Human milk oligosaccharides in breast milk and 2-year outcome in preterm infants: An exploratory analysis.

BACKGROUND & AIMS: The health benefit of human milk (HM) for preterm infant development is known but the role of human milk oligosaccharides (HMOs) contained in HM remains underexplored. We explored the relationship between exposure to HMOs contained in mother's milk and growth and neurodevelopment at 2-years corrected age in preterm infants. METHODS: Exclusively breastfed preterm infants born between 27 and 34 weeks of gestation were enrolled in a monocentric prospective observational study, LACTACOL. Samples of breast milk were collected once a week for 7 weeks after birth. HMOs and sialic acid were measured by liquid chromatography. Age and Stages questionnaire (ASQ) version 2 was used to assess 2-year neurodevelopmental outcome. We analyzed the relationship between HMO content and (i) infant neurodevelopment at 2-years, and (ii) growth outcome at discharge and at 2 years. A secondary analysis was performed among Secretor(+) Lewis(+) mothers. Only associations with a false discovery rate of 10% or less according to the Benjamini-Hochberg procedure were considered significant. RESULTS: 137 preterm infants (mean gestational age of 31.3 ± 1.7 weeks, mean birth weight of 1494 g ± 336 g) born to 117 mothers (mean age of 30.8 ± 5.0 years) were enrolled. Total HMOs and most individual HMOs and sialic acid concentrations decreased with advancing postnatal age, except for lacto-N-fucopentaose-III and 3-fucosyllactose, which increased. Total HMOs were positively correlated with neonatal length growth (adjusted p = 0.012). Neither total HMOs nor any individual HMO correlated with ASQ score in the overall cohort. However, lacto-N-fucopentaose-III (LNFP-III) was significantly associated with total ASQ score (adjusted p ≤ 0.015) among the 104 infants born to Secretor(+) Lewis(+) mothers. CONCLUSIONS: In this exploratory study in very preterm infants, total HMOs and most individual HMOs, except LNFP-III, decreased with advancing postnatal age. Neither the concentration of total HMOs nor that of any individual HMO were associated with ASQ score at 2 years, except for LNFP-III in Secretor(+) Lewis(+) mothers.

Perinatal Administration of C-Phycocyanin Protects Against Atherosclerosis in apoE-Deficient Mice by Modulating Cholesterol and Trimethylamine-N-Oxide Metabolisms.

OBJECTIVE: Gestational hypercholesterolemia concomitantly with a highly oxidative environment is associated with higher atherosclerosis in human and animal offspring. This work aimed to determine whether perinatal administration of a C-phycocyanin concentrate, a powerful antioxidant, can protect against atherosclerosis development in genetically hypercholesterolemic mice in adult life. Approach and Results: C-Phycocyanin was administered during gestation solely or gestation and lactation to apolipoprotein E-deficient mice. Male and female offspring were studied until 25 weeks old. Progenies born to supplemented mothers displayed significantly less atherosclerotic root lesions than control group in all groups excepted in male supplemented during gestation and lactation. Female born to supplemented mothers had a greater gallbladder total bile acid pool, lower secondary hydrophobic bile acid levels such as lithocholic acid, associated with less plasma trimethylamine N-oxide at 16 weeks old compared with control mice. Regarding male born to C-Phycocyanin administrated mothers, they expressed a higher high-density lipoprotein cholesterol level, more soluble bile acids such as ß-muricholic acids, and a decreased plasma trimethylamine at 16 weeks old. Liver reduced-to-oxidized glutathione ratio were increased and liver gene expression of superoxide dismutase and glutathione peroxidase were significantly decreased in male born to gestational supplemented mothers. No difference in the composition of cecal microbiota was found between groups, regardless of sex. CONCLUSIONS: Our findings suggest a protective effect of perinatal antioxidant administration on atherosclerosis development in apolipoprotein E-deficient mice involving sex-specific mechanisms.

Maternal Microbiota Transfer Programs Offspring Eating Behavior.

