The PROMOTE Study, a Pilot: The Characterization of the Microbiome in Pregnancy and Prediction of Pregnancy Outcomes
Conditions: Obesity, Maternal, Pregnancy Complications, Gut Microbiota
Sponsor: Erasmus Medical Center

NL80155.078.22 / OZBS72.21318/OMB No: 0925-0586   
PROMOTE  
 
 
 
 
 
 
 
 
 
 
 
 
 
The PROMOTE study, a pilot: The 
characterization of the microbiome in 
pregnancy and prediction of pregnancy 
outcomes. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Version 2.2 
June 15th, 2022 
 

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PROMOTE  
 
 
 
 
PROTOCOL TITLE ‘The PROMOTE study, a pilot: The characterization of the microbiome in 
pregnancy and prediction of pregnancy outcomes. 
 
Protocol ID 
NL80155.078.22 
OZBS72.21318 
Short title 
PROMOTE = Pregnancy Obesity Microbiome 
OuTcomEs 
EudraCT number  
Not applicable 
Version 
2.2 
Date 
June 15th, 2022 
Coordinating investigator/project 
leader 
 
S. Schoenmakers, MD, PhD 
Department of Obstetrics and Gynaecology 
Erasmus MC, Sophia 
E-mail: s.schoenmakers@erasmusmc.nl 
Principal investigator(s) (in 
Dutch: hoofdonderzoeker/ 
uitvoerder) 
Prof. R.P.M. Steegers-Theunissen, MD, PhD. 
Department of Obstetrics and Gynaecology 
Erasmus MC 
E-mail: r.steegers@erasmusmc.nl 
Sponsor  
Erasmus MC, Theme Sophia 
Department of Obstetrics and Gynaecology 
Subsidising party 
ZonMw grant; dossier number: 09120011910046 
Independent expert (s) 
Marijn Vermeulen 
Laboratory sites  
Clinical chemistry Laboratory Erasmus MC 
Pharmacy  
Not applicable 
 
 

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PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Head of Department of Obstetrics & 
Gynaecology: 
Prof. dr. E.A.P. Steegers 
 
 
 
Coordinating Investigator/Project 
leader: 
Dr. S. Schoenmakers 
 
 
 
Principal Investigator: 
Prof. dr. R.P.M. Steegers-Theunissen 
 
 
 
 
 

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TABLE OF CONTENTS 
 
1. 
INTRODUCTION AND RATIONALE ................................................................................. 9 
2. 
OBJECTIVES .................................................................................................................. 10 
3. 
STUDY DESIGN ............................................................................................................. 10 
4. 
STUDY POPULATION .................................................................................................... 10 
4.1 
Population (base) ..................................................................................................... 10 
4.2 
Inclusion criteria ....................................................................................................... 11 
4.3 
Exclusion criteria ...................................................................................................... 11 
4.4 
Sample size calculation ........................................................................................... 11 
5. 
TREATMENT OF SUBJECTS ........................................................................................ 12 
5.1 
Investigational product/treatment ............................................................................. 12 
5.2 
Use of co-intervention (if applicable) ........................................................................ 12 
5.3 
Escape medication (if applicable) ............................................................................ 12 
6. 
INVESTIGATIONAL PRODUCT ..................................................................................... 12 
6.1 
Name and description of investigational  product(s) ................................................ 13 
6.2 
Summary of findings from non-clinical studies ......................................................... 13 
6.3 
Summary of findings from clinical studies ................................................................ 13 
6.4 
Summary of known and potential risks and benefits ................................................ 13 
6.5 
Description and justification of route of administration and dosage ......................... 13 
6.6 
Dosages, dosage modifications and method of administration ................................ 13 
6.7 
Preparation and labelling of Investigational Medicinal Product ................................ 13 
6.8 
Drug accountability .................................................................................................. 13 
7. 
NON-INVESTIGATIONAL PRODUCT ............................................................................ 14 
7.1 
Name and description of non-investigational product(s) .......................................... 14 
7.2 
Summary of findings from non-clinical studies ......................................................... 14 
7.3 
Summary of findings from clinical studies ................................................................ 14 
7.4 
Summary of known and potential risks and benefits ................................................ 14 
7.5 
Description and justification of route of administration and dosage ......................... 14 
7.6 
Dosages, dosage modifications and method of administration ................................ 14 
7.7 
Preparation and labelling of Non Investigational Medicinal Product ........................ 14 
7.8 
Drug accountability .................................................................................................. 14 
8. 
METHODS ...................................................................................................................... 14 
8.1 
Study parameters/endpoints .................................................................................... 14 
8.1.1 
Main study parameter/endpoint ........................................................................ 14 
8.1.2 
Secondary study parameters/endpoints (if applicable) ..................................... 14 
8.1.3 
Other study parameters (if applicable) .............................................................. 15 
8.2 
Randomisation, blinding and treatment allocation ................................................... 15 
8.3 
Study procedures ..................................................................................................... 15 
8.4 
Withdrawal of individual subjects ............................................................................. 18 
8.4.1 
Specific criteria for withdrawal (if applicable) .................................................... 18 
8.5 
Replacement of individual subjects after withdrawal ............................................... 18 
8.6 
Follow-up of subjects withdrawn from treatment ...................................................... 18 

