The Impact of Amniotic Fluid on the Development and Microbial Colonization of the Preterm Intestinal Tract: the AMFIBIE Study
Conditions: Chorioamnionitis, Chorioamnionitis Affecting Fetus or Newborn, Necrotizing Enterocolitis of Newborn, Neonatal Sepsis, Early-Onset, Neonatal Sepsis, Late-Onset, Fetal Growth Restriction (FGR), Preterm Birth Complication, Prematurity Complications
Sponsor: Maxima Medical Center

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The impact of AMniotic Fluid on the development 
and microBIal colonization of the prEterm 
intestinal tract: the AMFIBIE study 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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SUMMARY 
 
Rationale: Preterm birth remains a major global health issue. Extremely preterm infants, 
born with gestational age below 28 weeks, are especially vulnerable to adverse health 
outcomes. Necrotizing enterocolitis, an inflammatory disease of the neonatal intestines, and 
sepsis, a systemic response to microbial invasion of the bloodstream, are more common in 
this population. The pathogenesis of both conditions has been linked to the immaturity of the 
preterm intestines and preclinical alterations in the gut microbiome. It has been hypothesized 
that amniotic fluid (AF) impacts the intra-uterine development of the fetal gastrointestinal tract 
as the intestines are exposed to amniotic fluid through swallowing in utero. Previous studies 
on the characterization of AF have demonstrated that the constitution of AF gradually 
changes over the course of pregnancy. However, current literature still lacks knowledge on 
the, potentially, critical changes, that occur in the content of AF between the 24th and 28th 
week of pregnancy, their effect on the development of the fetal gastrointestinal tract and 
impact on the microbial colonization of the neonatal gut. 
Objective: The aim of this study is to characterize the composition of AF, collected during 
preterm delivery, of extremely preterm infants (24-28 weeks), using novel molecular 
techniques. These approaches include microbial (e.g. IS-pro analysis) and metabolomic (e.g. 
(un)targeted metabolomics) techniques. The impact of AF on neonatal gut colonization will 
be assessed through analyses of neonatal stool samples, which are collected according to 
the ongoing eMINDS study. 
 
Study design: In this multicenter observational cohort study, AF will be isolated from 
mothers delivering their infants extremely preterm. Two subgroups are of particular interest 
due to their association with morbidity and mortality in the neonatal phase: 1) preterm infants 
exposed to intra-uterine inflammation, and 2) preterm infants with fetal growth restriction 
(FGR). To assess the impact of the composition of AF on the colonization of the neonatal 
gastrointestinal tract, neonatal stool samples are collected daily for the first 28 days of life, 
according to the ongoing eMINDS study. 
 
Study population: This study involves the inclusion of mother-infant pairs. Obstetric patients 
expected to deliver extremely preterm and their infants are eligible to participate in the study. 
Obstetric patients with pregnancies complicated with major fetal congenital or chromosomal 
abnormalities will be excluded. A reference cohort consisting of AF samples derived during 
amniocentesis (< 24 weeks) or during planned cesarean sections (C-sections) or vaginal 

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delivery is included to allow for adequate interpretation of findings. For this reference cohort, 
AF samples are collected from early midtrimester (< 24 weeks), very early and moderate to 
late preterm (28 + 0 – 36 + 6/7 weeks), and full term pregnancies (37 + 0 – 41 + 6/7 weeks). 
 
Study parameters: The main outcome is the characterization of the bacterial and metabolic 
composition of AF derived from obstetric patients delivering their infants extremely preterm 
using advanced biomedical techniques. Secondary outcomes include analysis of AF profiles 
of extremely preterm infants exposed to, respectively, inflammation in utero and FGR; 
assessment of key metabolites found in AF of infants exposed to inflammation in utero or 
FGR over the course of gestation; correlation of AF profiles to neonatal inflammatory 
diseases and identification of potential biomarkers in AF for premature infants at risk to 
develop these diseases; correlation of AF profile to microbial colonization of neonatal 
intestinal gut microbiome in first month of life.  
 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: Collection of AF, which is generally discarded as biological waste, is 
non-invasive, posing no additional strain on the obstetric patient. AF can safely be isolated 
without interfering with the practice of a C-section or a vaginal delivery. 
 
 
 
 
 
 
 
 
 

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1. INTRODUCTION AND RATIONALE 
 
Introduction 
Preterm birth, defined as gestational age (GA) < 37 weeks, is the leading cause of neonatal 
death and the second leading cause of death in children under five years old worldwide (1). 
Infants born extremely preterm (GA < 28 weeks) are most susceptible to developing adverse 
short-term and long-term health outcomes (2). Clinical outcomes of extremely preterm infants 
have significantly improved over the past decade due to major advancements in medical 
care. Studies demonstrated that a shift has taken place in the causes of neonatal mortality 
and morbidity. While previously death and disease was mostly related to pulmonary illness, 
extremely preterm infants are now more likely to survive the first days of life and are 
challenged by other, non-pulmonary, diseases, including necrotizing enterocolitis (NEC) and 
sepsis (3). These developments highlight the need to focus our efforts on improving the 
identification of preterm infants at risk of developing NEC and sepsis and optimizing 
diagnosis and treatment. 
 
NEC is a potentially life-threatening disease of the neonatal intestines typically affecting 
preterm infants and infants with extremely low birth weight (BW). Clinical presentation ranges 
from abdominal distension and bloody stools to intestinal perforation, sepsis, and circulatory 
insufficiency. Severe cases NEC can also have a fatal outcome (4). While early detection is 
essential in determining the course and prognosis of NEC, clinical signs are often subtle and 
non-specific, particularly at disease onset. Radiological findings and biochemical alterations 
are often only evident in more advanced stages (5). The pathogenesis of NEC is 
multifactorial, most likely caused by an inappropriate inflammatory response of the immature 
intestinal epithelial cells as a result of enteral feeding in the presence of intestinal hypoxia-
ischemia-reperfusion combined with intestinal dysbiosis (6-9). While changes in the intestinal 
microbiome composition, characterized by increased relative abundances of Proteobacteria 
and decreased relative abundances of Firmicutes and Bacteroidetes, are associated with the 
pathogenesis of NEC, a distinctive microbial signature for NEC has not yet been 
characterized [9]. Besides BW, NEC incidence largely depends on GA: a lower GA is 
associated with higher incidence of NEC. In other words, evidence suggests that each 
gestational week spent in utero reduces the risk of NEC (10). 
 
Another important cause of neonatal morbidity and mortality is neonatal sepsis, which refers 
to microbial invasion of the bloodstream in infants less than 28 days old. Signs and 
symptoms of sepsis are often non-specific, while failure to recognize sepsis and commence 

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antibiotic treatments in an early stage may lead to serious complications such as 
disseminated intravascular coagulation, congestive heart failure, shock, and death (11). 
Preterm infants are more susceptible to develop LOS than term neonates due to their 
immature immune system and exposure to invasive devices including vascular access, 
endotracheal tubes, and feeding tubes (12, 13). The pathogenesis of LOS, similar to NEC, is 
associated with alterations in the gut microbiome. While intravascular catheters are 
considered the most important source for LOS, recent studies have shown genetic 
similarities between the causative pathogens of LOS, as detected through blood culture, and 
bacterial isolates derived from the gastrointestinal tract (GIT). Several days prior to clinical 
onset of LOS, the causative pathogens can be detected in the GIT. The finding that the GIT 
serves as a reservoir for pathogens responsible for, at least a part of LOS cases, is known as 
the “gut-derived sepsis” hypothesis (14-16). 
 
