Background:

Necrotizing enterocolitis (NEC) and sepsis in preterm infants have been linked to intestinal immaturity and preclinical gut microbiota alterations. An important yet understudied contributor in the development of the gastrointestinal tract (GIT) is amniotic fluid (AF). Knowledge is lacking on the critical shifts that may occur in AF in extremely preterm birth. The aim of the current study is to assess the composition of AF using advanced biomedical techniques. Secondary objectives are to assess AF profiles of infants with chorioamnionitis (CAM) and/or fetal growth restriction (FGR), assess key metabolites across gestation, correlate AF profiles with neonatal outcomes, and explore associations with early gut microbiota.

Methods:

ln this multicenter, prospective, cohort study, AF (~5 mL) will be collected from obstetric patients delivering their infants extremely preterm (gestational age (GA) 24+0/7-27+6/7 weeks, n=125), either during vaginal delivery or cesarean section (CS). Additionally, AF samples will be collected from a reference group (n=150), including early midtrimester (GA <23+/7 weeks), very early and moderate to late preterm (GA 28+0/6-36+6/7 weeks), and full-term pregnancies (GA 37+0/7-41+6/7 weeks). Thorough characterization of AF will be conducted, including microbial profiling and metabolomics. Microbiota profiling of neonatal fecal samples will be conducted to assess the association between AF and early neonatal gut colonization patterns.

Discussion and expected results:

AF profiles associated with CAM and/or FGR in extremely preterm infants are expected to be identified, as well as relevant associations with neonatal health outcomes (including NEC and sepsis) and early neonatal gut colonization patterns. The current study will not only increase the understanding of the GIT development and the pathogenesis of NEC and sepsis but may also aid in the identification of high-risk infants. In the future, these findings may facilitate early targeted microbiota-based interventions to prevent disease progression and ultimately improve clinical outcomes.

Inclusion Criteria:

• Maternal age ≥16 years
• Written informed consent
• Successful collection of amniotic fluid

Exclusion Criteria:

• Pregnancies complicated by fetal congenital and/or chromosomal abnormalities.
• Insufficient proficiency of Dutch or English language

Infants included in the study group must be born between 24+0/7 and 27+6/7 weeks. AF samples are also collected from a reference group, including early midtrimester (GA ≤23+6/7 weeks), very and moderate to late preterm (GA 28+0/7-36+6/7 weeks), and full-term pregnancies (GA 37+0/7-41+6/7 weeks).