Influencia del procesamiento de la leche humana donada sobre la microbiota intestinal, la expresión genómica y el equilibrio oxidativo en recién nacidos pretérmino menores de 32 semanas de edad gestacional

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Worldwide, 15 million babies are born prematurely each year, which is to be estimated at approximately 11% of all births, with an increasing tendency in most of the countries. The neonatal period is an exceptionally vulnerable period of life during which not only full-term newborns but also, and especially premature ones have, compared to other stages of life, an increased risk of mortality and morbidity that can lead to permanent neurocognitive, motor and sensory sequelae, thus constituting a major economic and social problem. An adequate microbial colonization during this period is essential for proper maturation of the immune system, metabolism as well as the development of the central nervous system, encouraging the maturation of cognitive and sensory functions, such as vision. Microbiota alterations can have important health consequences. Full-term newborns have an antioxidant defense system that will protect them against increased production of oxygen-free radicals (ROS) and nitrogen (RNS). Nevertheless, premature newborns have an immature antioxidant defense system. Breast milk (BM) has been recognized as the gold standard for human nutrition. Those bioactive compounds are considered not only protectors but also stimulators of growth and neurodevelopment along with the maturation of the immature immune system. Pasteurized donated human milk (DHM) is the best alternative in premature children under 32 weeks of gestational age, lighter than 1.500 grams, when mother's own milk (OMM) is not available. The proven benefits of feeding newborns with DHM versus artificial formulas are for a short-term: protection against necrotizing enterocolitis, nosocomial infection and better enteral tolerance. For the long term, they have a better neurodevelopment than those fed with formula and a lower cardiovascular risk during adolescence. Pasteurization causes the loss of some of the biological, structural and functional properties. OBJECTIVES: Our objective was to determine the impact of DHM on the gut microbiota, oxidative stress in urine and genomic expression in exfoliated epithelial intestinal cells (EEIC), in premature newborns admitted to a reference neonatal intensive care unit. POPULATION AND METHODS: A cohort, prospective, observational and unicentric study was carried out where all newborns are included for a period of 12 months, ≤ 32 weeks gestational age, ≤ 1.500 grams, admitted in the Neonatology Unit of the University and Polytechnic Hospital La Fe (Valencia, Spain). Fecal and urine samples of 69 preterm were collected, when the neonates reached complete enteral feeding (defined as ≥150 cc/ kg / day). There were 3 groups: neonates fed with OMM, DHM or premature formula (FP). The composition of the gut microbiota was analyzed by sequencing the 16S RNAr gene. The determination of biomarkers of oxidative damage to DNA and proteins in urine samples of premature newborns, as well as lipid peroxidation biomarkers mediated by individual free radicals, was performed following a pre-validated Ultra Performance liquid chromatography method - tandem mass spectrometry (UPLC-MS/MS). The total RNA of CIEE was processed with the Clariom S Human micro-matrix. RESULTS: Despite greater variability, no differences in diversity and microbial wealth were found, even though the type of diet significantly influenced the composition of the gut microbiota in preterm ones and in the predictive functional profiles. Moreover, the non-invasive in vivo evaluation of oxidative stress revealed no statistically significant differences in any of the 22 biomarkers in urine, between the two groups (OMM vs. DHM) when they reached full enteral nutrition (150 ml / kg / day). The OMM group overexpressed the lactalbumin alpha gene (LALBA), the cytochrome C oxidase subunit I gene (COX1) and the casein kappa gene (CSN3), the beta gene (CSN2) and the alpha gene (CSN1S1) and the neutrophil cytosolic factor gene 1 (NCF1) was down-expressed in comparison with the DHM group. This explains the lack of activation of inflammatory pathways, the formation of non-inflammatory cytokines and the blocking of oxygen-free radical generation (ROS). CONCLUSIONS: 1. Donor human milk favors the development of a gut microbiome that resembles the microbiome acquired by infants nourished by their own mother’s milk more than microbiome in formula fed infants. Nonetheless, preterm infants nourished donor human milk, have a distinct microbial profile but shortage in the microbial diversity and richness present in the gut microbiome of infants fed with own mother’s milk. 2. Despite these differences, donor human milk confers beneficial potential effects upon intestinal functionality, immune system and metabolic activities. 3. Preterm infants nourished with donor human milk have similar antioxidant capacity as preterm fed own mother's milk as reflected in the level of urinary biomarkers of oxidative damage to proteins, DNA or lipids. 4. The variability in terms of transcriptomic expression in exfoliated intestinal cells was 12.1% between own mother´s milk and donor human milk. Nevertheless, 1629 genes were differentially expressed between both groups. 5. The candidate genes with different expression between preterm nourished with owns mother milk and donor human milk were Lactalbumin alpha (LALBA),)Casein kappa (CSN3), Casein beta (CSN2), Casein alpha-1 (CSN1S1), Cytochrome C oxidase subunit 1 (COX1), Neutrophil cytosolic factor 1 (NCF1). 6. The biological analysis of these genes indicates that donor human milk possesses a lower anti-inflammatory potential than its own mother´s milk. This is important in terms of its ability to protect against pathogens and the development of necrotizing enterocolitis. 7. Notwithstanding the differences, our studies show many data in favor of important advantages of donor human milk over formula milk. Conversely, new pasteurization methods that preserve the integrity of donor human milk should be developed.
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