Understanding the link between mother's obesity and regulation of the child's appetite is a prerequisite for the design of successful preventive strategies. Beyond the possible contributions of genetic heritage, family culture, and hormonal and metabolic environment during pregnancy, we investigate in the present paper the causal role of the transmission of the maternal microbiotas in obesity as microbiotas differ between lean and obese mothers, maternal microbiotas are the main determinants of a baby's gut colonization, and the intestinal microbiota resulting from the early colonization could impact the feeding behavior of the offspring with short- and long-term consequences on body weight. We thus investigated the potential role of vertical transfers of maternal microbiotas in programming the eating behavior of the offspring. Selectively bred obese-prone (OP)/obese-resistant (OR) Sprague-Dawley dams were used since differences in the cecal microbiota have been evidenced from males of that strain. Microbiota collected from vagina (at the end of gestation), feces, and milk (at postnatal days 1, 5, 10, and 15) of OP/OR dams were orally inoculated to conventional Fischer F344 recipient pups from birth to 15 days of age to create three groups of pups: F-OP, F-OR, and F-Sham group (that received the vehicle). We first checked microbiotal differences between inoculas. We then assessed the impact of transfer (from birth to adulthood) onto the intestinal microbiota of recipients rats, their growth, and their eating behavior by measuring their caloric intake, their anticipatory food reward responses, their preference for sweet and fat tastes in solutions, and the sensations that extend after food ingestion. Finally, we searched for correlation between microbiota composition and food intake parameters. We found that maternal transfer of microbiota differing in composition led to alterations in pups' gut microbiota composition that did not last until adulthood but were associated with specific eating behavior characteristics that were predisposing F-OP rats to higher risk of over consuming at subsequent periods of their life. These findings support the view that neonatal gut microbiotal transfer can program eating behavior, even without a significant long-lasting impact on adulthood microbiota composition.

Breast milk protein content at week 3 after birth and neurodevelopmental outcome in preterm infants fed fortified breast milk.

BACKGROUND: Feeding supplemented mother milk during hospital stay improves neurodevelopment in preterm infants. Yet the composition of mother milk varies widely between subjects. The relationship between this variation and outcome is unknown. OBJECTIVE: To determine whether the protein content in native breast milk (BM) correlates with 2-year infant outcome. DESIGN: In a monocentric prospective observational study, LACTACOL, preterm infants born between 28 and 34 weeks of gestation, whose mothers decided to exclusively breastfeed, were enrolled during the first week of life. Samples of expressed breast milk obtained at several times of the day were pooled over a 24-h period, and such pool was used for macronutrient analysis, using mid-infrared analyzer. Age and Stages questionnaire (ASQ) was used to assess 2-year neurodevelopmental outcome. We analyzed the relationship between protein content in BM, and (i) infant neurodevelopment at 2-year (primary outcome), and (ii) growth until 2-year (secondary outcome). RESULTS: 138 infants were enrolled. The main analysis concerned 130 infants (including 40 twin infants) and 110 mothers with BM samples collected at week 3 after birth. Native BM samples were ranked in three tertiles of protein content (g/100 ml): 0.91 ± 0.09 (lower), 1.14 ± 0.05 (middle) and 1.40 ± 0.15 (upper); 48, 47 and 35 infants were ranked, respectively, in these three tertiles. Infants in the upper tertile were more often singleton (P = 0.012) and were born with lower birth weight and head circumference Z-scores (P = 0.005 and 0.002, respectively). Differences in weight and head circumference were no longer observed at 2-year. ASQ score at age 2 did not differ between the three tertiles (P = 0.780). Sensitivity analyses with imputations, including all 138 infants, confirmed the main analysis as well as analyses based on fortified BM as exposure. CONCLUSIONS: Protein content of BM (native or fortified) is not associated with preterm infant neurodevelopment at 2-year. Higher protein content was associated with a lower birth weight.

Correction: Alexandre-Gouabau et al. "Comprehensive Preterm Breast Milk Metabotype Associated with Optimal Infant Early Growth Pattern", Nutrients, 2019, 11, 528.

The authors wish to make a correction to Section 2 [...].

Simultaneous exploration of nutrients and pollutants in human milk and their impact on preterm infant growth: An integrative cross-platform approach.

