International Milk Composition (IMiC) Consortium

Study Overview
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Brief Summary
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The IMiC Consortium will analyze milk from 1000 mother-infant dyads across 4 diverse settings (Tanzania, Pakistan, Burkina Faso and Canada). Samples will be stored centrally at the Manitoba Interdisciplinary Lactation Centre (MILC) biorepository and distributed to multiple laboratories for analysis of macronutrients, micronutrients, oligosaccharides, growth factors, immunoglobulins, cytokines, metabolites and microbes. Data will be harmonized and stored in a central database, and diverse statistical methods will be applied for data integration and analysis.

Detailed Description
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Human Milk Composition: Milk is a highly complex biofluid that has evolved over millions of years to nourish infants and protect them from infection while their immune system matures. In addition to delivering complete nutrition (i.e. macronutrients and micronutrients), milk provides bioactive components that further support infant growth, development and health. These include immunoglobulins, antibodies, hormones, growth factors, prebiotic oligosaccharides, and probiotic bacteria. Milk composition is specifically adapted to each mammalian species depending on the growth requirements of their young offspring. For example, the average energy content of human milk is around 70 kcal/100g, compared to 38 kcal/100g in donkey milk and 171 kcal/100g in mouse milk. Mice produce just 2 milk oligosaccharides, while humans produce over 150. Even among humans, milk composition is highly variable - for example, energy content can range from 57-83 kcal/100g and oligosaccharide concentrations range from 5-25 g/L. Surprisingly little is known about the determinants and consequences of this variation. The investigators will study the following milk components in the IMiC Consortium to understand variability between individual women and across different geographic settings, and their associations with infant growth. In addition, to guide these analyses, a review of human milk components and infant growth will be undertaken by the IMiC members during Year 1 of the Project. Priority Components (to be analyzed in all samples): Macronutrients include carbohydrates (primarily lactose), proteins and lipids. Lipids provide about 50% of the energy content in human milk. The vast majority (98%) of milk lipids are triacylglycerides, with the remainder consisting of diacylglycerides, monoacylglycerides, free fatty acids, phospholipids and cholesterol. The fatty acid profile of human milk varies in relation to maternal diet and genetics, particularly in the long-chain polyunsaturated fatty acids (LCPUFAs), such as arachidonic and docosahexaenoic acids, which contribute to immune function and neurodevelopment. Micronutrient quality and concentrations can be compromised by maternal malnutrition. Micronutrients in milk include minerals (e.g. Zinc, Calcium, Phosphorus, Magnesium, Iodine, Selenium) and vitamins (A, B1, B2, B6, B12, C, D, E; folate, choline). Immunoglobulins (Ig) are transferred in human milk, including IgA, IgM and IgG. Infants are born with immature adaptive immunity, and rely on these maternal antibodies for defense against pathogens. Soluble IgA (sIgA) is the predominant antibody of human milk; sIgA-antigen complexes are taken up by intestinal dendritic cells, allowing for antigen recognition. Cytokines are multifunctional peptides can cross the intestinal barrier, where they influence immune activity. Milk-borne cytokines include anti-inflammatory transforming growth factor (TGF)-b, interleukins (IL)-10 and IL-7, and proinflammatory tumor necrosis factor (TNF)-a, IL-6, IL-8, and interferon (IFN)-g. Lactoferrin is an iron binding glycoprotein with antimicrobial activity against many bacteria, viruses, and fungi. Osteopontin is an extensively phosphorylated acidic glycoprotein that is present at high concentrations in human milk. It affects immune functions, intestinal development, and brain development. Growth factors and hormones in human milk have wide-ranging effects on the infant intestinal tract, vasculature, nervous system, and endocrine system. Some act locally on the neonatal intestine and many are absorbed into systemic circulation through the 'leaky' infant gut. Epidermal growth factor (EGF) is critical to the maturation and healing of the intestinal mucosa. Insulin-like growth factor (IGF) promotes tissue growth. The metabolic hormones leptin, insulin, adiponectin and ghrelin regulate energy conservation, appetite and infant BMI. Human milk oligosaccharides (HMOs) are the third most abundant component of human milk. Over 100 different HMOs have been identified. These structurally diverse carbohydrates are not digested by the infant, but are metabolized by the infant's gut bacteria, providing a selective substrate to help shape the developing microbiome. In addition, HMOs serve as soluble decoy receptors and prevent pathogen attachment to infant mucosal surfaces, lowering the risk for viral and bacterial infections. HMOs may also modulate epithelial and immune cell responses and provide the infant with sialic acid, an important nutrient for brain development. In the CHILD cohort the investigators have observed that, beyond genetic secretor status, HMO composition is associated with ethnicity, lactation stage, parity, geographic location, season of collection, and breastfeeding exclusivity. Omics approaches will be applied to broadly assess the complete spectrum of peptides, proteins, lipids, and metabolites in human milk. Targeted metabolomics analyses to be conducted using the Biocrates platform ( 500 metabolites), untargeted metabolomic analyses to be conducted by Sapient Bioanalytics via mass spectrometry. Microbes are present in human milk. Culture-dependent and independent (sequencing-based) studies have confirmed the presence of bacteria and fungi in milk from healthy mothers. In the CHILD cohort, the investigators have found that milk microbiota composition differs by infant sex, method of feeding, maternal BMI, and maternal atopy. It is estimated that breastfed infants receive 10^4-10^6 bacteria per day, providing a source of live microbes to seed the infant gut, oral cavity and airways. Studies demonstrating strain similarities between maternal gut, milk, and infant gut support this hypothesis, and find that Bifidobacterium spp. constitute the majority of shared taxa between maternal milk and infant stool. Given the central role of the gut microbiome in infant growth, metabolism and protection from infectious disease, including in low to middle income (LMIC) settings, it is critical to understand the origins of these fundamentally important gut microbes early in life. A secondary objective of IMiC will be to support data integration across sites to answer important questions related to 1) the impact of maternal health and nutrition interventions on breast milk composition, and 2) its relation to infant health, growth and development. Each site will own its own data and will also be independently addressing these same questions by site, as originally intended in their own grants/studies.