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8.7 
Premature termination of the study .......................................................................... 18 
9. 
SAFETY REPORTING .................................................................................................... 19 
9.2 
AEs, SAEs and SUSARs ......................................................................................... 19 
9.2.1 
Adverse events (AEs) ....................................................................................... 19 
9.2.2 
Serious adverse events (SAEs) ........................................................................ 19 
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) .......................... 19 
9.3 
Annual safety report ................................................................................................. 19 
9.4 
Follow-up of adverse events .................................................................................... 19 
9.5 
[Data Safety Monitoring Board (DSMB) / Safety Committee] .................................. 19 
10. STATISTICAL ANALYSIS ............................................................................................... 20 
10.1 
Primary study parameter(s) ..................................................................................... 20 
10.2 
Secondary study parameter(s) ................................................................................. 21 
10.3 
Other study parameters ........................................................................................... 21 
10.4 
Interim analysis (if applicable) .................................................................................. 21 
11. ETHICAL CONSIDERATIONS ........................................................................................ 21 
11.1 
Regulation statement ............................................................................................... 22 
11.2 
Recruitment and consent ......................................................................................... 22 
11.3 
Objection by minors or incapacitated subjects (if applicable) .................................. 23 
11.4 
Benefits and risks assessment, group relatedness .................................................. 23 
11.5 
Compensation for injury ........................................................................................... 24 
11.6 
Incentives (if applicable) .......................................................................................... 24 
12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ............................. 24 
12.1 
Handling and storage of data and documents ......................................................... 24 
12.2 
Monitoring and Quality Assurance ........................................................................... 27 
12.3 
Amendments ............................................................................................................ 27 
12.4 
Annual progress report ............................................................................................ 27 
12.5 
End of study report ................................................................................................... 27 
12.6 
Public disclosure and publication policy ................................................................... 28 
13. STRUCTURED RISK ANALYSIS ................................................................................... 29 
13.1 
Potential issues of concern ...................................................................................... 29 
13.2 
Synthesis ................................................................................................................. 30 
14. REFERENCES ................................................................................................................ 30 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
General Assessment and Registration form (ABR form), the application form 
that is required for submission to the accredited Ethics Committee; in Dutch: 
Algemeen Beoordelings- en Registratieformulier (ABR-formulier) 
AE 
Adverse Event 
CV 
Curriculum Vitae 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
IC 
Informed Consent 
METC  
Medical research ethics committee (MREC); in Dutch: medisch ethische 
toetsing commissie (METC) 
(S)AE 
(Serious) Adverse Event  
SUSAR Suspected Unexpected Serious Adverse Reaction 
GDM 
Gestational Diabetes Mellitus 
BMI 
Body Mass Index 
 
 

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SUMMARY 
 
Rationale: Our research aims to elucidate an underlying mechanism of maternal obesity 
(MOB)-related pregnancy and long-term health complications for mothers and their offspring. 
With the rising global prevalence of obesity, MOB-related pregnancy problems are 
increasingly occurring. Microbial gut symbiosis plays an important role in health, where 
dysbiosis is associated with diseases as obesity. Of interest, pregnancy, dietary patterns and 
pre- or probiotics affect the composition of the gut microbiome. The microbiome itself can 
influence many physiological processes, such as immune responses (production of microbial 
products) and the nutrient dependent one-carbon metabolism. We hypothesize that gut 
dysbiosis in MOB can be regarded as an endogenous chronic stressor inducing deranged 
immune responses and one-carbon metabolism. Both processes result in excessive 
oxidative stress, detrimental for cell multiplication, differentiation and epigenetic programming 
of maternal and offspring tissues. Together, these biological derangements contribute to 
placental and vascular dysfunction resulting in increased risks of preeclampsia or gestational 
diabetes mellitus. Vertical (during pregnancy) and horizontal (during delivery) transfer of gut 
dysbiosis from mother to new-born, and epigenetic placental and fetal changes can 
ultimately accumulate in macrosomia and childhood obesity.  
 
