Impact of Breast Milk miRNAs on the Infant Gut Microbiota

The first 1,000 days of life are a critical period that significantly contributes to the programming of an individual's health. It is increasingly evident that establishing a healthy gut microbiota (GM) plays an important role in preventing both short- and long-term health issues.

Among the factors influencing the establishment of the infant's gut microbiota, breast milk (BM) plays a fundamental role. Unlike formula milk, breast milk contains miRNAs that are potential mediators of the interactions between the host and the microbiota. Thus, we hypothesize that miRNAs from breast milk may be present in the infant's luminal content and interact with their microbiota, thereby modulating it.

Main Objective: Explore the effect of breast milk on the composition of fecal miRNAs in infants.

Primary Evaluation Criterion: Profiles and quantities of miRNAs at different time points (D10, D30, D60, M6, and M12) in the stools of exclusively breastfed infants compared to those fed with formula milk.

Secondary Objectives:

Study the effect of miRNAs from breast milk on the composition of the infant's fecal microbiota, considering environmental confounding factors, including the composition of maternal microbiota.

Investigate the effect of breast milk microbiota on the miRNA profiles present in the milk.

Study the effect of breast milk microbiota on the gut microbiota of infants. Explore the links between breast milk miRNAs, the infant's gut microbiota, and intestinal inflammation in infants.

Secondary Evaluation Criteria:

Correlations between breast milk miRNA profiles from D1 to D60 and the gut microbiota of breastfed infants from D1 to M12.

Correlations between the breast milk microbiota and the breast milk miRNA profiles from D1 to D60.

Correlations between breast milk microbiota from D1 to D60 and the infant fecal microbiota from D1 to M12.

Direct correlations between (1) breast milk miRNAs and the measurement of fecal calprotectin in infants from D1 to D60, and (2) the infant's gut microbiota and fecal calprotectin in infants from D1 to D60.

To address the research question, we plan to include 200 mother-infant dyads. Our goal is to have 30 dyads with exclusive breastfeeding for at least two months, and 30 dyads where the infant will not have received any breast milk. Each dyad will participate in the study for a period of 12 months, and the recruitment phase will last for 18 months.

At the infant's birth, we will collect stool samples from both the mother and the infant, as well as a breast milk sample from the mother.

At 10, 30, and 60 days after the infant's birth, we will request both a breast milk sample from the mother and a stool sample from the infant.

At 6 and 12 months, only a stool sample from the infant will be collected. During each visit, a clinical research coordinator will contact the mothers to gather information about both their health and diet, as well as their infant's health and diet.

Analysis of miRNAs in Breast Milk and Stool Samples: miRNAs will be extracted from infant stool samples and breast milk using next-generation Illumina sequencing (NGS). The top 10 most abundant miRNAs will then be validated through qPCR. The overall miRNA profiles will be analyzed using Principal Coordinate Analysis (PCoA).

Analysis of Gut and Breast Milk Microbiota: Stool and breast milk samples will be used for 16S rRNA gene sequencing to analyze the microbial composition. Microbiota profiles will also be examined using Principal Coordinate Analysis (PCoA).

Inclusion criteria

• Pregnant women in the 3rd trimester of pregnancy (3T) with a normal course of pregnancy up to 39 weeks consulting for the monitoring visit at the Robert-Debré hospital.
• Patients who did not receive antibiotic treatment or probiotics in 3T.
• Patient with a negative PV for Strepto B
• Patients who do not have complications such as pre-eclampsia or gestational diabetes.
• Breastfeeding (+/- milk pump) or exclusive artificial breastfeeding.
• No opposition from the mother
• Ability to master the French language required.
• Freezer at home.
• Affiliation to a social security scheme.

Exclusion criteria

• Cesarean delivery
• Perinatal pathologies (Anoxia, IUGR, etc.)
• Neonatal pathologies (FGR, malformations, etc.)
• Initial choice of mixed breastfeeding
• Patient under AME (State Medical Aid)Critères d'exclusions :
• Antibiotics for the mother or child before day 60
• Antifungal for the mother or child before day 60
• Complete cessation of breastfeeding before day 60