Fact-based nutrition for infants and lactating mothers-The NUTRISHIELD study.

Background: Human milk (HM) is the ideal source of nutrients for infants. Its composition is highly variable according to the infant's needs. When not enough own mother's milk (OMM) is available, the administration of pasteurized donor human milk (DHM) is considered a suitable alternative for preterm infants. This study protocol describes the NUTRISHIELD clinical study. The main objective of this study is to compare the % weight gain/month in preterm and term infants exclusively receiving either OMM or DHM. Other secondary aims comprise the evaluation of the influence of diet, lifestyle habits, psychological stress, and pasteurization on the milk composition, and how it modulates infant's growth, health, and development. Methods and design: NUTRISHIELD is a prospective mother-infant birth cohort in the Spanish-Mediterranean area including three groups: preterm infants <32 weeks of gestation (i) exclusively receiving (i.e., >80% of total intake) OMM, and (ii) exclusively receiving DHM, and (iii) term infants exclusively receiving OMM, as well as their mothers. Biological samples and nutritional, clinical, and anthropometric characteristics are collected at six time points covering the period from birth and until six months of infant's age. The genotype, metabolome, and microbiota as well as the HM composition are characterized. Portable sensor prototypes for the analysis of HM and urine are benchmarked. Additionally, maternal psychosocial status is measured at the beginning of the study and at month six. Mother-infant postpartum bonding and parental stress are also examined. At six months, infant neurodevelopment scales are applied. Mother's concerns and attitudes to breastfeeding are registered through a specific questionnaire. Discussion: NUTRISHIELD provides an in-depth longitudinal study of the mother-infant-microbiota triad combining multiple biological matrices, newly developed analytical methods, and ad-hoc designed sensor prototypes with a wide range of clinical outcome measures. Data obtained from this study will be used to train a machine-learning algorithm for providing dietary advice to lactating mothers and will be implemented in a user-friendly platform based on a combination of user-provided information and biomarker analysis. A better understanding of the factors affecting milk's composition, together with the health implications for infants plays an important role in developing improved strategies of nutraceutical management in infant care. Clinical trial registration: https://register.clinicaltrials.gov, identifier: NCT05646940.

Joint Microbiota Activity and Dietary Assessment through Urinary Biomarkers by LC-MS/MS.

Accurate dietary assessment in nutritional research is a huge challenge, but essential. Due to the subjective nature of self-reporting methods, the development of analytical methods for food intake and microbiota biomarkers determination is needed. This work presents an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method for the quantification and semi quantification of 20 and 201 food intake biomarkers (BFIs), respectively, as well as 7 microbiota biomarkers applied to 208 urine samples from lactating mothers (M) (N = 59). Dietary intake was assessed through a 24 h dietary recall (R24h). BFI analysis identified three distinct clusters among samples: samples from clusters 1 and 3 presented higher concentrations of most biomarkers than those from cluster 2, with dairy products and milk biomarkers being more concentrated in cluster 1, and seeds, garlic and onion in cluster 3. Significant correlations were observed between three BFIs (fruits, meat, and fish) and R24h data (r > 0.2, p-values < 0.01, Spearman correlation). Microbiota activity biomarkers were simultaneously evaluated and the subgroup patterns detected were compared to clusters from dietary assessment. These results evidence the feasibility, usefulness, and complementary nature of the determination of BFIs, R24h, and microbiota activity biomarkers in observational nutrition cohort studies.

Metabolomic Diversity of Human Milk Cells over the Course of Lactation-A Preliminary Study.

Human milk (HM) is a complex biofluid containing a wide cell variety including epithelial cells and leukocytes. However, the cellular compositions and their phenotypic properties over the course of lactation are poorly understood. The aim of this preliminary study was to characterize the cellular metabolome of HM over the course of lactation. Cells were isolated via centrifugation and the cellular fraction was characterized via cytomorphology and immunocytochemical staining. Cell metabolites were extracted and analyzed using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QqTOF-MS) in the positive and negative electrospray ionization modes. Immunocytochemical analysis revealed a high variability of the number of detected cells with relative median abundances of 98% of glandular epithelial cells, 1% of leukocytes, and 1% of keratinocytes. Significant correlations between the milk postnatal age with percentage of epithelial cells and leukocytes, and with total cell count were observed. Results from the Hierarchical Cluster Analysis of immunocytochemical profiles were very similar to those observed in the analysis of the metabolomic profiles. In addition, metabolic pathway analysis showed alterations in seven metabolic pathways correlating with postnatal age. This work paves the way for future investigations on changes in the metabolomic fraction of the cellular compartment of HM.