The immaturity of the intestines and dysbiosis plays a prominent role in the pathogenesis of 
both NEC and sepsis. An important yet understudied player in the development of the fetal 
intestines is amniotic fluid (AF) (17). Reduced exposure to trophic factors in AF, as is the 
case for preterm infants, may compromise healthy development of the intestines and impact 
intestinal colonization. However, little is still known about the constitution of AF in extremely 
preterm infants and its effect on the initiation of the gut microbiome. We hypothesize that 
elucidating the composition of AF and discerning its effect on the fetal intestines and its 
potential impact on the initiation of the gut microbiome will increase our understanding of 
NEC and LOS and aid in the identification of biomarkers and clinical interventions. 
 
Amniotic fluid impacts development of fetal intestines 
Amniotic fluid (AF) is a dynamic biological fluid, which plays a pivotal role in fetal 
development. AF has multiple functions, ranging from functioning as a protective fluid against 
physical trauma, to contributing to fetal nutrition and aiding in the protection against fetal 
infection through the presence of immune cells, microbial peptides, and enzymes (18). AF is 
also known to be an important contributor to the development of the fetal GIT as the fetal 
intestines are exposed to AF through swallowing, starting around the 12th week of gestation. 
Trophic factors impacting the fetal intestinal epithelium include epidermal growth factor 
(EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF)-1, erythropoietin 
(EPO), and various interleukins (IL) (18, 19). AF reflects the health of both mother and fetus, 
highlighting the value of AF as a tool in the diagnosis of a wide spectrum of both maternal 
and fetal clinical conditions (20, 21). Previous studies on the characterization of AF through 
metabolomic, proteomic, and transcriptomic analyses have demonstrated that the 
constitution of AF gradually changes over the three trimesters of pregnancy (22, 23). 

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However, current literature still lacks knowledge on the, potentially critical, changes that 
occur in the content of AF between the 24th and 28th week of pregnancy, their effect on the 
development of the fetal intestines and correlation to morbidity and mortality in the neonatal 
period. Our aim is to address this gap in knowledge through the thorough characterization of 
AF of premature infants using various novel molecular techniques. To accomplish this we 
aim to isolate AF from mothers delivering their infants extremely preterm (24 to 28 weeks). 
Two subgroups are of particular interest due to their association with morbidity and mortality 
in the neonatal phase: 1) preterm infants exposed to intra-uterine inflammation (IUI), and 2) 
preterm infants with fetal growth restriction (FGR).  
 
Background: Intra-uterine inflammation 
The first group of interest consists of preterm infants exposed to chorioamnionitis (CAM). 
While the intra-uterine environment is thought to lack a microbiome in a physiological state 
(24), microbial invasion of the amniotic cavity (MIAC) can cause CAM or intra-uterine inflam-
mation (IUI), which is defined as an inflammatory or infectious disorder of either the chorion, 
the amnion or both. MIAC is the most commonly found cause of IUI but other causes such as 
sterile inflammation have also been described. Various factors are associated with IUI, in-
cluding preterm premature rupture of membranes (PPROM), prolonged rupture of mem-
branes (PROM), and maternal colonization of group B streptococcus (GBS) (25). 
 
CAM is often challenging to diagnose due to non-specific clinical presentation. Manifestation 
of CAM can also occur subclinically without any clinical signs and present as preterm labor 
with PROM or even with intact fetal membranes. Suspicions of CAM can be confirmed by 
histopathology of the placenta by a trained pathologist. The placenta can demonstrate signs 
of neutrophilic infiltration in the decidua or even the presence of micro-abscesses as a result 
of IUI. However, obtaining the placenta report may take up to six weeks (26). In a subset of 
patients with CAM, pathogens can be isolated from AF. In up to 65% of microbially proven 
cases two or more pathogens can be isolated from AF. The most commonly isolated micro-
organisms (MO) from AF and the placenta are Ureaplasma spp. but also Gardnerella vagi-
nales, Bacteroides, GBS and Escherichia coli. The presence of Ureaplasma spp. is associ-
ated with chronic and clinically silent intra-uterine infections. From the second trimester on-
wards Ureaplasma spp. can be isolated from AF and can remain clinically silent for up to sev-
eral months (27). Traditional culturing of many of these pathogens has proven to be difficult. 
Novel molecular methods, such as 16S-based sequencing, yield more promising results (28). 
Diagnosis of CAM can be difficult due to a variety of challenges, while identification of infants 
exposed to IUI may provide novel insights on various neonatal diseases, highlighting the 
need to study the composition of AF of infants exposed to CAM.  

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IUI is one of the major causes of preterm birth. IUI can induce a fetal inflammatory response 
(FIRS), which can initiate preterm labor (29, 30). Fetal exposure to IUI is associated with ne-
onatal morbidity in various organ systems, including NEC and sepsis (31, 32). In vivo studies 
in animal models have shown that in utero exposure of the fetal GIT to pathogens and in-
flammatory mediators in AF is associated with altered development of the intestinal barrier, 
including increased intestinal permeability and increased bacterial translocation (33). Another 
study using a mice model also underlined these findings, while demonstrating that exposure 
to IUI results in increased susceptibility to injury later in life (34), providing a possible expla-
nation for the correlation found between NEC and CAM. Noteworthy, a recent study in a rat 
model demonstrated that exposure to IUI not only resulted in intestinal injury but also in sig-
nificant changes in the neonatal intestinal microbiome. These changes were characterized by 
increased Proteobacteria and decreased Firmicutes 7 days after birth (35). To our knowledge 
so far only one study focused on identifying the impact of CAM on neonatal gut colonization 
in humans. The fecal samples of preterm infants < 28 weeks exposed to CAM with funisitis 
demonstrated a higher relative abundance of Mycoplasmataceae, Prevotella and Sneathia 
compared to a healthy control group. In addition, exposure to CAM was associated with 
higher incidence of LOS, which correlated with the presence of Fusobacteria, Sneathia, and 
Mycoplasmatacea (36).  
 
Background: Fetal growth restriction 
The second group of interest consists of preterm infants with fetal growth restriction (FGR). 
FGR is defined as an inability of the fetus to grow to its expected biological potential in utero 
(estimated fetal weight (EFW) or abdominal circumference (AC)) < 10th percentile for GA) 
and is one of the most common pregnancy complications. FGR has various known causes 
and can be multifactorial. Causes include maternal factors, fetal factors, and causes 
involving placental insufficiency (37, 38). FGR can be classified depending on the time of 
onset; early FGR commences < 32 weeks, while late FGR is diagnosed ≥ 32 weeks. Early 
onset FGR is associated with preterm birth and a variety of neonatal morbidities, including 
increased incidence of LOS and NEC (39, 40).  
 
In response to the lack of oxygen and nutrients, fetal cardiovascular adaption will take place 
to ensure brain sparing and altered development of various organ systems may take place, 
including altered development of the fetal GIT (41). Animal studies have shown that FGR not 
only results in decreased BW but also impacts the development of the small intestines and 
may result in increased susceptibility to gastrointestinal disorders (42). Infants with FGR are 
characterized by the disrupted constitution of gut microbiota and metabolic profiles compared 

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to non-growth restricted infants. The gut microbiome of growth-restricted infants is 
characterized by decreased relative abundance of Enterococcus and Acinetobacter, while 
the metabolic profile is characterized by downregulated methionine and cysteine levels (43). 
 