Early nutritional management including fortified human breastmilk is currently recommended to fulfil the energy demands and counterbalance risks associated to preterm birth. However, little is known about the potential adverse effects of exposure to persistent organic pollutants (POPs) carried in human milk on preterm infant growth. We conducted a pilot study proving the application of an integrative analytical approach based on mass spectrometry (MS) coupled to advanced statistical models, favouring the comprehensive molecular profiling to support the identification of multiple biomarkers. We applied this workflow in the frame of a preterm infants' cohort to explore environmental determinants of growth. The combination of high resolution gas and liquid chromatography MS platforms generated a large molecular profile, including 102 pollutants and nutrients (targeted analysis) and 784 metabolites (non-targeted analysis). Data analysis consisted in a preliminary examination of associations between the signatures of POPs and the normalised growth of preterm infants, using multivariate linear regression adjusting for known confounding variables. A second analysis aimed to identify multidimensional biomarkers using a multiblock algorithm allowing the integration of multiple datasets in the growth model of preterm infants. The preliminary results did not suggest an impairment of preterm growth associated to the milk concentrations of POPs. The multiblock approach however revealed complex interrelated molecular networks of POPs, lipids, metabolites and amino acids in breastmilk associated to preterm infant growth, supporting the high potential of biomarkers exploration of this proposed workflow. Whereas the present study intended to identify simultaneously pollutant and nutrient exposure profiles associated to early preterm infant growth, this workflow may be easily adapted and applied to other matrices (e.g. serum) and research settings, favouring the functional exploration of environmental determinants of complex and multifactorial diseases.

Neonatal Consumption of Oligosaccharides Greatly Increases L-Cell Density without Significant Consequence for Adult Eating Behavior.

Oligosaccharides (OS) are commonly added to infant formulas, however, their physiological impact, particularly on adult health programming, is poorly described. In adult animals, OS modify microbiota and stimulate colonic fermentation and enteroendocrine cell (EEC) activity. Since neonatal changes in microbiota and/or EEC density could be long-lasting and EEC-derived peptides do regulate short-term food intake, we hypothesized that neonatal OS consumption could modulate early EECs, with possible consequences for adult eating behavior. Suckling rats were supplemented with fructo-oligosaccharides (FOS), beta-galacto-oligosaccharides/inulin (GOS/In) mix, alpha-galacto-oligosaccharides (αGOS) at 3.2 g/kg, or a control solution (CTL) between postnatal day (PND) 5 and 14/15. Pups were either sacrificed at PND14/15 or weaned at PND21 onto standard chow. The effects on both microbiota and EEC were characterized at PND14/15, and eating behavior at adulthood. Very early OS supplementation drastically impacted the intestinal environment, endocrine lineage proliferation/differentiation particularly in the ileum, and the density of GLP-1 cells and production of satiety-related peptides (GLP-1 and PYY) in the neonatal period. However, it failed to induce any significant lasting changes on intestinal microbiota, enteropeptide secretion or eating behavior later in life. Overall, the results did not demonstrate any OS programming effect on satiety peptides secreted by L-cells or on food consumption, an observation which is a reassuring outlook from a human perspective.

Comprehensive Preterm Breast Milk Metabotype Associated with Optimal Infant Early Growth Pattern.

Early nutrition impacts preterm infant early growth rate and brain development but can have long lasting effects as well. Although human milk is the gold standard for feeding new born full-term and preterm infants, little is known about the effects of its bioactive compounds on breastfed preterm infants' growth outcomes. This study aims to determine whether breast milk metabolome, glycome, lipidome, and free-amino acids profiles analyzed by liquid chromatography-mass spectrometry had any impact on the early growth pattern of preterm infants. The study population consisted of the top tercile-Z score change in their weight between birth and hospital discharge ("faster grow", n = 11) and lowest tercile ("slower grow", n = 15) from a cohort of 138 premature infants (27⁻34 weeks gestation). This holistic approach combined with stringent clustering or classification statistical methods aims to discriminate groups of milks phenotype and identify specific metabolites associated with early growth of preterm infants. Their predictive reliability as biomarkers of infant growth was assessed using multiple linear regression and taking into account confounding clinical factors. Breast-milk associated with fast growth contained more branched-chain and insulino-trophic amino acid, lacto-N-fucopentaose, choline, and hydroxybutyrate, pointing to the critical role of energy utilization, protein synthesis, oxidative status, and gut epithelial cell maturity in prematurity.