Effects of Sepsis on Immune Response, Microbiome and Oxidative Metabolism in Preterm Infants.

This is a narrative review about the mechanisms involved in bacterial sepsis in preterm infants, which is an illness with a high incidence, morbidity, and mortality. The role of the innate immune response and its relationship with oxidative stress in the pathogenesis are described as well as their potential implementation as early biomarkers. Moreover, we address the impact that all the mechanisms triggered by sepsis have on the dysbiosis and the changes on neonatal microbiota.

GC-MS analysis of short chain fatty acids and branched chain amino acids in urine and faeces samples from newborns and lactating mothers.

BACKGROUND: Short chain fatty acids (SCFAs) and branched chain amino acids (BCAAs) are frequently determined in faeces, and widely used as biomarkers of gut-microbiota activity. However, collection of faeces samples from neonates is not straightforward, and to date levels of these metabolites in newborn's faeces and urine samples have not been described. METHODS: A targeted gas chromatography - mass spectrometry (GC-MS) method for the determination of SCFAs and BCAAs in both faeces and urine samples has been validated. The analysis of 210 urine and 137 faeces samples collected from preterm (PI), term infants (TI) and their mothers was used to report faecal and urinary SCFA and BCAA levels in adult and neonatal populations. RESULTS: A significant correlation among five SCFAs and BCAAs in faeces and urine samples was observed. Reference ranges of SCFAs and BCAAs in mothers, PI and TI were reported showing infant's lower concentrations in faeces and higher concentrations in urine. CONCLUSION: This method presents a non-invasive approach for the simultaneous assessment of SCFAs and BCAAs in faecal and urine samples and the results will serve as a knowledge base for future experiments that will focus on the study of the impact of nutrition on the microbiome of lactating mothers and their infants.

The effect of Holder pasteurization on the lipid and metabolite composition of human milk.

Human milk (HM) is the gold standard for newborn nutrition. When own mother's milk is not sufficiently available, pasteurized donor human milk becomes a valuable alternative. In this study we analyzed the impact of Holder pasteurization (HoP) on the metabolic and lipidomic composition of HM. Metabolomic and lipidomic profiles of twelve paired HM samples were analysed before and after HoP by liquid chromatography-mass spectrometry (MS) and gas chromatography-MS. Lipidomic analysis enabled the annotation of 786 features in HM out of which 289 were significantly altered upon pasteurization. Fatty acid analysis showed a significant decrease of 22 out of 29 detectable fatty acids. The observed changes were associated to five metabolic pathways. Lipid ontology enrichment analysis provided insight into the effect of pasteurization on physical and chemical properties, cellular components, and functions. Future research should focus on nutritional and/or developmental consequences of these changes.

Feeding the preterm infant: an overview of the evidence.

Feeding from own mother's milk is not always an option for preterm infants, and choosing between alternative means of feeding should be made in light of their effect on infants' health. In this work, we aimed to present evidence regarding feeding pre-term infants with human milk, either own mother's or donor milk (DM), and the potential effects on growth and other health related outcomes. Exclusive breastfeeding (BF) remains the optimal option, whereas feeding with DM as a sole diet or supplemental to maternal milk confers immunological advantages and fewer rates of necrotising enterocolitis against preterm formula feeding, yet the latter results in greater growth velocity. Literature gaps in the use of DM, practical suggestions for choosing suitable feeding means (i.e. continuous support of BF, adequate education regarding feeding preterm infants, including DM), and future perspectives on the potential effects of dietary manipulations of the maternal diet, are also discussed.

Effect of donor human milk on host-gut microbiota and metabolic interactions in preterm infants.

BACKGROUND & AIMS: Human milk is the gold standard for infant nutrition. Preterm infants whose mothers are unable to provide sufficient own mother's milk (OMM), receive pasteurized donor human milk (DHM). We studied metabolic signatures of OMM and DHM and their effect on the interplay of the developing microbiota and infant's metabolism. METHODS: Metabolic fingerprinting of OMM and DHM as well as infant's urine was performed using liquid chromatography-mass spectrometry and the infant's stool microbiota was analyzed by 16S rRNA sequencing. RESULTS: Significant differences in the galactose and starch and sucrose metabolism pathways when comparing OMM and DHM, and alterations of the steroid hormone synthesis and pyrimidine metabolism pathways in urine were observed depending on the type of feeding. Differences in the gut-microbiota composition were also identified. CONCLUSION: The composition of DHM differs from OMM and feeding of DHM has a significant impact on the metabolic phenotype and microbiota of preterm infants. Our data help to understand the origin of the observed changes generating new hypothesis: i) steroid hormones present in HM have a significant influence in the activity of the steroid hormone biosynthesis pathway in preterm infants; ii) the pyrimidine metabolism is modulated in preterm infants by the activity of gut-microbiota. Short- and long-term implications of the observed changes for preterm infants need to be assessed in further studies.

Biomonitoring of bisphenols A, F, S and parabens in urine of breastfeeding mothers: Exposure and risk assessment.

In the present study we used human biomonitoring to assess the internal exposure and the risk to four parabens and three bisphenols in 103 Spanish breastfeeding mothers participating in the BETTERMIILK project. Urinary methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP) presented detection frequencies ranging from 12% (BP) to 92% (MP), while bisphenol A (BPA), bisphenol F (BPF) and bisphenol S (BPS) were detected in 76% (BPA) and 20% (BPF, BPS) of the mothers. Average paraben concentrations (geometric mean) ranged from 0.021 ng mL-1 (BP) to 17.7 ng mL-1 (MP), whereas bisphenols had geometric means concentrations from 0.042 ng mL-1 (BPF) to 0.927 ng mL-1 (BPA). Except for BPA, the estimated daily intakes (EDI) were calculated in order to interpret urinary levels in a risk assessment context. The obtained EDIs ranged from 0.00042 mg/kg/day for PP to 0.0434 mg/kg/day for MP and EP. A hazard quotient (HQ) was calculated for BPA (0.0049) and parabens (0.001-0.004), showing no risk in the studied population. Sociodemographic characteristics, food consumption, and usage patterns of personal care products (PCPs) were investigated as possible determinants of exposure. Use of makeup and skincare products were associated with higher concentrations of MP and PP, respectively. Regarding dietary habits, MP was also associated with the consumption of packaged and bakery products.

The Evolving Microbiome from Pregnancy to Early Infancy: A Comprehensive Review.

Pregnancy induces a number of immunological, hormonal, and metabolic changes that are necessary for the mother to adapt her body to this new physiological situation. The microbiome of the mother, the placenta and the fetus influence the fetus growth and undoubtedly plays a major role in the adequate development of the newborn infant. Hence, the microbiome modulates the inflammatory mechanisms related to physiological and pathological processes that are involved in the perinatal progress through different mechanisms. The present review summarizes the actual knowledge related to physiological changes in the microbiota occurring in the mother, the fetus, and the child, both during neonatal period and beyond. In addition, we approach some specific pathological situations during the perinatal periods, as well as the influence of the type of delivery and feeding.

Current Practice in Untargeted Human Milk Metabolomics.

Human milk (HM) is considered the gold standard for infant nutrition. HM contains macro- and micronutrients, as well as a range of bioactive compounds (hormones, growth factors, cell debris, etc.). The analysis of the complex and dynamic composition of HM has been a permanent challenge for researchers. The use of novel, cutting-edge techniques involving different metabolomics platforms has permitted to expand knowledge on the variable composition of HM. This review aims to present the state-of-the-art in untargeted metabolomic studies of HM, with emphasis on sampling, extraction and analysis steps. Workflows available from the literature have been critically revised and compared, including a comprehensive assessment of the achievable metabolome coverage. Based on the scientific evidence available, recommendations for future untargeted HM metabolomics studies are included.

Biomonitoring of parabens in human milk and estimated daily intake for breastfed infants.

In this study, we assessed the presence of four parabens in human milk of 120 mothers from Valencia (Spain) which took part in a human biomonitoring project (BETTERMILK). The detection frequency ranges of parabens were 41-60% and 61-89% for unconjugated- and total (unconjugated + conjugated)-parabens, respectively. The concentrations ranged from

Impact of Donor Human Milk in the Preterm Very Low Birth Weight Gut Transcriptome Profile by Use of Exfoliated Intestinal Cells.

BACKGROUND: Own mother's milk (OMM) is the optimal nutrition for preterm infants. However, pasteurized donor human milk (DHM) is a valid alternative. We explored the differences of the transcriptome in exfoliated epithelial intestinal cells (EEIC) of preterm infants receiving full feed with OMM or DHM. METHODS: The prospective observational study included preterm infants ≤ 32 weeks' gestation and/or ≤1500 g birthweight. Total RNA from EEIC were processed for genome-wide expression analysis. RESULTS: Principal component analysis and unsupervised hierarchical clustering analysis revealed two clustered groups corresponding to the OMM and DHM groups that showed differences in the gene expression profile in 1629 transcripts. The OMM group overexpressed lactalbumin alpha gene (LALBA), Cytochrome C oxidase subunit I gene (COX1) and caseins kappa gene (CSN3), beta gene (CSN2) and alpha gene (CSN1S1) and underexpressed Neutrophil Cytosolic Factor 1 gene (NCF1) compared to the DHM group. CONCLUSIONS: The transcriptomic analysis of EEIC showed that OMM induced a differential expression of specific genes that may contribute to a more efficient response to a pro-oxidant challenge early in the postnatal period when preterm infants are at a higher risk of oxidative stress. The use of OMM should be strongly promoted in preterm infants.

Does Pasteurized Donor Human Milk Efficiently Protect Preterm Infants Against Oxidative Stress?

Pasteurized donor human milk (DHM) is the preferred alternative for infant nutrition when own mother's milk (OMM) is unavailable. Whether DHM is an efficient means for protecting preterm infants from oxidative stress remains unknown. We quantified a panel of oxidative stress biomarkers in urine samples from preterm infants (≤32 weeks of gestation and a birth weight ≤1500 g) receiving ≥80% of feeding volume as either DHM or OMM. The noninvasive in vivo assessment of oxidative stress showed no statistically significant difference between both groups at the time when full enteral nutrition (150 mL/kg body weight) was achieved and until hospital discharge. In addition, the changes of urinary biomarker levels with time were assessed. This is the first longitudinal study on oxidative stress levels in preterm infants fed with DHM in comparison with OMM. There is no statistically significant difference in urinary oxidative stress levels of preterm infants from both groups indicating that despite the effects of pasteurization, DHM is a valid alternative when OMM is not available. Based on the results, we raise the hypothesis that pasteurized DHM protects preterm infants from oxidative stress as good as OMM, and consequently, its use could prevent oxidative stress-related diseases. Antioxid. Redox Signal. 31, 791-799.

Biomonitoring of bisphenols A, F, S in human milk and probabilistic risk assessment for breastfed infants.

The present study addresses the presence of bisphenols A (BPA) and its analogs bisphenol F (BPF) and S (BPS) in milk of 120 mothers living in Valencia (Spain) and participating in the BETTERMILK project (year 2015). We also studied the factors that could influence the BPA levels and estimated the exposure and the risk for breast fed infants. The frequency of detection of total (conjugated + unconjugated) and unconjugated-BPA were 83% and 77%, with a geometric mean of 0.29 ng/mL and 0.15 ng/mL, respectively. The frequency of detection was much lower for total-BPF (22%) and total-BPS (1.1%). The place of residence of the mother and the use of personal care products showed significant association with BPA concentrations. The estimated daily intake of total-BPA for breastfed infants amounted to a geometric mean of 0.04 µg/kg bw and a 95th percentile of 1.0 µg/kg bw, below the tolerable daily intake of 4 µg/kg bw-day established by EFSA. To our knowledge, this is the largest biomonitoring study of bisphenols in human milk in Europe.