Official Title
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International Milk Composition (IMiC) Consortium

Conditions
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Infant Growth, Maternal Health, Infant Nutrition

Intervention / Treatment
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* Dietary Supplement: Vitamin B3
* Biological: Azithromycin
* Dietary Supplement: Fortified food supplement


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: CHILD Study: Full term healthy infant; singleton pregnancy; English literary ELICIT Study: Adult mothers, child <14 days, lives within 25km of Haydom VITAL Study: Lactating, biological mothers MISAMEIII Study: Confirmed (pregnancy test/ultrasound) pregnant women 15-40 years old, informed consent, self, parents or husband (for minors; assent). Exclusion Criteria: CHILD Study: IVF; congenital abnormality; preterm delivery ELICIT Study: Multiple gestation, birth defects/neonatal illness, weight <1.5g, no intention to breastfeed, will move from area within 18 months VITAL Study: Use of any ointments or topical solutions immediately prior to sampling, non-consent MISAMEIII Study: Peanut allergy, gestational age <20weeks, women who will not deliver babies in or live in study area by delivery date.

Ages Eligible for Study
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Minimum Age: 15 Years

Sexes Eligible for Study
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All

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Canadian Healthy Infant Longitudinal Development (CHILD) Cohort Study<br>The CHILD Cohort Study is a prospective longitudinal birth cohort study. It is an observational study of healthy term infants in Canada (Vancouver, Edmonton, Manitoba, Toronto). The birth years were between 2009-2012, and is currently at the 8 year postnatal follow up phase. IMiC will receive 400 breast milk samples from 400 dyads (100/site) that were taken between 3-4 months postnatal. | |
| The Early Life Interventions for Childhood Growth and Development in Tanzania (ELICIT) Study<br>(NCT03268902). ELICIT is a randomized controlled trial (RCT) evaluating the efficacy of antimicrobials and nicotinamide in increasing growth in the setting of Rural Tanzania. It is factorial design RCT of nicotinamide (vitamin B3) to mothers and infants, and antimicrobial prophylaxis to infants. IMiC will receive 400 breast milk samples from 200 dyads, 2 samples per dyad taken at 1 & 5 months postnatal. | Dietary Supplement: Vitamin B3<br>* ELICIT Study: Factorial design RCT of nicotinamide (vitamin B3) to mothers and infants, and antimicrobial prophylaxis (Azithromycin) to infants.<br>* Other names: Nicotinamide;Biological: Azithromycin<br>* ELICIT Study: Factorial design RCT of nicotinamide (vitamin B3) to mothers and infants, and antimicrobial prophylaxis (Azithromycin) to infants. VITAL Pakistan Study: 3-arm RCT of fortified food supplement (protein energy) during lactation, with or without azithromycin prophylaxis for infant.<br>|
| VITAL Pakistan<br>Two Randomized Controlled Trials: Mumta (Nutritional support for lactating women with or without azithromycin)PW - NCT04012177 and MumtaLW - NCT03564652 VITAL is a community-based, randomized control, assessor blinded trial in peri-urban settings of Karachi, Pakistan to study the impact of Lipid-based Nutritional Supplement for Pregnant and Lactating women which is balanced energy-protein (BEP) dietary supplement, a locally produced ready-to-use nutritional product for lactating women (LW) and single prophylaxis dose of Azithromycin for infants, on growth of infants over the period of six months since birth compared to current standard of care. IMiC will receive 600 breast milk samples from 200 dyads, 3 samples per dyad taken at 0-1, 1-2 & 2-3 months. | Biological: Azithromycin<br>* ELICIT Study: Factorial design RCT of nicotinamide (vitamin B3) to mothers and infants, and antimicrobial prophylaxis (Azithromycin) to infants. VITAL Pakistan Study: 3-arm RCT of fortified food supplement (protein energy) during lactation, with or without azithromycin prophylaxis for infant.<br>Dietary Supplement: Fortified food supplement<br>* VITAL Study: 3-arm RCT of fortified food supplement (protein energy) during lactation, with or without azithromycin prophylaxis for infant. MISAMEIII Study: 2x2 cross-over efficacy RCT of fortified food supplement (folic acid/iron +/- peanut spread) during pregnancy and/or lactation; unmasked (open label).<br>|