Previous studies on the composition of AF have shown that various metabolites demonstrate 
a significant change in abundance associated with the second-to-third-trimester transition. 
These changes are hypothesized to correlate with the stabilization of fetal growth during this 
period (23), raising our interest in the characterization of AF in growth-restricted infants. 
However, studies on composition and characterization of AF of infants diagnosed with FGR, 
specifically in the context of preterm birth, are still lacking from current literature and of 
particular interest due to the impact of AF on the development of the intestines, the initiation 
of the gut microbiome and correlation with dysbiosis-related disease in the neonatal period.  
 
Conclusion  
Being born extremely preterm lays a pivotal foundation for short-term and long-term 
consequences, in which diseases due to inflammation and infection play an important role. 
We argue that we must increase our understanding of the role of microbiota and 
inflammation before and after preterm birth to ensure better outcomes for preterm infants in 
the long run. To achieve this we must cross the bridge between fetal and neonatal research. 
We argue that this exploratory cohort focusing on the characterization of AF in preterm 
pregnancies between 24 and 28 weeks of gestation is uniquely suited to make a step in the 
right direction. Thoroughly studying the content of AF in relationship to the initiation of the 
neonatal gut microbiome and metabolome in extremely preterm infants will not only increase 
our understanding of the pathogenesis of NEC and sepsis but will also aid in the 
identification of subgroups of infants at risk to develop such morbidities. In the future, this 
research will potentially guide strategies to prevent NEC and sepsis and associated long-
term morbidities.  
 
 
 
 
 
 
 
 
 
 
 

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2. OBJECTIVES 
 
Primary objectives:  
To assess the bacterial and metabolic composition of AF derived from mothers delivering 
extremely preterm infants (duration of pregnancy 24 + 0 – 27 + 6/7 weeks) using advanced 
biomedical techniques (e.g. 16S-based sequencing, IS-pro, (un)targeted metabolomics). 
 
Secondary objectives:  
1. To analyze and compare the AF profiles of extremely preterm infants exposed to in-
flammation in utero vs. infants who were not exposed to inflammation, taking into ac-
count clinical signs and symptoms of CAM as well placental histopathology reports.  
2. To analyze and compare the AF profiles of extremely preterm infants diagnosed with 
FGR, according to the consensus definition by Gordijn et al. (2016) vs. non-growth 
restricted infants.  
3. To assess the course of key metabolites in AF identified in objective 2 (infants ex-
posed to inflammation) and 3 (infants with FGR) over the course of gestation.   
4. Relate AF profiles to neonatal disease in which inflammation plays a role (including 
NEC, sepsis, and bronchopulmonary dysplasia (BPD) and identify potential bi-
omarkers in AF for premature infants at risk to develop these diseases.  
5. To correlate the bacterial and metabolic composition of AF to microbial colonization of 
neonatal intestinal gut microbiome in the first month of life. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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3. STUDY DESIGN 
 
This prospective observational cohort study will be conducted in the Amsterdam UMC 
(Amsterdam, the Netherlands) and Máxima Medisch Centrum (Veldhoven, the Netherlands), 
commencing in 2024 until the desired amount of mother-infant pairs (pregnancy duration 
between 24 + 0 and 27 + 6/7 weeks) have been included (n = 125). In addition, AF will be 
collected from a reference cohort (n = 150). 
 
 
 
Figure 1 – schematic overview of the study population (24 + 0 to 27 + 6/7 weeks), consisting 
of the obstetric phase and the neonatal phase. Created with Biorender.com.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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4. STUDY POPULATION 
4.1 Population (study population)   
 
AF will be collected from the study population (pregnancy duration of 24 + 0 – 27 + 6/7 
weeks) and from a reference cohort (respectively, early midtrimester (≤ 23 + 6/7 weeks), very 
and moderate to late preterm (28 + 0 – 36 + 6/7 weeks), and full-term pregnancies (37 + 0 – 
41 + 6/7 weeks). Table 1 contains an overview of the inclusions for each category.  
 
Study population: Patients admitted to the obstetric unit of the participating center, either 
the Amsterdam-UMC (Amsterdam, The Netherlands) or the Máxima Medical Center 
(Veldhoven, The Netherlands), who are expected to deliver extremely preterm (pregnancy 
duration 24 + 0 – 27 + 6/7 weeks) will be briefed on participation the study. In short, patients 
will be asked for permission for the following: 1) collection of AF during extremely preterm 
delivery (24 + 0 – 27 + 6/7 weeks of gestation), and 2) collection of clinical data from the 
electronic patient record (EPR) by researchers, as will be elaborated below. To be included 
in the AMFIBIE study, an AF sample at time of delivery must successfully be collected. In 
case of a failed attempt, participants will not be included in the study.  
 
Inclusion criteria study population: If the obstetric patient is > 16 years of age and mentally 
competent, AF can be collected at time of delivery between 24 and 27 + 6/7 weeks of 
gestation and informed consent is obtained from the obstetric patient, the participant can be 
included.  
 
Exclusion criteria study population: Obstetric patients < 16 years of age and/or mentally 
incompetent or with pregnancies complicated with major fetal congenital or chromosomal 
comorbidities will be excluded from the study.  
 
Reference cohort: Patients admitted to the obstetric unit of the participating center, either 
the Amsterdam-UMC (Amsterdam, The Netherlands) or the Máxima Medical Center 
(Veldhoven, the Netherlands), who are delivering their infants between 28 + 0 – 36 + 6/7 
weeks (very and moderate to late preterm) or 37 + 0 – 41 + 6/7 weeks (full-term) can be 
included in the reference cohort. Patients will be asked for permission for collection of AF 
during delivery and the collection of clinical data from the EPR. Patients can also be included 
in the reference cohort if there is a clinical indication for amniocentesis, which are generally 
performed before the 24th week of gestation (usually around the 15th/16th week) and a few 
milliliters of additional AF can be collected during this procedure. In the unlikely event that 
diagnostic amniocentesis is performed > 24 weeks, the sample will not be included in the 

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reference cohort. The reference cohort is explained in more detail in section 8.3 “Study 
procedures – reference cohort”.  
 
Inclusion criteria reference cohort: If the obstetric patient is > 16 years of age and mentally 
competent and AF can be collected at time of clinically indicated amniocentesis (during early 
midtrimester pregnancies) or during delivery in very preterm and moderate to late preterm 
pregnancies or full-term pregnancies, the participant can be included in the study. 
 
Exclusion criteria reference cohort: Obstetric patients < 16 years of age and/or mentally 
incompetent or with pregnancies complicated with major fetal congenital or chromosomal 
comorbidities will be excluded from the study. 
 
Table 1: overview of collection of amniotic fluid samples  
 
Study population 
Reference population 
Methods  
≤ 23 + 6/7 weeks  
(early midtrimester) 
- 
60 
Amniocentesis (clinical 
indication) 
24 + 0 – 27 + 6/7 weeks 
(early preterm pregnancies) 
125 infants* 
- 
Vaginal delivery, 
cesarean section 
28 + 0 – 36 + 6/7 weeks  
(very, moderate and late 
preterm pregnancies) 
- 
60 
Vaginal delivery, 
cesarean section 
37 + 0 – 41 + 6/7 weeks 
(full-term pregnancies) 
- 
30 
Vaginal delivery,  
cesarean section 
Total 
125 
150 
 
*Amniotic fluid is collected from all obstetric patients during extremely preterm delivery. The study group is 
expected to include approximately 30 infants exposed to CAM and 25 infants exposed to FGR based on 
incidence of respective pregnancy complications.  
 
