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.

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.

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

Residence in Aarhus area is necessary and Aarhus University Hospital has to be the place of giving birth. This is due to samples being collected by midwifes during labor and when study personnel collect samples in study participants' homes.