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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.

Background Human milk is the most genuine form of personalized nutrition, whereby its nutritional and bioactive constituents support the changing needs of the growing infant. Personalized proteome profiling strategies may provide insights into maternal-infant relationships. Proteins and endogenous peptides in human milk play an important role as nutrients for growth and have distinct functionality such as immune defense. Comprehensive monitoring of all of the human milk proteinaceous components, including endogenous peptides, is required to fully understand the changing role of the human milk proteome throughout lactation. Objective We aimed to investigate the personalized nature of the human milk proteome and peptidome for individual mother-infant dyads. Methods Two individual healthy milk donors, aged 29 and 32 y and both of a normal BMI, were longitudinally observed over weeks 1, 2, 3, 4, 6, 8, 10, 12, and 16 postpartum. Milk collection was standardized. Comprehensive variations in the human milk proteinaceous components were assessed using quantitative LC-MS/MS methods. Results We longitudinally profiled the concentrations of >1300 milk proteins and 2000 endogenous milk peptides spanning 16 wk of lactation for 2 individual donors. We observed many gradual and alike changes in both donors related to temporal effects, for instance early lactation was marked by high concentrations of proteins and peptides involved in lactose synthesis and immune development. Uniquely, in 1 of the 2 donors, we observed a substantial anomaly in the milk composition, exclusively at week 6, likely indicating a response to inflammation and/or infection. Conclusions Here, we provide a resource for characterizing the lactational changes in the human milk proteome, encompassing thousands of proteins and endogenous peptides. Further, we demonstrate the feasibility and benefit of personalized profiling to monitor the influence of milk on the development of the newborn, as well as the health status of each individual mother-infant pair.

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 .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.

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.

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.

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant’s best start at a healthy life. One key component of human milk is β-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk’s endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of β-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of β-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in β-casein’s known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of β-casein may be important as it resides on known β-casein-derived antimicrobial peptide sequences.

Immunoglobulins are the primary protective products in human milk and are responsible for transferring maternal pathogen memory to the infant, providing protection by binding to recognized pathogens and inhibiting virulence. To better understand potentially protective/anti-infective compounds in human milk, the establishment of human milk–tailored analytical approaches is crucial, as most contemporary analytical methods have been optimized for plasma or serum. One of the most prominent immunoglobulins in human milk is secretory immunoglobulin A (sIgA), which may be relevant for the protection of breastfed infants from harmful pathogens. Advanced sIgA detection methods can help monitor the immune status and development of the mother-infant dyad. We therefore developed an enzyme-linked immunosorbent assay (ELISA) sIgA method for the quantitative analysis of IgA plus secretory component (SC), validated with sIgA standards and substantiated by mass spectrometry (MS)–based proteomics. A very strong correlation was observed between the MS-detected IgA1 and the human milk–specific sIgA ELISA (r = 0.82). Overall, the MS data indicate that the developed human milk sIgA ELISA does not differentiate between sIgA1 and sIgA2 and is, therefore, a reflection of total sIgA. Furthermore, our MS data and the human milk–derived sIgA ELISA data are better correlated than data derived from a standard serum IgA ELISA kit (relative to MS IgA1 r = 0.82 and r = 0.42, respectively). We therefore propose our human milk–specific sIgA ELISA as an ideal quantitative indicator of total sIgA with advantages over current serum IgA ELISA kits. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03468-4.

Human milk fatty acid composition varies during lactation and is influenced by maternal diet, maternal lifestyle-related factors and genetic background. This is one of the first studies to investigate a period effect, i.e. the impact of lifestyle-related changes on human milk fatty acid composition, in two different cohorts. Lactating women were recruited from the general population a decade apart in Ulm, Germany, using similar methodology. Human milk samples collected 6 weeks post-partum were analysed [Ulm Birth Cohort Study (UBCS (2000)), n=567; Ulm SPATZ Health Study (SPATZ (2012)), n=458)]. Centred log ratio transformation was applied to fatty acid data. Principal component analysis (PCA) was used to determine study-dependent fatty acid profiles. A general linear model was used to determine the study (or period) effect on fatty acid profiles adjusting for duration of gestation, age, education, delivery mode, smoking and pre-pregnancy BMI. Two principal components were retained (PC1 and PC2). PC1 was associated with UBCS, while PC2 was associated with SPATZ. PC1 comprised high saturated fatty acids (SFAs), and low monounsaturated fatty acids (MUFAs), n-6 and n-3 long-chain polyunsaturated fatty acids (LCPUFAs). The inverse was true for PC2. Although human milk remains a source of essential fatty acids, infants could be at risk of inadequate n-3 and n-6 LCPUFA intake through human milk. The differences in the human milk fatty acid profiles also reflect changes in maternal dietary habits in the more recent cohort, which may comprise lower intakes of dietary TFAs, SFAs and higher intakes of vegetable oils.