Neonatal population: Both parents and/or legal guardians of extremely preterm infants (GA 
24 + 0 – 27 + 6/7 weeks) born to mothers included in the study will be asked for permission 
for the collection of both clinical data and neonatal fecal samples during the first month of life, 
in line with the eMINDS study (reference number: Metc2014.386(A2020.190)). eMINDS is an 
acronym for “Enteral Microbiota and metabolomics in Infants for prediction of Necrotizing 
enterocolitis, neuromotor Development and late-onset Sepsis”. The eMINDS study is a 
research project that has been ongoing in seven intensive care units (NICUs) in the 
Netherlands since March 2021. The overarching aim of this project is the characterization of 
the intestinal microbiome and metabolome for the early detection of NEC and LOS in 
extremely preterm infants. For a detailed explanation of the eMINDS study, including a 
comprehensive description of collection, storage, and analysis of fecal samples as well as 

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collection of clinical neonatal data, the eMINDS study protocol can be consulted. The most 
recent version of the protocol is added as attachment. 
 
If permission is obtained for the eMINDS study and AF is adequately collected during 
delivery, both parents and/or legal guardians will sign an additional form to give permission to 
link the data from the AMFIBIE study to the data from the eMINDS study. In the following 
sections, we will refer to the participants included in the study population and their infants as 
the “mother-infant pairs” (Table 2). In both the Amsterdam-UMC as well as the Máxima 
Medical Center, the eMINDS study has a very high inclusion rate (>95%) and the expectation 
is that the vast majority of patients that will give permission for the AMFIBIE study, will also 
give permission for participation in the eMINDS study. In the unlikely case that no permission 
is granted on behalf of the infant for participation in the eMINDS study but informed consent 
has been provided for the AMFIBIE study, the obstetric patients will not be excluded from the 
study and the sample can be included in the analyses. Noteworthy, the eMINDS study also 
includes a follow-up study (known as generation P), which requires additional consent. 
However, follow-up data will not be linked to the data of the AMFIBIE study, so consent for 
the generation P study is not required in our study setup.  
 
Tabel 2: overview mother-infant pairs in the study population 
Study group: 24 + 0 – 27 + 67 weeks (very preterm pregnancies) 
 
Obstetric participants 
Neonatal participants 
Number of inclusions 
125 
 
125* 
Sample type 
Amniotic fluid sample during delivery 
Daily fecal sample during the first 28 
days of life  
Clinical data 
Extensive clinical and demographic 
data (8.3 Study procedures) 
Extensive clinical and demographic data 
(8.3 Study procedures) 
*In the unlikely case that no informed consent is provided by parents and/or guardians for participation in the 
eMINDS study and therefore fecal samples cannot be collected, obstetric patients will not be excluded from the 
AMFIBIE study and microbial and metabolic analysis on AF will still be performed. 
 
4.2 Sample size calculation 
 
A formal power analysis cannot be performed as there is currently insufficient literature 
available on the composition of AF in extremely preterm infants and its impact on the 
development of the neonatal gut microbiome. The incidence of CAM and FGR varies greatly 
in literature, respectively ± 25-40% for CAM and ± 20-30% for FGR in preterm infants (44, 
45). Due to the exploratory nature of this study, we aim to include approximately 30 infants 

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exposed to inflammation in utero and 25 infants with FGR. Taking into account the incidence 
of CAM and FGR in preterm infants, we aim to collect AF samples from 125 mother-infant 
pairs with a pregnancy duration between 24 and 28 weeks. In this cohort, we expect to 
include 31 infants with a sepsis episode and 9 infants with a NEC episode, based on the 
incidence of 25% of sepsis and 7% of NEC in extremely preterm infants (46, 47).  
 
5. TREATMENT OF SUBJECTS  
Not applicable 

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6. INVESTIGATIONAL PRODUCT 
 
Not applicable 
7. NON-INVESTIGATIONAL PRODUCT 
 
Not applicable  
 
8. METHODS 
8.1 Study parameters/endpoints 
8.1.1 Main study parameter/endpoint 
 
Characterization of bacterial and metabolic composition of AF derived from mothers 
delivering extremely preterm infants (duration of pregnancy 24 + 0 – 27 + 6/7 weeks) using 
advanced biomedical techniques (e.g. 16S-based sequencing, IS-pro, (un)targeted 
metabolomics). 
8.1.2 Secondary study parameters/endpoints  
 
• 
Analysis of microbial constitution of AF by 16S rRNA sequencing and/or IS-pro. 
• 
Analysis of neonatal intestinal microbiota by 16S rRNA sequencing and/or IS-pro. 
• 
(Un)targeted metabolomics of isolated AF samples.  
• 
Assessment of longitudinal course throughout gestation of key metabolites. 
• 
Correlation of demographic and clinical maternal as well as neonatal data to identified 
AF profile, as elaborated on above.   
• 
Correlation of neonatal intestinal microbiome to AF profile.  
8.1.3 Other study parameters  
 
Not applicable  
8.2 Randomisation, blinding and treatment allocation 
 
Not applicable 
 
 

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8.3 Study procedures 
 
The study consists of two phases for each mother-infant pair in the study group (extremely 
preterm pregnancies 24 + 0 – 27 + 6/7 weeks, n = 125): phase 1 (the obstetric phase) and 
phase 2 (the neonatal phase) (Figure 1). Additionally, a reference database will be 
constructed of AF collected from early midtrimester (≤ 23 + 6/7 weeks), very preterm and 
moderate to late preterm (28 + 0 – 36 + 6/7 weeks), and full -term pregnancies (37 + 0 – 41 + 
6/7 weeks) (n = 150).  
 
Phase 1: the obstetric phase   
 
The obstetric phase consists of the following components: collection of maternal clinical data, 
isolation of AF during delivery, and subsequent short-term storage, laboratory analyses of AF 
samples and long-term storage for future research.   
 
Clinical data collection  
 
Maternal clinical data to be collected include:   
• 
Standard demographic information (e.g. maternal age, medical history, gravidity, par-
ity, date of delivery, mode of delivery (e.g. vaginal delivery, primary or secondary C-
section), duration of pregnancy).  
• 
Maternal complications during pregnancy and/or delivery, including the occurrence of 
infectious diseases or inflammatory diseases.  
• 
Medication before, during, and after delivery, including oral/intravenous antibiotics 
use, insulin, and antihypertensives.  
• 
Vaccinations received during pregnancy. 
• 
Clinical data (e.g. signs of intra-uterine infection, including maternal fever (> 38OC), 
tachycardia, abdominal pain, elevated white blood cell count and/or elevated C-reac-
tive protein, positive blood culture results, fetal tachycardia or other signs of fetal dis-
tress in cardiotocography (CTG)).  
• 
Placenta pathology reports (e.g. signs of maternal vascular malperfusion, fetal vascu-
lar malperfusion, acute or chronic chorioamnionitis, and villitis of unknown etiology 
(VUE)).   
• 
Fetal ultrasound reports (e.g. head circumference (HC), AC, EFW, femur length (FL), 
HC/AC and FL/HC ratios, uterine artery Doppler velocimetry (UADV)).  

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• 
Results of any microbial swabs that were taken prior/after delivery (e.g. vaginal swab 
to assess maternal colonization with GBS).  
 