Preterm Gut Microbiome Depending on Feeding Type: Significance of Donor Human Milk.

Preterm microbial colonization is affected by gestational age, antibiotic treatment, type of birth, but also by type of feeding. Breast milk has been acknowledged as the gold standard for human nutrition. In preterm infants breast milk has been associated with improved growth and cognitive development and a reduced risk of necrotizing enterocolitis and late onset sepsis. In the absence of their mother's own milk (MOM), pasteurized donor human milk (DHM) could be the best available alternative due to its similarity to the former. However, little is known about the effect of DHM upon preterm microbiota and potential biological implications. Our objective was to determine the impact of DHM upon preterm gut microbiota admitted in a referral neonatal intensive care unit (NICU). A prospective observational cohort study in NICU of 69 neonates <32 weeks of gestation and with a birth weight ≤1,500 g was conducted. Neonates were classified in three groups according to feeding practices consisting in their MOM, DHM, or formula. Fecal samples were collected when full enteral feeding (defined as ≥150 cc/kg/day) was achieved. Gut microbiota composition was analyzed by 16S rRNA gene sequencing. Despite the higher variability, no differences in microbial diversity and richness were found, although feeding type significantly influenced the preterm microbiota composition and predictive functional profiles. Preterm infants fed MOM showed a significant greater presence of Bifidobacteriaceae and lower of Staphylococcaceae, Clostridiaceae, and Pasteurellaceae compared to preterm fed DHM. Formula fed microbial profile was different to those observed in preterm fed MOM. Remarkably, preterm infants fed DHM showed closer microbial profiles to preterm fed their MOM. Inferred metagenomic analyses showed higher presence of Bifidobacterium genus in mother's milk group was related to enrichment in the Glycan biosynthesis and metabolism pathway that was not identified in the DHM or in the formula fed groups. In conclusion, DHM favors an intestinal microbiome more similar to MOM than formula despite the differences between MOM and DHM. This may have potential beneficial long-term effects on intestinal functionality, immune system, and metabolic activities.

Recomendaciones para la creación y el funcionamiento de los bancos de leche materna en España / Recommendations for the creation and operation of maternal milk banks in Spain

La mejor alimentación para un recién nacido es la leche de su propia madre. En aquellos casos en los que esta no está disponible, especialmente en los recién nacidos muy prematuros o de muy bajo peso al nacer, así como en otros recién nacidos enfermos, el alimento de elección es la leche materna de donantes seleccionadas, antes que la fórmula artificial. Esta indicación está respaldada por los máximos organismos internacionales dedicados a la salud de la población infantil, como la Organización Mundial de la Salud, así como las principales sociedades científicas nacionales e internacionales en el ámbito de la Pediatría. Los bancos de leche surgen como instituciones sanitarias responsables de la gestión de las donaciones, del procesamiento y de la distribución de leche materna donada. Actualmente existen 14 bancos de leche materna en España, agrupados en la Asociación Española de Bancos de Leche Humana, creada en septiembre de 2008. Con el fin de homogeneizar los criterios y unificar los métodos de trabajo, la Asociación Española de Bancos de Leche Humana ha elaborado unos estándares para armonizar los protocolos de los diferentes bancos y para que sirvan de guía para la puesta en marcha de nuevos bancos de leche en el territorio español. Dichos estándares, presentados en este artículo, abarcan desde el proceso de selección y evaluación de la donante hasta la recogida, el procesamiento, el almacenamiento y la distribución de leche materna de donante