| Micronutriments pour la Santé de la Mère et de l'Enfant (MISAME)-3 study (NCT03533712)<br>MISAME-3 is a randomized controlled clinical trial in the setting of Rural Burkina Faso. A BEP supplement provides less than 25% of protein of the total energy content, and includes different vitamins and minerals. The first part of an exploratory study will determine which type of BEP supplement (bar, drink, biscuit, soup or paste) is most accepted by pregnant women. Subsequently, two products will be tested for longer-term acceptability and at-home use (phase 1). The effect of the most suitable supplement will be tested in a controlled clinical trial (phase 2). The intervention group will receive the dietary supplement during pregnancy and/or lactation, while the control group complies with the standard iron and folic acid tablets following the national guidelines. IMiC will receive 600 breast milk samples from 200 dyads, 3 samples per dyad taken at 0-1, 1-2 & 3-4 months. | Dietary Supplement: Fortified food supplement<br>* VITAL Study: 3-arm RCT of fortified food supplement (protein energy) during lactation, with or without azithromycin prophylaxis for infant. MISAMEIII Study: 2x2 cross-over efficacy RCT of fortified food supplement (folic acid/iron +/- peanut spread) during pregnancy and/or lactation; unmasked (open label).<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Create an international consortium of 4 birth cohorts in Tanzania, Pakistan, Burkina Faso and Canada (Field Site Partners), human milk scientists (Laboratory Partners), and data scientists and biostatisticians (Data Science Partners). | The result of this outcome will be measured by the completion of governance structure and data sharing agreements with all partners. | This outcome is anticipated to reach completion in December 2021. | 
| Create a centralized biorepository of human milk samples from the Field Site Partners, housed at the Manitoba Interdisciplinary Lactation Centre (MILC) at the University of Manitoba in Winnipeg, Manitoba, Canada. | The result of this outcome will be measured by the completion of all milk samples collected, stored and catalogued at MILC. | This outcome is anticipated to reach completion in April 2022. | 
| Create a standardized protocol for the comprehensive analysis of human milk composition using state-of-the-art methods in expert laboratories | The result of this outcome will be measured by the completion of list of target milk components; agreements with laboratories; standard operating procedures (SOPs) for sample collection, processing, shipping and analysis. | This outcome is anticipated to reach completion in December 2021. | 
| Create a harmonized dataset of human milk composition and relevant maternal, infant and environmental data from 1000 dyads | The result of this outcome will be measured by complete, clean, accessible, dataset meeting FAIR (Findable, Accessible, Interoperable, Reusable: www.gofair.org) Guiding Principles, including milk composition data and relevant metadata for all included dyads. | This outcome is anticipated to reach completion in October 2023. | 
| Create an integrated analysis of this dataset | Create an integrated analysis of this dataset, addressing research questions such as: What are the ranges and distributions of analytes in human milk in different geographic settings? How are various components in human milk correlated with each other? How is breast milk composition influenced by maternal, environmental and sociodemographic factors? The result of this outcome will be measured by answering the above research questions, compiled in reports and open access academic publications | This outcome is anticipated to reach completion in October 2023. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| A secondary objective of IMiC will be to support data integration across sites to answer important questions. | A secondary objective of IMiC will be to support data integration across sites to answer important questions related to 1) the impact of maternal health and nutrition interventions on breast milk composition, and 2) its relation to infant health, growth and development. Each site will own its own data and will also be independently addressing these same questions by site, as originally intended in their own grants/studies. | This outcome is anticipated to reach completion in October 2023. |