Relevant definitions: chorioamnionitis and fetal growth restriction  
 
Maternal clinical data will be collected for all participants included in the study. To study the 
primary and secondary research objectives, participants in the study group of extremely pre-
term pregnancies will be assigned to three groups: 1) CAM, 2) FGR, and 3) no suspicion of 
CAM or FGR. The definition used for FGR is the consensus definition by Gordijn et al. 
(2016), which agreed upon three solitary parameters (AC < 3rd centile, EFW < 3rd centile, and 
absent end-diastolic flow in the umbilical artery (UA)) for early FGR (48, 49). The definition 
for CAM is more complex. For example, CAM can be defined as a clinical or histopathologic 
entity (50). For clinical CAM, the definition is based on the clinical signs of CAM (e.g. mater-
nal fever, uterine tenderness, odorous discharge, and maternal-fetal tachycardia) as well as 
laboratory findings (e.g. leukocytosis) (49). For histologic CAM, the diagnosis is based on 
histopathologic examination of the placenta assessing the morphologic features of acute his-
tologic CAM, which are characterized by diffuse infiltration of neutrophils into the chorioamni-
otic membranes (50). In both the Máxima Medical Center and Amsterdam-UMC assessment 
of the placenta by a pathologist is standard practice, so pathologic reports of the placenta will 
be available. Since there is no single consensus definition, both definitions will be utilized in 
the analyses of the samples. All information required to adequate assign participants to the 
three groups are collected during standardized data collection, as explained in detail above.  
 
Collection of amniotic fluid during (preterm) delivery 
 
AF will be collected from a cohort of obstetric patients delivering their infants extremely 
preterm by the obstetrician of the participating center. AF can be easily, safely, and non-
invasively collected by the treating doctor during C-section or vaginal delivery, posing no 
additional strain on the obstetric patient.  
 
Vaginal delivery  
While AF is often lost during vaginal delivery, inclusion of mothers delivering vaginally is 
encouraged, if AF can be collected by the obstetric health care provider, for example when 
the amniotic membranes are manually ruptured in the case of induction of delivery or during 
routine cervical exams. The different methods of collection are described in detail in 
Appendix A. Noteworthy, when AF is collected during vaginal delivery, there is a high 
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assessing the samples with the various microbial and metabolomic approaches, the mode of 
collection should be taken into account when interpreting the findings. 
 
C-section 
The rate of C-section for delivery of preterm infants is between 31.0% and 36.7%. If a C-
section is indicated, AF, which is generally discarded as biological waste, can be safely 
collected in multiple ways. After abdominal and uterine incision, which will be performed 
according to standard practice, approximately 5 mL AF can be collected in a sterile conical 
tube. AF can be drained from the amniotic cavity before rupturing of the membranes using a 
sterile catheter, for example, a blunt plastic canula or catheter, which is inserted through the 
amniotic membranes and attached to a needleless sterile syringe. If collection of AF before 
rupturing of the amniotic membranes is not deemed feasible by the obstetrician, AF can be 
collected in a sterile tube after rupturing of the amniotic membranes. The different methods 
of collection are described in the Standard Operating Procedure (SOP) in Appendix A. 
 
The collection of AF through intact amniotic membranes has been described in various 
publications, none of which have reported complications (such as damaging the skin or eyes 
of the neonate or hampering the progress of the C-section). The amniotic membranes are 
transparent, making the position of the neonate in the uterus clearly visible. A selection of 
these studies, a description of their cohort, the used techniques for AF collection, and any 
reported complications is provided below (Tabel 3).  
 
The various approaches to collect AF during C-section were extensively reviewed with the 
obstetric department of the Amsterdam-UMC and the Máxima Medical Center. The staff 
members of the Amsterdam-UMC prefer using a cannula to collect AF due to previous 
positive experiences, while the staff members of the Máxima Medical Center prefer to use a 
container to collect AF after rupturing of the membranes, due to less experiences with 
cannula collection and a preference of “en caul” delivery of the infant. Both methods are 
deemed safe according to the available literature. Therefore, the physician performing the C- 
section can choose their preferred method. Arguably the collection through intact amniotic 
membranes minimizes contamination risk the most. The success rate of collection of AF (for 
both C- section and vaginal delivery) has to be closely monitored and evaluated at least 
yearly. If necessary, the number of intended inclusions may be altered based on the 
percentage of successful AF collection. 
 
 
 

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Table 3: an overview of studies that collected amniotic fluid through intact amniotic 
membranes prior to or during C-section.  
Referentie 
Cohort 
Methode  
Sample volume 
Complications 
(51) 
n = 17 samples, healthy 
term pregnancies. 
“Blunt end insertion 
with a catheter into 
amnion membrane” 
Median 70 mL, 
range 10-815 mL 
1 failed attempt, 
no complications 
(52) 
n = 79 samples, GA 37-42 
weeks (planned C-section = 
35 samples, secondary C-
section = 44 samples)   
“Direct syringe 
aspiration at time of 
cesarean section”” 
Unknown 
5 attempts without 
enough material, 
no complications 
(53) 
n = 47 samples (healthy 
pregnancies = 40 samples, 
mean GA 37.9 ± 1.8; 
preeclampsia = 7 samples, 
mean GA 36.1 ± 2.6).  
“Sterile acupuncture 
after myometrial 
incision before 
incision of amniotic 
membranes” 
Unknown 
No complications 
(54) 
n = 8 samples, GA 37 – 39 
weeks 
“Collected at 
cesarean delivery 
after entry into the 
uterus prior to rupture 
of amniotic 
membranes with blunt 
plastic cannula 
attached to 20 mL 
syringe’” 
10 mL 
No complications 
(55) 
N = 50 samples (preterm 
pregnancies = 25 samples, 
mean GA 32.6 ± 3.2; term 
pregnancies = 25 samples, 
mean GA = 39.9 ± 0.5).  
“Puncture of intact 
membranes using a 
22-gauge needle 
through dilated 
cervical cavity prior to 
artificial rupture of 
dilated cervical cavity” 
Unknown 
No complications 
(56) 
n = 24 samples, healthy 
term pregnancies. 
“At time of C-section 
by aspirating through 
intact amniotic 
membranes” 
Unknown 
No complications 
(57) 
n = 65 samples, GA 35 – 41 
weeks.  
“After uterotomy, by 
aspiration of amniotic 
fluid through intact 
amniotic membranes 
using a sterile 19G 
needle and 10mL 
syringe” 
Unknown 
No complications 
  

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All methods of collecting AF during preterm delivery have been described in the designated 
informed consent form (respectively, E1-E2b_PIF_1_preterm delivery) in both the Dutch and 
English language. AF samples will immediately be stored at 4-8 oC. Within 7 days after 
collection, AF will be aliquotted in 2 mL tubes and stored at -20oC for short term storage 
before being transferred to -80oC for permanent storage.  
 
IS-pro analysis  
 
The main approach to assess to the microbial composition of AF is the intergenic space 
profiling (IS-pro) method, which will be conducted in collaboration with the external party 
inBiome (Amsterdam, The Netherlands). The IS-pro technique utilizes the length and 
sequence variations within the 16S-23S rDNA interspace region (58). A standardized 
procedure will be performed. In short, approximately 1 to 2 ml of aliquotted AF is needed for 
IS-pro analysis. After bacterial DNA isolation, 10 µl of eluted DNA will be amplified using a 
standardized PCR reaction. Using two appropriate fluorescently-labeled forward primers and 
three universal unlabeled reverse primers, the phylum-specific interspace region will be 
amplified. The forward primers, contained within the FIRBAC mastermix 2.0 (inBiome, 
Amsterdam, The Netherlands) and PROTEO+IC mastermix 2.0 (inBiome, Amsterdam, the 
Netherlands), are targeted towards, respectively, the Firmicutes, Actinobacteria and 
Bacteroidetes phyla as well as the Proteobacteria phylum. Following the amplification 
process, 2.5 µl of the PCR product is prepared for DNA fragment analysis through addition of 
20 µl of eMix (inBiome, Amsterdam, the Netherlands). During the final step, IS fragments are 
separated and detected by capillary gel electrophoresis (CGE) using ABI Prism 3130XL 
Genetic Analyzer (Applied Biosystems). The resulting data consists of peak profiles with 
different colors relating to the different phyla and length signatures corresponding to specific 
species. Preprocessing and preliminary analysis will be performed using IS-pro software 
(Spotfire).  
 
