO characterization combineion mobility (IM) with LC-MS for enhanced structural resolution butare typically lacking the robustness necessary for application toHM cohorts with hundreds of samples. To overcome these challenges,we introduce a novel, robust all-ion fragmentation (AIF) LC-ESI-IM-MSmethod integrating four analytical dimensions: high-resolution LCseparation, IM drift time, accurate mass precursor, and fragment ionmeasurements. This four-dimensional (4D) analytical characterizationis sufficient for resolving various HMO structural isomers in an efficientway. Thereby, up to 200 HMO compounds with a maximum degree of polymerizationof 13 could be simultaneously identified and relatively quantified.We devised two methods using this 4D analytical approach. One intendedfor in-depth characterization of multiple known but also novel HMOstructures and the second is designed for robust, increased-throughputanalyses. With the first approach, five trifucosyl-lacto-N-tetraose isomers (TF-LNTs), four of which were never detected beforein HM, as well as additional difucosyl-lacto-N-heaoseisomers (DF-LNHs), were revealed and structures fully elucidated byAIF and IM. This exemplifies the potential of our method for in-depthcharacterization of novel complex HMO structures. Furthermore, theincreased-throughput method featuring a shorter LC gradient was appliedto real-world HM samples. Here, we could differentiate the HM typesI–IV based on a broader range of partly new marker HMOs. Wecould also derive valuable new insights into variations of multipleand rare HMOs up to DP 11 across lactational stages. Overall, ourAIF LC-ESI-IM-MS approach facilitates in-depth monitoring and confidentidentification of a broad array of distinct and simple to very complexHMOs. We envision this robust AIF LC-ESI-IM-MS approach to advanceHMO research by facilitating the characterization of a broad rangeof HMOs in high numbers of HM samples. This may help to further extendour understanding about HMOs structure–function relationshipsrelevant for infants’ healthy development