Breast Milk Lipidome Is Associated with Early Growth Trajectory in Preterm Infants.

Human milk is recommended for feeding preterm infants. The current pilot study aims to determine whether breast-milk lipidome had any impact on the early growth-pattern of preterm infants fed their own mother's milk. A prospective-monocentric-observational birth-cohort was established, enrolling 138 preterm infants, who received their own mother's breast-milk throughout hospital stay. All infants were ranked according to the change in weight Z-score between birth and hospital discharge. Then, we selected infants who experienced "slower" (n = 15, -1.54 ± 0.42 Z-score) or "faster" (n = 11, -0.48 ± 0.19 Z-score) growth; as expected, although groups did not differ regarding gestational age, birth weight Z-score was lower in the "faster-growth" group (0.56 ± 0.72 vs. -1.59 ± 0.96). Liquid chromatography-mass spectrometry lipidomic signatures combined with multivariate analyses made it possible to identify breast-milk lipid species that allowed clear-cut discrimination between groups. Validation of the selected biomarkers was performed using multidimensional statistical, false-discovery-rate and ROC (Receiver Operating Characteristic) tools. Breast-milk associated with faster growth contained more medium-chain saturated fatty acid and sphingomyelin, dihomo-γ-linolenic acid (DGLA)-containing phosphethanolamine, and less oleic acid-containing triglyceride and DGLA-oxylipin. The ability of such biomarkers to predict early-growth was validated in presence of confounding clinical factors but remains to be ascertained in larger cohort studies.

Calibration Adjustment of the Mid-infrared Analyzer for an Accurate Determination of the Macronutrient Composition of Human Milk.

BACKGROUND: Human milk composition analysis seems essential to adapt human milk fortification for preterm neonates. The Miris human milk analyzer (HMA), based on mid-infrared methodology, is convenient for a unique determination of macronutrients. However, HMA measurements are not totally comparable with reference methods (RMs). OBJECTIVE: The primary aim of this study was to compare HMA results with results from biochemical RMs for a large range of protein, fat, and carbohydrate contents and to establish a calibration adjustment. METHODS: Human milk was fractionated in protein, fat, and skim milk by covering large ranges of protein (0-3 g/100 mL), fat (0-8 g/100 mL), and carbohydrate (5-8 g/100 mL). For each macronutrient, a calibration curve was plotted by linear regression using measurements obtained using HMA and RMs. RESULTS: For fat, 53 measurements were performed, and the linear regression equation was HMA = 0.79RM + 0.28 (R(2) = 0.92). For true protein (29 measurements), the linear regression equation was HMA = 0.9RM + 0.23 (R(2) = 0.98). For carbohydrate (15 measurements), the linear regression equation was HMA = 0.59RM + 1.86 (R(2) = 0.95). A homogenization step with a disruptor coupled to a sonication step was necessary to obtain better accuracy of the measurements. Good repeatability (coefficient of variation < 7%) and reproducibility (coefficient of variation < 17%) were obtained after calibration adjustment. CONCLUSION: New calibration curves were developed for the Miris HMA, allowing accurate measurements in large ranges of macronutrient content. This is necessary for reliable use of this device in individualizing nutrition for preterm newborns.

MiRNA Analysis by Quantitative PCR in Preterm Human Breast Milk Reveals Daily Fluctuations of hsa-miR-16-5p.

BACKGROUND AND AIMS: Human breast milk is an extremely dynamic fluid containing many biologically-active components which change throughout the feeding period and throughout the day. We designed a miRNA assay on minimized amounts of raw milk obtained from mothers of preterm infants. We investigated changes in miRNA expression within month 2 of lactation and then over the course of 24 hours. MATERIALS AND METHODS: Analyses were performed on pooled breast milk, made by combining samples collected at different clock times from the same mother donor, along with time series collected over 24 hours from four unsynchronized mothers. Whole milk, lipids or skim milk fractions were processed and analyzed by qPCR. We measured hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-146-5p, and hsa-let-7a, d and g (all -5p). Stability of miRNA endogenous controls was evaluated using RefFinder, a web tool integrating geNorm, Normfinder, BestKeeper and the comparative ΔΔCt method. RESULTS: MiR-21 and miR-16 were stably expressed in whole milk collected within month 2 of lactation from four mothers. Analysis of lipids and skim milk revealed that miR-146b and let-7d were better references in both fractions. Time series (5H-23H) allowed the identification of a set of three endogenous reference genes (hsa-let-7d, hsa-let-7g and miR-146b) to normalize raw quantification cycle (Cq) data. We identified a daily oscillation of miR-16-5p. PERSPECTIVES: Our assay allows exploring miRNA levels of breast milk from mother with preterm baby collected in time series over 48-72 hours.