16S rRNA analysis 
 
In addition to the IS-pro method, 16S rRNA sequencing will be conducted for a subset of the 
samples for validation purposes. After preprocessing, consisting of centrifugation at 12.000 x 
g for 20 min at 4oC to pellet the cells, the pellet will be resuspended in 1 mL sterile PBS, 
while the supernatant is saved and stored for analysis by other techniques. A standardized 
procedure will be performed, briefly explained below. Approximately 1 to 2 ml of aliquotted 
AF is needed for 16S analysis. After DNA extraction, the 16s rRNA gene sequences will be 
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preparation, sequencing, and analysis to identify and classify the bacterial strains present in 
the AF sample. Noteworthy, taking the main pathogens found in AF from existing literature 
into account, forward primers with adequate coverage of Ureaplasma spp. and Mycoplasma 
spp. will be used (59).  
 
(Un)targeted metabolomics  
 
To characterize the metabolic content, all AF samples will be analyzed using liquid 
chromatography-mass spectrometry (LC-MS) in collaboration with the Department of 
Laboratory Medicine (Amsterdam-UMC, location AMC, Amsterdam, The Netherlands). 
Dependent on the technique, up to 2 mL AF has to be available to perform analysis. Prior to 
analysis the AF samples are centrifuged at 3000xg for 20 minutes to separate the cells from 
the supernatant, the latter will be used to assess the metabolic content. The metabolite 
profiles of AF will be detected using an LC-MS/MS system. Either untargeted metabolomics 
will conducted on the collected samples or targeted metabolomics on a specific subgroup of 
interest drawn from current literature, for example amino acids and amino alcohols (20). 
 
Storage 
 
The remainder of the AF samples will be frozen at -80oC and kept for possible future studies, 
in line with the overarching aim of the present study, to increase our understanding of the 
impact of AF on the development of the fetal GIT and the initiation of the gut microbiome in 
extremely preterm infants. AF samples can potentially be studied  models of the fetal GIT.  
 
Phase 2:  the neonatal phase 
 
Neonatal clinical data and daily fecal samples during the first 28 days of life will be collected 
according to the protocol of the eMINDS study. In short, for all preterm infants born below 28 
weeks informed consent is obtained from both parents or legal guardians. Neonatal stool 
samples are collected daily in the first month of life. Subsequently, the stool samples are 
stored at  -20oC for short-term storage before they are transferred to -80oC for long-term 
storage until analysis. The following clinical data, as drawn from the eMINDS study protocol, 
will be collected during the first 28 days of life.    
 
• Standard demographic data, such as sex, gestational age, mode of delivery, birth 
 
month, APGAR score (1 and 5 min).  
• Information on maternal complications and medication during gestation. 

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• Neonatal medication (including antibiotics, surfactant, steroids, enema). 
• Total duration and type of parenteral nutrition. 
• Feeding type (breastfeeding or formula). 
• Invasive medical devices, type and duration (including respiratory tubes, peripheral 
 
iv’s, umbilical arterial or venous line, central venous catheter and/or arterial lines). 
• Anthropometrics (including length, weight, and skull circumference). 
• Potential diagnosis of Infant respiratory distress syndrome (IRDS), sepsis,  
 
intraventricular hemorrhage (IVH), BPD, and modified Bell’s staging for NEC will be 
 
noted. 
 
Relevant definitions: necrotizing enterocolitis and sepsis  
 
Of particular interest in this study are infants diagnosed with NEC or sepsis. The definition 
that is used for NEC is the modified Bell’s staging (60). All infants with NEC suspected by the 
treating clinician will retrospectively be reviewed and staged by two experts. At least the clini-
cal, radiographic, biochemical and, in some cases, pathological data will be thoroughly as-
sessed. All NEC cases with modified Bell stage > 2A will be considered a true NEC case. 
The definition for neonatal sepsis is used is according to the Vermont Oxford Criteria. Early 
and late-onset sepsis cases are defined as isolation of pathogen from blood culture drawn, 
respectively < 72h and  ≥ 72h postnatally and pathogen-specific antibiotic treatment that was 
continued for at least 5 consecutive days. An exception to this definition is sepsis caused by 
coagulase negative staphylococci (CoNS), which is often treated with shortened duration of 
antibiotics and may include removal of (central) line. CoNS sepsis is defined as a positive 
blood culture with CoNS species with clinical signs of infectious disease and/or a CRP value 
of at least 10 mg/L within 24 hours of suspicion of disease.  
 
The fecal samples are analyzed using various methods, including IS-pro and 16S rRNA 
analysis to assess the composition of the gut microbiome and LC-MS to assess the 
composition of the gut metabolome. For detailed explanation of the eMINDS study, the study 
protocol can be consulted (reference number: Metc2014.386(A2020.190)).  
 
Reference cohort 
 
While the goal of this study is not to determine GA-dependent changes in the metabolite 
composition of AF, the composition of AF is highly dependent on the GA and the construction 
of a database with AF samples from a reference population is necessary to adequately 
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the previously referenced studies by Bhatti et al. (2022) and Orczyk-Pawilowicz et al. (2016) 
important components of AF have been described in detail over the course of gestation, 
including the proteome and the metabolome (22, 23). While these studies serve as important 
reference publications for our reference cohort, we argue that these studies differ too much 
from our methodology. For example, no samples from very preterm and moderate to late 
preterm pregnancies are collected (28 + 0 – 36 + 6/7 weeks) and crucial information 
regarding maternal-fetal health is lacking. For that reason, we argue that setting up our own 
reference cohort is required for adequate interpretation of the findings regarding the 
composition of AF in extremely preterm pregnancies.  
 
AF will be collected from early midtrimester (< 23 + 6/7 weeks), very to moderate and late 
preterm (28 + 0 – 36 + 6/7 weeks), and full-term pregnancies (37 + 0 – 40 + 6/7 weeks). An 
overview of the samples that will be collected is provided in Table 1. In addition to the 
collection of AF, extensive maternal clinical data will be collected from the EPD to assess 
whether the sample is collected from a healthy pregnancy or, for example, a pregnancy 
complicated by FGR. This will be relevant for further analyses of the reference samples 
explained in more detail in section 10 “Statistical analysis”. No formal power analysis can be 
conducted due to limited knowledge on this topic. To get an idea of the changing composition 
over the course of gestation of potential discriminatory metabolites associated with extremely 
preterm birth, specifically in case of FGR and CAM, we aim to collect approximately 60 early 
midtrimester, 60 very preterm and moderate to late preterm, and 30 full term AF samples (n 
= 150). The in- and exclusion criteria are described in detail in section 4 “Study population”.  
 
