Human Milk and Its Effect on Infant's Metabolism and Infant Gut Microbiome

Study Overview
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Brief Summary
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This protocol explains the MAINHEALTH cohort. The study examine the influence of maternal health parameters on human breast milk composition and integrates milk phenotype with infant metabolism and infant gut microbial content and metabolism.

Detailed Description
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The birth of a living human being is the result of an approximately nine-month pregnancy in which the developing foetus has taken exactly the building blocks necessary to grow and develop from its mother. However, growth and development continues in multiple dimensions at an increasing pace after birth. The nutrition in the first 1,000 days from conception to the child's 2nd birthday plays a pivotal role in shaping the future health of the child. Yet, little is known of how breast milk components vary due to maternal factors or of the biological mechanisms behind the beneficial actions of many breast milk nutrients. The investigators propose to overcome these obstacles by combining specialties to give a more complete account of what breast milk is (major and minor milk constituents and microbiota), how it affects the infants directly or indirectly through breast milk-gut microbiome interactions and by which mechanisms. In this study longitudinal samples from 200 mother-infant dyads during the first year of life across three groups of pregestational maternal BMI; normal weight (BMI 18.5-24.99), overweight (BMI 25-30), and obese (BMI >30) are collected. The samples give a comprehensive record of what the infant has ingested (milk samples) and how the infant and infant gut microbiome responds to this (infant urine and feces). Maternal diet in pregnancy and at milk sample deliveries are recorded through a 24h online food recall and diary system. Maternal health attributes will, besides BMI, be analysed through clinical blood biochemistry parameters. Follow-up samples and infant dietary intake as the infant grows allow investigating how early life diet shaped infant growth and gut colonization more long term. The investigators have formed an experienced team of scientists within metabolomics, microbiology and medicine, holding leading positions within their respective fields in Denmark. The novelty in the study is the interdisciplinarity, unique study design and the emphasis to integrate a number of dynamic measurements thereby offering the ability to identify the factors in breast milk affecting infant metabolism and gut colonization. Knowing this enable the optimization of infant formula. The research questions asked in this project are three-fold. First, the investigators want to determine the variability of breast milk nutrients by application of multi-omics (metabolomics, proteomics and glycomics). The production of human breast milk has a high maternal metabolic cost. Thus, hypothesis is that maternal health attributes (metabolic dysfunction or obesity) influence which breast milk nutrients are made available to the infant. Second, the investigators want to establish the microbiome of breast milk. Maternal obesity can lead to an apparent gut microbial ecology and increases the risk of obesity for the child. Thus, the hypothesis is that maternal obesity confers distinct microorganisms to the infant. Third, the investigators want to identify biological mechanisms for how breast milk nutrients are metabolised in the infants. Infants exclusively breast-feeding offer total compliance. Thus, the hypothesis is that by deconstructing breast milk components and markers of infant metabolism through comprehensive analysis of infant urine and feces, bioactivity of breast milk nutrients can be elucidated.

Official Title
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The Influence of Maternal Health on Human Breast Milk Composition With Potential Downstream Effects on Infant Metabolism and Gut Colonization

Conditions
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Breast Milk Collection, Human Milk/Breastfeeding, Human Milk Microbiome, Infant Gut Microbiome

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria related to mother: Residing in Aarhus area Above 18 years of age BMI above 18.5 kg/m2 Intention to breastfeed the first four to six months following birth Be able to communicate in Danish Exclusion Criteria related to mother: Smoking Multiple gestation Suffering from the following chronic diseases that demands medical treatment: diabetes mellitus, celiac disease, inflammatory bowel disease (Chrohn's disease or ulcerosa colitis) Taking medicaments for irritable bowel syndrome Taking medicine for metabolic disorders Taking medicine for psychological disorders Have had gastric bypass surgery Planned caesarean section Received antibiotics after week 12 in their pregnancy Utilizing significant amount of infant formula following birth Inclusion Criteria related to infant: Infants born after gestational age 37 weeks Infants with a birth weight between 2500 g and 5000 g Exclusion Criteria related to infant: • Inborn errors of metabolism
Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Maternal pre-gestational BMI 18.5<25<br> | |
| Maternal pre-gestational BMI 25<30<br> | |
| Maternal pre-gestational BMI >30<br> | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Metabolite profile of human milk | Human milk metabolites by Nuclear Magnetic Resonance (NMR) spectroscopy. Data will be analysed as absolute concentrations of milk metabolites; how milk metabolite profiles are related to mother's blood chemistry, milk microbial profiles, milk oligosaccharides, infant urine metabolome, and infant fecal microbiomes will be explored using multivariate analyses. | Birth to 3 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Metabolite profile of human milk by Liquid Chromatography-Mass Spectrometry (LC-MS) analysis | Human milk metabolites by LC-MS-based metabolomics. Data will be analysed as absolute concentrations of milk metabolites; how milk metabolite profiles are related to milk microbial profiles, milk oligosaccharides, infant urine metabolome, and infant fecal microbiomes will be explored using multivariate analyses. | Birth to 3 months | 
| Milk proteome profile | Human milk proteome by LC-MS-based, bottom-up proteomics. Data will be analysed as relative abundances of milk proteins. | Birth to 3 months | 
| Milk protein post-translational modification (PTM) profile | Post-translational modifications of human milk proteins is analysed by LC-MS-based and 2D- gel-based proteomics. Data will be analysed as relative abundances of milk protein PTMs. | Birth to 3 months | 
| Milk glycome profile | Human milk glycome by LC-MS-based glycomics. Data will be analysed as relative abundances of milk glycans. | Birth to 3 months | 
| Microbial structure of human milk | Human milk microbiome by nanopore sequencing. Data will be analysed as relative abundances of bacteria from phylum to genus levels. | Birth to 3 months | 
| Microbial structure of infant feces | Infant fecal microbiome by nanopore sequencing. Data will be analysed as relative abundances of bacteria from phylum to genus levels. | Birth to 5 years of age | 
| Microbial structure of infant oral cavity | Oral cavity microbiome by nanopore sequencing. Data will be analysed as relative abundances of bacteria from phylum to genus levels. | 30 days postpartum | 
| Microbial structure of mother's skin microbiome | Skin microbiome by nanopore sequencing. Data will be analysed as relative abundances of bacteria from phylum to genus levels. | 30 days postpartum | 
| Metabolite profile of infant fecal material | Infant fecal metabolome by NMR-based metabolomics. Data will be analysed as absolute concentrations of fecal metabolites | Birth to 5 years of age | 
| Infant metabolism investigated by infant urine metabolomics | Infant urine metabolome by NMR-based metabolomics. Data will be analysed as absolute concentrations of urine metabolites | Birth to 3 months of age | 
| Microbial structure of maternal vagina and rectum to investigate vertical transmission of bacteria to infant during birth | Vertical transmission of microbiome from mother to infant. Vaginal and rectal microbiome by nanopore sequencing. Data will be analysed as relative abundances of bacteria from phylum to genus levels. | During birth | 
| Mother's 24-h dietary recall (myfood24) | Aggregated nutrient intake data (e.g. proteins, vitamins, fibers, omega-3-fatty acids) | During pregnancy (Gestational age 30), 30, 60, and 90 days postpartum. In each case two times within a week (one weekday and one weekend day). | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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milk metabolites, multi-omics, gut microbiome