Free amino acids (FAAs) are important regulators of key pathways necessary for growth, development, and immunity. Data on FAAs in human milk (HM) and their roles in infant development are limited. We investigated the levels of FAAs and total amino acids (TAA, i.e., the sum of conjugated amino acids and FAAs) in HM in relation to infant and maternal characteristics and immunological conditions. FAA and TAA levels in HM sampled at 6 weeks (n = 671) and 6 months (n = 441) of lactation were determined using high-performance liquid chromatography. Child growth was ascertained at 4–5 weeks and at 6–7 months of age. Child allergy and lower respiratory tract infections were assessed in the first years of life. Associations of amino acid (AA) levels in HM with child growth and health outcomes were determined by Spearman correlation and modified Poisson regression, respectively. Free glutamine, glutamate, and serine in 6-week HM positively correlated with infant weight gain in the first 4–5 weeks of age. Maternal pre-pregnancy weight and body mass index (BMI) were negatively correlated with free glutamine and asparagine in 6-week and 6-month HM and positively correlated with the sum of TAAs in 6-month HM, but significance was lost following confounder adjustment. Free glutamine was lower in 6-month HM of mothers with an allergy (either active or non-active). No consistent associations were found between FAAs in HM and child health outcomes. However, potential negative associations were observed between specific FAAs and the risk of food allergy. These results suggest that specific FAAs play a role in infant growth. Moreover, these findings warrant further investigations into the relation of FAAs in HM with infant health outcomes and maternal allergy., publishedVersion

Human milk oligosaccharides (HMOs) have specific dose‐dependent effects on child health outcomes. The HMO profile differs across mothers and is largely dependent on gene expression of specific transferase enzymes in the lactocytes. This study investigated the trajectories of absolute HMO concentrations at three time points during lactation, using a more accurate, robust, and extensively validated method for HMO quantification. We analyzed human milk sampled at 6 weeks (n = 682), 6 months (n = 448), and 12 months (n = 73) of lactation in a birth cohort study conducted in south Germany, using label‐free targeted liquid chromatography mass spectrometry (LC‐MS2). We assessed trajectories of HMO concentrations over time and used linear mixed models to explore the effect of secretor status and milk group on these trajectories. Generalized linear model‐based analysis was used to examine associations between HMOs measured at 6 weeks of lactation and maternal characteristics. Results: Overall, 74%, 18%, 7%, and 1% of human milk samples were attributed to milk groups I, II, III, and IV, respectively. Most HMO concentrations declined over lactation, but some increased. Cross-sectionally, HMOs presented high variations within milk groups and secretor groups. The trajectories of HMO concentrations during lactation were largely attributed to the milk group and secretor status. None of the other maternal characteristics were associated with the HMO concentrations. The observed changes in the HMO concentrations at different time points during lactation and variations of HMOs between milk groups warrant further investigation of their potential impact on child health outcomes. These results will aid in the evaluation and determination of adequate nutrient intakes, as well as further (or future) investigation of the dose‐dependent impact of these biological components on infant and child health outcomes.