Prior to collection of AF, the obstetric patient will be informed on the study, ideally by the 
member of the research team, and informed consent has to be obtained. AF will be 
withdrawn either transabdominally while monitoring with ultrasound (amniocentesis) or during 
delivery, as described in more detail in the section 8.3 (Collection of amniotic fluid during 
(preterm) delivery) and Appendix A. If there is a clinical indication to conduct an 
amniocentesis, a few milliliter of additional AF can be collected for research. In the case of 
left-over AF, which is not required for clinical diagnostics, the residual fluid can also be used 
as reference material. Amniocentesis will be performed conform the guideline of the medical 
center where the collection is taking place, posing no additional stress or risks for the 
participant of the study. Amniocentesis is generally performed in early midtrimester, between 
the 16th and 20th week of gestation. All AF samples that can be obtained through 
amniocentesis before the 24th week of gestation will be included in the reference cohort. After 
24 weeks of gestation, amniocentesis is generally not performed anymore as the infant is 
considered viable and any diagnostic findings do not have clinical implications anymore (for 

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example, termination of the pregnancy). In the unlikely case that amniocentesis is performed 
after the 24th week of gestation, this sample will not be included in the reference cohort. For 
very preterm to moderate and late preterm and full-preterm pregnancies, AF will be collected 
during vaginal delivery or C-section, similar to the collection in the study group with extremely 
preterm infants. Noteworthy, the amniocentesis procedure is only performed in the 
Amsterdam-UMC. Hence, all AF samples in the reference population from the Máxima 
Medical Center will be collected during vaginal or cesarean delivery. The 150 samples that 
are included in the reference cohort do not need to be evenly distributed across the two 
participating centers.  
 
Noteworthy, two separate informed consent forms have been constructed for collection of AF 
from the reference population as opposed to collection during preterm delivery (respectively, 
“E1-E2b_PIF_2_delivery reference group” for collection during delivery and “E1-
E2b_PIF_3_amniocentesis reference group” for collection by means of amniocentesis). Both 
forms are available in the Dutch and English language.  
8.3.1 Specific criteria for withdrawal  
 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. The investigator can decide to withdraw a subject from the study for urgent 
medical reasons. 
8.4 Replacement of individual subjects after withdrawal 
 
Not applicable 
8.5 Follow-up of subjects withdrawn from treatment 
 
Not applicable 
8.6 Premature termination of the study 
 
Due to the non-invasive nature of this study, we expect that premature termination of the 
study is highly unlikely. In the case of premature termination, the investigator will notify the 
accredited METC within 15 days, including the reasons for the premature termination (as 
described in detail in section 12.5). 
 
9. SAFETY REPORTING 
9.1 Temporary halt for reasons of subject safety 
 

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In accordance to section 10, subsection 4, of the WMO, the investigator will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise subject health 
or safety. The investigator will notify the accredited METC without undue delay of a 
temporary halt including the reason for such an action. The study will be suspended pending 
a further positive decision by the accredited METC. The investigator will take care that all 
subjects are kept informed.  
9.2 AEs, SAEs and SUSARs 
9.2.1 Adverse events (AEs) 
 
Due to the non-invasive nature of the study, which is limited to the collection of AF, which is 
generally discarded as biological waste, AEs or SAEs are not expected.  
9.2.2 Serious adverse events (SAEs) 
 
In the unlikely case of SAEs surrounding the collection of AF during delivery, the investigator 
will report this without undue delay after obtaining knowledge of the events. Since collection 
of AF is the only procedure in this study, there is no indication for registering SAE’s in the 
neonatal period (such as feeding intolerance or invasive ventilation). The investigator will 
report the SAEs through the web portal ToetsingOnline to the accredited METC that 
approved the protocol, within 7 days of first knowledge for SAEs that result in death or are 
life-threatening followed by a period of maximum of 8 days to complete the initial preliminary 
report. All other SAEs will be reported within a period of maximum 15 days after the 
investigator has first knowledge of the serious adverse events. 
9.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
 
Not applicable 
9.3 Annual safety report 
 
Not applicable 
9.4 Follow-up of adverse events 
 
All AEs will be followed until they have abated, or until a stable situation has been reached. 
Depending on the event, follow up may require additional tests or medical procedures as 
indicated, and/or referral to the general physician or a medical specialist. SAEs need to be 
reported till end of study within the Netherlands, as defined in the protocol  

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9.5 Data Safety Monitoring Board (DSMB) / Safety Committee 
 
Not applicable 
 
10. STATISTICAL ANALYSIS 
 
Clinical and demographical characteristics will be analyzed using Statistical Package for the 
Social Science (SPSS) version 26.0 (IBM, Armonk, NY). When deemed appropriate, either 
independent t-test (for normally distributed continuous variables), Mann-Whitney U test (for 
non-normally distributed continuous variables) or χ2 test (for categorical variables) will be 
used for the analysis of the clinical and demographical data of both maternal and neonatal 
data.  
10.1 Primary study parameter(s) 
 
For bacterial analysis, relative abundances will be assessed and compared between the 
groups of interest. Alpha-diversity will be calculated as the Shannon diversity index for each 
phylum as well as all phyla combined. Beta-diversity will be calculated using analysis of 
similarity in Bray-Curtis distance. For both bacterial as well as metabolomic data, principle 
coordinate analysis (PCoA) will be used to assess the variation in the composition of AF 
between samples and to visualize potential clusters of samples by metadata, specifically for 
the subgroups of infants exposed to IUI and infants with FGR. To give further insight into the 
key metabolites and bacterial species that are identified within the study population for FGR 
and CAM, the levels of these metabolites in the reference cohort will be assessed. The 
changes in the metabolite composition over the course of duration of pregnancy will be 
analyzed using mixed-effect models to account for both fixed effects (e.g. time) and random 
effects (e.g. individual variability). We aim to determine the temporal behavior of the 
proposed key metabolites in both healthy pregnancies as well as pregnancies complicated 
with FGR as these are heavily impacted by both duration of pregnancy as well as disease 
state. 
10.2 Secondary study parameter(s)  
 
To determine whether the clinical groups of interest can be distinguished from each other 
using the principle components an independent t-test will be conducted. To check for 
potential confounders (e.g. GA, BW, antibiotic use, etc.), the association between potential 
confounders and outcomes of interest will be assessed. If a potential confounder is identified, 
the association between the confounder and the content of interest will be tested by Fishers 
Exact test (for categorical variables) and ANOVA (for continuous variables). Logistic 

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regression models will be used to adjust for confounding variables. To assess the 
composition of AF in relation to the neonatal gut microbiome (e.g. Shannon diversity, 
microbial genera) for both microbial and bacterial components, associations will be tested by 
fitting generalized linear models of the microbiome feature on each component of interest, 
adjusting for confounding variables. Noteworthy, multivariate regression analyses require an 
adequate sample size and should be used cautiously due to the risk of overfitting and 
reduced statistical power, which can lead to biased findings.  
10.3 Other study parameters 
 
Not applicable 
10.4 Interim analysis  
 
Not applicable  
 
11.  ETHICAL CONSIDERATIONS 
11.1 Regulation statement 
 
The study will be conducted according to the principles of the Declaration of Helsinki 
(version, date, see for the most recent version: www.wma.net) and in accordance with the 
Medical Research Involving Human Subjects Act (WMO).  
11.2 Recruitment and consent 
 
The study will be conducted in the Amsterdam-UMC (Amsterdam, the Netherlands) and the 
Máxima Medisch Centrum (Veldhoven, the Netherlands), commencing in 2024 until the 
desired amount of mother-infant pairs as well as participants of the reference cohort have 
been included. A member of the research team, including research nurses, will approach the 
obstetric patients eligible for this study. Besides receiving a participant information form 
(PIF), patients will receive an oral explanation in either Dutch or English on all aspects 
deemed necessary to give informed consent. Information on collection of AF during delivery 
and use of personal and clinical data by researchers will be discussed. If it is not possible to 
obtain informed consent by a member of the research team, for example when the delivery 
occurs at night or on the weekends, the obstetric patients will be approached and informed 
by an obstetric doctor. If feasible, the informed consent will be asked by another obstetric 
doctor rather than their primary obstetrician.  
 