Optimization of a synthetic mixture composed of major Trichoderma reesei enzymes for the hydrolysis of steam-exploded wheat straw.

BACKGROUND: An efficient hydrolysis of lignocellulosic substrates to soluble sugars for biofuel production necessitates the interplay and synergistic interaction of multiple enzymes. An optimized enzyme mixture is crucial for reduced cost of the enzymatic hydrolysis step in a bioethanol production process and its composition will depend on the substrate and type of pretreatment used. In the present study, an experimental design was used to determine the optimal composition of a Trichoderma reesei enzyme mixture, comprising the main cellulase and hemicellulase activities, for the hydrolysis of steam-exploded wheat straw. METHODS: Six enzymes, CBH1 (Cel7a), CBH2 (Cel6a), EG1 (Cel7b), EG2 (Cel5a), as well as the xyloglucanase Cel74a and the xylanase XYN1 (Xyl11a) were purified from a T. reesei culture under lactose/xylose-induced conditions. Sugar release was followed in milliliter-scale hydrolysis assays for 48 hours and the influence of the mixture on initial conversion rates and final yields is assessed. RESULTS: The developed model could show that both responses were strongly correlated. Model predictions suggest that optimal hydrolysis yields can be obtained over a wide range of CBH1 to CBH2 ratios, but necessitates a high proportion of EG1 (13% to 25%) which cannot be replaced by EG2. Whereas 5% to 10% of the latter enzyme and a xylanase content above 6% are required for highest yields, these enzymes are predicted to be less important in the initial stage of hydrolysis. CONCLUSIONS: The developed model could reliably predict hydrolysis yields of enzyme mixtures in the studied domain and highlighted the importance of the respective enzyme components in both the initial and the final hydrolysis phase of steam-exploded wheat straw.

Non-invasive exploration of neonatal gastric epithelium by using exfoliated epithelial cells.

BACKGROUND & AIMS: In preterm infants, exfoliated gastric epithelial cells can be retrieved from aspirates sampled through the naso-gastric feeding tube. Our aims were to determine (1) whether the recovery of exfoliated cells is feasible at any time from birth through the removal of the nasogastric tube, (2) whether they can be grown in culture in vitro, and (3) whether the physiological state of exfoliated cells expressing H+/K+ -ATPases reflects that of their counterparts remaining in situ at the surface of the gastric epithelium in neonatal rat pups. METHODS: In infants, gastric fluid aspirates were collected weekly after birth or every 3 hours over 24-h periods, and related to clinical parameters (Biocollection PROG/09/18). In rat pups submitted to a single fasting/refeeding cycle, we explored circadian exfoliation with the cellular counter-parts in the gland. All samples were analyzed by confocal imaging and Enzyme-Linked Immunosorbent Assay. RESULTS: Epithelial cells were identified by microscopy using membrane-bound anti-H+/K+ ATPases antibody, assessed for nucleus integrity, and the expression of selected proteins (autophagy, circadian clock). On 34 infants, the H+/K+-ATPase-positive cells were consistently found quiescent, regardless of gestational age and feeding schedule from day-5 of life to the day of removal of the naso-gastric tube. By logistic regression analysis, we did find a positive correlation between the intensity of exfoliation (cellular loss per sample) and the postnatal age (p<0.001). The H+/K+ ATPase-positive cells established in culture retained the expression of a biomarker of progenitor status (Pouf5F1-Oct4). In rat pups, the expression pattern of Survivin in H+/K+ ATPase-positive exfoliated cells paralleled that observed in cells remaining at the surface of the gastric gland. CONCLUSIONS: Tracking parietal cells can improve clinical monitoring and understanding of the autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway.