Background: Although breastfeeding has known protective effects, such as preventing childhood obesity, the specific mechanisms remain unclear. Idaho has a high breastfeeding initiation rate (92%) but a significant prevalence of childhood obesity (30.5% overweight/obese). Limited research exists on the impact of maternal inflammation, maternal body mass index (BMI), C-reactive protein (CRP), and interleukin-6 (IL-6) concentrations in breastmilk on infant health outcomes, especially in healthy full-term infants.

Objective: This study aims to expand understanding of the role of maternal inflammation on breastmilk composition and its effect on infant immune development. The investigators seek to investigate the relationship between maternal health status, breastmilk inflammatory concentrations, and balanced immune development in infants. Additionally, the investigators aim to explore the potential influence of early diet exposure, including maternal inflammatory status, on the risk of obesity and other inflammatory conditions.

Methods: Healthy full-term infants (breastfed/formula-fed) and their mothers will be recruited. Maternal inflammation markers (BMI, CRP, IL-6) and immune markers in infants will be analyzed. Flow cytometry will assess immune populations. Correlations between maternal systemic inflammation, infant inflammation, and breastmilk inflammatory markers will be examined for breastfeeding mothers.

Outcomes: The investigators hypothesize breastfed infants will display a more favorable anti-inflammatory profile. This study will identify factors influencing immune development and potential pathways linking early-life exposures to long-term health outcomes. Findings will inform strategies for promoting balanced immune development and elucidate the role of early diet exposure, including maternal inflammation, as a protective or risk factor for obesity and inflammatory conditions.

The immune system of newborns is immature and undergoes rapid development during the first few months of life. Inflammation is a natural response to infection or tissue injury, but excessive or prolonged inflammation can be detrimental to immune development. Understanding the relationship between inflammation and immune development in healthy full-term infants is critical to developing effective interventions to promote healthy immune function in early life.

This project will collect blood samples from healthy full-term infants at 2 and 4 months of age. The samples will be analyzed using flow cytometry to quantify the proportions and activation status of different immune cell populations, such as T, B, natural killer, and monocytes. The analysis will also include quantifying intracellular cytokines to assess the functional capacity of the immune cells. The samples will be analyzed using established methods to measure biomarkers of inflammation, such as CRP, TNF-alpha, and other inflammatory interleukins and cytokines. This project will expand upon biomarkers previously examined to aid in the overall understanding of the role of inflammation in healthy immune development. Fecal samples will be collected from infants five times during the first 6 to 18 weeks of life. Fecal CRP and calprotectin levels will be quantified to detect low-level inflammation and enhance systemic results. Breastmilk will also be collected from lactating women at corresponding collection times to assess breastfeeding's impact on immune development. In addition to data collected on maternal BMI, maternal blood will be collected at 2 and 4 months postpartum to enhance investigations into maternal inflammatory status. Using existing clinical partnerships, 30 maternal-infant dyads will be recruited during the first 6 weeks of life and followed for 12 weeks. Dyads will include 15 breastfeeding infants, to be matched with 15 infants receiving formula or other non-human-based milk products (i.e., plant based formula). Dyads will be matched on infant sex and maternal BMI. A human milk sample will be collected at 6, 8, 12, 16 and 18 weeks postpartum in connection with infant fecal samples (Figure 1). Mothers can opt-in to collecting their own feces and saliva. Blood will be obtained from infants during recurring pediatric wellness visits. Mothers will also be asked to complete reoccurring surveys regarding their diet, perceived stress and anxiety, infant diet, food introduction, and general infant behavior or level of fussiness. Maternal demographics, including education level, household income, and food security, will be collected upon enrollment. Electronic medical records will be accessed related to prenatal care; maternal health and pre-existing conditions; birth records; and infant health status, including growth and incidence of infection and use of antibiotics. The investigators will measure the concentrations of inflammatory cytokines in fecal and blood samples from infants and in milk and blood samples from their mothers. Flow cytometry will analyze infant and maternal blood samples for circulating immune cell populations. When available, saliva samples will be analyzed for cortisol concentration. Remaining infant and maternal fecal samples will be retained for future microbiome investigations.

Inclusion Criteria:

• Mother at least 18 years of age
• Mother is in third trimester (week 27 of gestation) or biological infant is 5 weeks of age or younger
• Mother plans to continue to provide your infant breastmilk (by breastfeeding or by pumping) for at least 18 weeks (4.5 months) from your delivery date or mother plans to continue to provide formula exclusively to infant for at least 18 weeks of life.
• Mother lives within a 45-mile radius of Study Site, or is willing to deliver samples for visits 2, 4, and 6.
• Mother willing to meet at (designated sample collection site) for visits 3 and 5 for sample collection and visit activities.
• Mother willing to consent and comply with all aspects of the study protocol and methods, save the optional activities and optional sample collections.
• Mother and infant are considered healthy by Principle Investigator.
• For Formula Fed Group: Mother-Infant dyad is able to match to a Breastfed dyad using maternal BMI and infant sex.

Exclusion Criteria:

• Mother or Infant have participated or are currently participating in an interventional drug or device (non-observational) research study before.
• Mother reports that they, or the infant, have had an adverse effect during a venous blood collection.
• Infant was born less than 36 weeks of gestation.
• Infant or mother have health conditions that increase the risk of study procedures.

This project is focused on the effects of maternal inflammation and health status on the developing infant, including the role of breastfeeding and early feeding practices.

Study population is postnatal women and their infant dyads.

Only deidentified data or samples will be shared with other researchers. The PI will ensure data confidentiality and deidentification of all data.