STUDY PROTOCOL article
Front. Pediatr.
Sec. Neonatology
The impact of AMniotic FluId on the development and microBIal colonization of the prEterm intestinal tract (AMFIBIE): study protocol for a multicenter, prospective cohort study
Provisionally accepted
Rimke Romee De Kroon1*
Sofie van Weelden2
Loes Moonen2
Petra Bakker3
Eva Pajkrt1
Eduard A Struys1Andries E Budding4
Tim De Meij1
Hendrik Niemarkt2
Mirjam Maria Van Weissenbruch1*- 1Amsterdam University Medical Center, Amsterdam, Netherlands
- 2Maxima Medisch Centrum, Eindhoven, Netherlands
- 3Amsterdam Universitair Medische Centra, Amsterdam, Netherlands
- 4inBiome BV, Amsterdam, Netherlands
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Abstract Introduction: Prematurity is associated with intestinal immaturity and gut microbiota alterations, both of which are linked to necrotizing enterocolitis (NEC) and sepsis. An important yet understudied contributor in the development of the gastrointestinal (GI) tract is amniotic fluid (AF). The aim of this study is to assess the composition of AF collected during extremely preterm birth. Secondary objectives are to identify AF profiles of pregnancies complicated with chorioamnionitis and/or fetal growth restriction, assess key AF components across gestation, correlate AF profiles with neonatal outcomes (e.g. NEC and sepsis), and explore associations with neonatal gut microbiota. Methods and analysis: In 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). AF can be collected safely and non-invasively during vaginal delivery or cesarean section. AF samples will also be collected from a reference group (GA <23+6/7 and 28+0/7-40+6/7 weeks, n=150). Characterization of AF will include microbial and metabolic profiling. By advancing our understanding of the role of AF in the GI development and neonatal disease, this study may contribute to the early identification of infants at risk to develop NEC and/or sepsis. Ultimately these findings may facilitate early targeted microbiota-based interventions to prevent disease progression and improve outcomes. While the current study provides clinical opportunities, simultaneously, several methodological challenges inherent to collecting AF at birth must be acknowledged, including variability in sampling method, maternal blood interference, and the risk of microbial contamination. Ethics and disseminations: Ethical approval was received by the METC of the Máxima Medical Center in Veldhoven, the Netherlands (W24.042). The study was registered in a public clinical trial registry (NCT07152106). Study findings will be disseminated through peer-reviewed journal articles as well as national and international conference presentations.
Keywords: Amniotic fluid1, chorioamnionitis7, metabolomics2, microbial profiling3, necrotizing enterocolitis5, neonatal gut colonization4, sepsis6
Received: 09 Oct 2025; Accepted: 19 Dec 2025.
Copyright: © 2025 De Kroon, van Weelden, Moonen, Bakker, Pajkrt, Struys, Budding, De Meij, Niemarkt and Van Weissenbruch. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Rimke Romee De Kroon
Mirjam Maria Van Weissenbruch
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