Human Milk Oligosaccharides' Impact on Maturation of the Gut Microbiota

Recruitment of study cohort and informed consent The mother-infants pair cohort have been recruited by Assoc. Prof. Lise Aunsholt (Copenhagen University Hospital, Rigshospitalet, Dept. Neonatology, Copenhagen, Denmark). We have advertised the study at the clinics of general practitioners in the Copenhagen area aiming to recruit pairs during the planned vaccination visit at an infant age of 3 month. Additionally, we have advertised the study via state employed health workers in conjunction with their standard visit at the mothers' residence 8 weeks after birth. The first contact with the subjects has been made either by the visiting nurses or the nurses of the general practitioner clinics.

Eligible participants were healthy mother-infant dyads, who met the inclusion criteria. Two enrolled pairs were disqualified due to intake of either formula or solid food during the study. Two mothers who had antibiotic at birth, otherwise fulfilled the inclusion criteria for both the mother and the infant, were enrolled towards the end of the study due to the low number of recruited volunteers. To assess eligibility, volunteers have answered a questionnaire provided in both English and Danish. For eligible volunteers, a personal meeting was arranged with the mothers and other accompanying persons (e.g. spouses or family members) to brief them on the study at Rigshospitalet Neonate Dept. The meeting was carried out by a nurse assigned by Assoc. Prof. Lise Aunsholt in a meeting room booked for this purpose at Rigshospitalet or online if that is more convenient for the volunteers. The assigned nurse provided oral information covering the purpose of the research, the scope of volunteer participation and general information regarding practicalities related to sample collection and sample handling. The nurse also reiterated the terms of participating in the study, especially its strictly voluntary nature and the rights to withdraw the consent to participate at any time without justification or consequences. The volunteers also got this information in a written form that includes the study purpose and background, the fate of their samples and the informed consent (available in Danish and English, provided as a separate participant information document) for signature. Additionally, the volunteers will also be informed on the possibility of signing separate consents to store their pseudonymized samples for potential future research.

A consideration time of 24-48 hours between the information meeting and the collection of the signed informed consents for both mothers and infants. After volunteers have given their consent, they were enrolled and provided with a study package containing written information regarding sample collection and handling, a feces collection kit, mother's milk collection tube, latex gloves and the study dairy. The stool and milk sample containers will be pre-labelled pseudonymously as to preclude traceability of samples to specific participants.

Sample collection and pseudonymization set-up The mothers were explicitly and thoroughly informed on the timing of the samples and were advised on how to estimate the timing for sample collection. The compliance of the mothers will be evaluated by including a table in the pseudonymous diary, where the mothers were asked to fill the numbers of breastmilk meals and the numbers of solid food means for the last ten meals prior to data collection.

Mothers in the enrolled infant-mother cohort were asked to collect stool and fill out the study diary at the time points indicated in the sampling scheme. The diary was also be pseudonymous to hinder traceability to specific donors. To ensure rapid collection and best quality samples, a person from the Technical University of Denmark (DTU), who is not affiliated with this study, was assigned sample pick-up from a collection site indicated by the mother subjects within the Copenhagen area. The sample collection containers will be pre-labelled with a QR code (generated at DTU) that encodes a letter identifier assigned to each mother-infant pair. In addition, the sample collection containers were pre-labelled with Arabic numerals (1, 2 or 3) to designate the sampling occasion as well as with the codes MF and IF, to designate mother's feces and infant feces, respectively.