Among the human milk oligosaccharides (HMOS), the galactosyllactoses (GLs) are only limitedly studied. This study aims to describe the presence and relative levels of HMOS, including GLs, in human milk (HM) according to maternal Secretor and Lewis (SeLe) phenotype and lactation stage. Relative levels of 19 HMOS were measured in 715 HM samples collected in the first 4 months postpartum from 371 donors participating in the PreventCD study. From a subset of 24 Dutch women (171 HM samples), samples were collected monthly up to 12 months postpartum and were additionally analyzed for relative and absolute levels of β6′-GL, β3′-GL and α3′-GL. Maternal SeLe phenotype or HM group was assigned based on the presence of specific fucosylated HMOS. Most HMOS, including β6′- and β3′-GL, were present in the vast majority (≥75%) of HM samples, whereas others (e.g., LNDFH II, 2′-F-LNH and α3′-GL) only occurred in a low number (&lt, 25%) of samples. Clear differences were observed between the presence and relative levels of the HMOS according to the maternal phenotype and lactation stage. Absolute concentrations of β6′-GL and β3′-GL were higher in HM group IV samples compared to samples of the other three HM groups. β3′-GL was also higher in HM group II samples compared to HM group I samples. β3′-GL and β6′-GL were stable over lactation stages. In conclusion, presence and levels of HMOS vary according to HM group and lactation stage. Not all HMOS behave similarly: some HMOS depend strongly on maternal phenotype and/or lactation stage, whereas others do not. β3′-GL and β6′-GL were present in low concentrations in over 75% of the analyzed HM samples and showed differences between HM groups, but not between the lactation stages.

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&prime, FL and 3-FL), neutral tetraoses (DFL, LNT, LNnT), neutral pentaoses (LNFP I, II, III, V), and acidic trioses (3&prime, SL and 6&prime, 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.

The aim of this study was to determine the differences in human milk fatty acid composition in relation to maternal allergy within a large birth cohort study using statistical methods accounting for the correlations that exist in compositional data. We observed marginal differences in human milk fatty acid composition of allergic and non-allergic mothers. However, our results do not support the hypothesis that human milk fatty acid composition is influenced by allergy or that it differs between mothers with or without allergy. Observed differences in our results between transformed and untransformed fatty acid data call for re-evaluation of previous, as well as future, studies using statistical methods appropriate for compositionality of fatty acid data.

The aim of this study was to determine the differences in human milk fatty acid composition in relation to maternal allergy within a large birth cohort study using statistical methods accounting for the correlations that exist in compositional data. We observed marginal differences in human milk fatty acid composition of allergic and non-allergic mothers. However, our results do not support the hypothesis that human milk fatty acid composition is influenced by allergy or that it differs between mothers with or without allergy. Observed differences in our results between transformed and untransformed fatty acid data call for re-evaluation of previous, as well as future, studies using statistical methods appropriate for compositionality of fatty acid data., publishedVersion

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.

There is a growing interest for investigating endogenous peptides from human biofluids which may provide yet unknown functional benefits or provide an early indication of disease states as potential biomarkers. A major technical bottleneck in the investigation of endogenous peptides from body fluids, e.g., serum, urine, saliva, and milk, is that each of these fluids seems to require unique workflows for peptide extraction and analysis. Thus, protocols optimized for serum cannot be directly translated to milk. One biofluid that is readily available, but which has not been extensively explored, is human milk, whose analysis could contribute to our understanding of the immune development of the newborn infant. Due to the occurrence of highly abundant lipids, proteins, and saccharides, milk peptidomics requires dedicated sample preparation steps. The aim of this study was to develop a time and cost-efficient workflow for the analysis of the human milk peptidome, for which we compared peptide extraction methodologies and peptide fragmentation methods. A method using strong acid protein precipitation and analysis by collision-induced dissociation fragmentation was found to be superior to all other test methods, allowing us qualitative and quantitative detection of about 4000 endogenous human milk peptides in a total analysis time of just 18 h. Electronic supplementary material The online version of this article (10.1007/s00216-018-01566-4) contains supplementary material, which is available to authorized users.

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.

One of the well-known features of human milk, is the capacity to protect against the risk and impact of neonatal infections, as well as to influence the onset of allergic and metabolic disease manifestations. The major objective of this review is to provide a detailed overview regarding the role of human milk, more specifically the diversity in human milk oligosaccharides (HMOS), on early life immune development. Novel insights in immune modulatory effects of HMOS obtained by in vitro as well as in vivo studies, adds to the understanding on how early life nutrition may impact immune development. Extensive description and analysis of single HMOS contributing to the diversity within the composition provided during breastfeeding will be discussed with specific emphasis on immune development and the susceptibility to neonatal and childhood infections.