As preterm delivery is often unexpected and requires acute handling with extensive 
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always be possible to immediately obtain written informed consent prior to the collection of 
the AF sample. Due to the non-invasive and safe nature of the study, if it is not possible to 
obtain full written informed consent prior to delivery, AF may be collected during delivery by 
the obstetric doctor. However, written informed consent must be obtained from the 
participant prior to commencing the study, which entails the collection of clinical data or 
microbial or metabolic analysis of the AF sample. At any time during the study period, the 
study participant can refuse further continuation of the study and the study will be stopped 
immediately, which means that no clinical data may be collected anymore and stored AF 
samples will not be analyzed in the laboratory Stored AF samples will be destroyed unless 
study participant agrees with further analysis of the samples.  
 
Receiving informed consent from both parents and/or legal guardians regarding the 
collection of neonatal clinical data and neonatal fecal samples will occur according to the 
existing protocol of the eMINDS study (reference number: Metc2014.386(A2020.190)). In 
addition to the form that is signed by both parents and/or legal guardians to consent to 
participation in the eMINDS study, they will give additional consent to link the data from the 
eMINDS study to the data from the AMFIBIE study.    
11.3 Objection by minors or incapacitated subjects  
 
Not applicable 
11.4 Benefits and risks assessment, group relatedness 
 
Through participation in this study, participants can contribute to improving our 
understanding of the impact of AF composition on the development and colonization of the 
premature intestines through consenting to the collection of AF during delivery, which would 
otherwise be discarded as medical waste. AF can safely and non-invasively be collected 
during the standard care procedures of obstetric patients, without interfering with daily clinical 
care. Similarly, neonatal feces are normally disposed of when changing diapers and can 
easily be collected without interfering with standard care of the preterm infant. When deemed 
feasible by the obstetric doctor, the obstetric patients will receive counseling from their 
caregiver regarding participating in the study prior to giving birth to their preterm infant.  
 
Part of obtaining consent is informing the obstetric patients delivering their infants extremely 
preterm about potential neonatal morbidities, such as NEC and sepsis, which may arise 
during the neonatal period. General practice already consists of a consultation between the 
obstetric patient and a neonatologist when extreme preterm birth is expected, providing, 
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Hence, no additional stress is posed on the obstetric patients when informing them prior to 
delivery about this study. However, thorough and empathetic counseling by the physician 
obtaining informed consent is always required.  
 
A potential theoretical risk includes delay of delivery due to collection of AF. However, these 
risks are deemed minimal due to the non-invasive nature of collection of AF. The treating 
obstetrician is responsible for making a well-informed decision on determining whether it is 
feasible to safely isolate AF. If deemed necessary by the treating physician due to urgent 
medical reasons, the subject can always be withdrawn from the study. Additionally, as is the 
case with all research where access is provided to personal and clinical data, there is a small 
chance that confidentiality could be compromised. However, adequate precautions will be 
taken to minimize this risk. 
11.5 Compensation for injury 
 
The investigator has a liability insurance which is in accordance with article 7 of the WMO. 
Because of the non-invasive observational character of the study, no additional risks are 
expected from participation in this study. Therefore, the sponsor has obtained dispensation 
from the statutory obligation to provide insurance for the subject participating in the study, 
because participating in the study is without risks and therefore no insurance for the subjects 
is necessary 
 
11.6 Incentives  
 
Not applicable 

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12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1 Handling and storage of data and documents 
 
Every obstetric patients and their infant participating in this study are assigned a unique 
study number. The key for description is kept at the center of birth. Only the main 
researchers are able to access this encrypted information. All pseudo-anonymized data is 
recorded into an electronic case report form on CASTOR EDC platform. Collection of AF 
samples will be stored at -80oC, until further analyses. The same is true for feces, in line with 
the eMINDS study (reference number: Metc2014.386(A2020.190)). 
 
Data will be collected and processed in accordance with the General Data Protection 
Regulation (EU) 2016/679 and General Data Protection Regulation (GDPR). Data processing 
will be done coded, as the researchers can only identify the subject by use of a key that is 
solely available to the local research team. Data will be collected using an accredited 
electronic data capture system (CastorEDC), which is an application system that enables the 
collection and clean-up of study data using the Internet. All data will be stored for 15 years in 
a secured database on a secured computer, to which only the investigators will have access. 
Coded human material that is identified by the same study code as mentioned above, will be 
stored temporarily at the participating center after sampling. 
 
Depending on the inclusion rate, samples may subsequently be stored at the study site 
(Amsterdam UMC), before being shared with an external laboratory (InBiome, Amsterdam), 
where further analyses will be performed. We intend to perform microbial and metabolomic 
analyses on a yearly basis, but this may depend on the inclusion rate and could equally 
affect storage time at either the participating center or at the sponsor site. No other data (e.g. 
name, date of birth, health data) will be shared with InBiome. Personal data will not be 
inserted in one of the machines or programs involved in any of the analyses. After the 
analysis on the collected AF at InBiome, isolated bacterial genetic material will be stored for 
5 years locally to allow quality control only within the context of this research project. The 
results of the analyses on the AF will not be shared with the care providers involved in the 
treatment of the individual study participants. Coded data can be shared with international 
authorities (e.g. FDA) when this is requested by these authorities. 
 
12.2 Monitoring and Quality Assurance  
 

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Monitoring of the coordinated investigator will be done by a GCP-certified person otherwise 
not involved in the study. The monitor will provide a yearly written report to the coordinated 
investigator after each visit including a summary of the significant findings, deviations and 
deficiencies, conclusions, actions take, or recommendations to secure compliance. The coor-
dinated investigator will run consistency checks on a monthly basis and produce queries to 
be resolved by the local investigator(s). The final database will be obtained after the resolu-
tion of all queries. Due to the nature of our study and the minimal risk associated with the col-
lection of amniotic fluid, the establishment of a Data Safety Monitoring Board (DSMB) is not 
deemed necessary. 
 
12.3 Amendments  
 
Amendments are changes made to the research after a favourable opinion by the accredited 
METC has been given. All amendments will be notified to the METC of the Máxima Medical 
Center that gave a favourable opinion. Non-substantial amendments will not be notified to 
the accredited METC and the competent authority, but will be recorded and filed by the 
investigator.  
12.4 Annual progress report 
 
The investigator will submit a summary of the progress of the trial to the accredited METC 
once a year. Information will be provided on the date of inclusion of the first subject, number 
of subjects included and number of subjects that have completed the trial, serious adverse 
events/ serious adverse reactions, other problems, and amendments.  
12.5 Temporary halt and (prematurely) end of study report 
 
The investigator will notify the accredited METC of the end of the study within a period of 8 
weeks. The end of the study is defined one the estimated amount of amniotic fluid samples is 
collected.  
 
The investigator will also notify the METC immediately of a temporary halt of the study, 
including the reason of such an action.  
 
In case the study is ended prematurely, the investigator will notify the accredited METC 
within 15 days, including the reasons for the premature termination. 
 
 Within one year after the end of the study, the investigator will submit a final study report 

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with the results of the study, including any publications/abstracts of the study, to the 
accredited METC.  
12.6 Public disclosure and publication policy 
 
Publication will be in accordance with the basic principles of Central Committee on Research 
Involving Human Subjects (CCMO) statement on publication policy. Amsterdam UMC has full 
clearance to publish the results and no constraints are in place in light of the collaboration 
with InBiome or any foreign participating centers. 
 
13. STRUCTURED RISK ANALYSIS  
Not applicable 
  
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