It is widely agreed that the best source of nutrition for the newborn is the milk of their own mothers. In those cases where it is not available, especially in very premature and/or very low birth weight infants, as well as other sick newborns, the preferred choice before formula is the human milk provided by selected donors. This indication is supported by the highest international bodies dedicated to the health of the child population, including the World Health Organisation as well as the main national and international scientific societies in the field of Paediatrics. Milk banks are health institutions responsible for the collection, processing and distribution of donated human milk. Currently, there are 14 human milk banks operating in Spain, grouped in the Spanish Association of Human Milk Banks, created in September 2008. In order to homogenise the criteria and to unify the working methods of the different milk banks, the Spanish Association of Human Milk Banks has developed standards to harmonise the protocols, and to serve as a guide for the start-up of new milk banks in the Spanish territory. These standards, set out in the present article, range from the donor selection and the evaluation process to the collection, processing, storage, and distribution of donor human milk

Biomarkers of oxidative stress derived damage to proteins and DNA in human breast milk.

Oxidative stress derived biomarkers have been extensively studied for assessing pathological situations in the neonatal period and their usefulness for an early outcome prediction of oxidative stress related diseases in non-invasive biofluids could be demonstrated. This is the first report on a method for the simultaneous detection of a panel of oxidative stress related biomarkers for quantifying damage to proteins and DNA in human breast milk samples. A straightforward sample clean-up procedure using 1.5 mL of milk was developed and extracts were analyzed by ultra-performance liquid chromatography coupled to tandem mass spectrometry. The analytical method was validated and applied to human milk samples from a cohort of mothers of preterm infants, as well as donor human milk samples before and after pasteurization at a human milk bank. The results demonstrate the feasibility of this method for the analysis of a panel of oxidative stress related biomarkers, reporting ranges found in human milk.

[Recommendations for the creation and operation of maternal milk banks in Spain]. / Recomendaciones para la creación y el funcionamiento de los bancos de leche materna en España.

It is widely agreed that the best source of nutrition for the newborn is the milk of their own mothers. In those cases where it is not available, especially in very premature and/or very low birth weight infants, as well as other sick newborns, the preferred choice before formula is the human milk provided by selected donors. This indication is supported by the highest international bodies dedicated to the health of the child population, including the World Health Organisation as well as the main national and international scientific societies in the field of Paediatrics. Milk banks are health institutions responsible for the collection, processing and distribution of donated human milk. Currently, there are 14 human milk banks operating in Spain, grouped in the Spanish Association of Human Milk Banks, created in September 2008. In order to homogenise the criteria and to unify the working methods of the different milk banks, the Spanish Association of Human Milk Banks has developed standards to harmonise the protocols, and to serve as a guide for the start-up of new milk banks in the Spanish territory. These standards, set out in the present article, range from the donor selection and the evaluation process to the collection, processing, storage, and distribution of donor human milk.

Nutritional practices in very low birth weight infants: a national survey.

BACKGROUND: Significant efforts have been made to improve the nutritional support of very preterm infants. Large surveys may help to know the nutritional practices for preterm infants in neonatal units and identify if they are in line with the current guidelines. METHODS: A multicentre nationwide web-based survey on clinical feeding practices in very low birth weight (VLBW) infants was conducted in tertiary neonatal hospitals that admit infants with a birth weight < 1,500 g and/or a gestational age of < 32 weeks. RESULTS: The questionnaire was completed by 53 units (response rate, 59%). Over 90% of the units surveyed start amino-acid administration immediately after birth and more than half use novel intravenous fish oil-based lipid emulsions. Enteral nutrition is started within 24 hours of birth in 65% of units and 86% of these are medium-sized or large. Feeding volumes are increased at a rate of 10-30 ml/kg/day in > 90% of units. Monitoring of serum phosphorus was measured more frequently than albumin (p = 0.009) or triglycerides (p = 0.037), but only 28% of centres regularly measure pre-albumin as a nutritional biomarker. Human milk fortification and iron supplementation, starting at four weeks of age, are almost universal. However, only 30% of units administer 800 IU/day of vitamin D. Nearly 50% of the units discharge infants on preterm formula. CONCLUSION: Most Spanish neonatology units use early amino-acid supplementation and over half use novel fish oil-based lipid emulsions. Post-discharge nutrition practices and vitamin administration vary greatly.