Objective:  
Research question: Is maternal microbiome dysbiosis in MOB an underlying mechanism in 
the pathophysiology of adverse maternal pregnancy and offspring outcome? 
 
Objectives:  
Analyse the differences between the gut and vaginal microbiome, maternal and fetal immune 
response and one-carbon metabolism in normal weight and obese pregnant women.  
 
Study design: A single center prospective longitudinal observational cohort pilot study 
embedded in the Rotterdam periconception cohort (Predict study, METC 2004-227) will be 
performed periconceptional/early in the first trimester and continue until the 
delivery/postpartum at the Erasmus MC. 
 
Study population: The Predict study population includes all pregnant women > 18 years 
and < 45 years who are willing to participate. Two participant groups will be included: 50 
(pregnant) women with a BMI > 30 kg/m2 (cases) and 50 (pregnant) women with a BMI: > 18 
and < 25 kg/m2 (controls) and their neonates (n = 100). In addition, 10 preconceptional 

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women with BMI > 30 kg/m2 and 10 preconceptional women with BMI 18-25 kg/m2 will be 
included. Making a total of 220 participants. 
 
Intervention: Not applicable. 
 
Main study parameters/endpoints: The composition of the maternal gut and vaginal 
microbiome (in obese women (BMI >30 kg/m2) and non-obese women (BMI 18-25 kg/m2)).  
 
Secondary study parameters: The associations between the composition of the 
microbiome and: 
1) Clinical maternal outcomes (e.g. pregnancy outcome, gestational age at delivery, 
preeclampsia, hypertension, gestational diabetes) 
2) Clinical fetal outcomes (e.g. fetal growth trajectories in first, second and third 
trimester, birthweight) 
3) Maternal (and fetal) immune response and one-carbon metabolism (e.g. inflammatory 
and biomarkers) 
4) Placental function (e.g. placental vascular architecture and volume during the 1st 
trimester, placental weight, inflammatory status) 
 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: For all cases, the risks involve primarily the burden of participating in a 
study, which usually means additional hospital visits and assessments. There will be a 
maximum of 6 hospital visits, which will take approximately 30-45 minutes each. There will 
be two/three first trimester vaginal ultrasound examinations, two abdominal ultrasound 
examinations during the second and third trimester and 1 postpartum visit (all part of the 
Predict study, METC 2004-227). At a maximum of 5 of the appointments (periconceptional, in 
the first, second, third trimester, postpartum) blood draws and rectal swabs will be obtained. 
The first blood draw is combined with the blood draw of the Predict study, the three/four 
consecutive blood draws are additional for the PROMOTE study, as are the rectal swabs. 
Maternal anthropometrics, including weight, height and blood pressure will be measured 
preconceptionally or during the first trimester (as part of the Predict study). The risks of 
participation are considered to be nil and the potential benefit outweighs the risks. From the 
above explanations, it is clear that there are no obvious risks associated with participation in 
the study. After each visit, the participant will receive a picture of their fetus obtained by 3D 
or 2D ultrasound, if it is possible to generate one.  

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1. INTRODUCTION AND RATIONALE 
 
Our research aims to elucidate the underlying mechanisms of maternal obesity (MOB)-
related pregnancy and long-term health complications for mothers and their offspring. In 
Europe around 16,5% of all people is obese (BMI > 30 kg/m2), in the Netherlands this is 
percentage is around 15%, and the region of Rotterdam around 18%-20% (RIVM, 2020). 
With a total of ~170.000 pregnancies in the Netherlands in 2020 (RIVM), and one in every 5-
6 pregnant woman being obese, this results in 25.000-35.000 MOB related pregnancies per 
year in the Dutch reproductive population (1,2). MOB is a significant risk factor for maternal 
complications, such as gestational diabetes mellitus (GDM), preeclampsia (3) and adverse 
birth outcomes, such as macrosomia with neonatal obesity extending in 2/3 of these babies 
into later life (4,5). MOB thus has a devastating impact on health, quality of life and 
healthcare costs (6-8). In order to break the vicious cycle of MOB, we will investigate the 
impact of derangements of the maternal gut microbiome (dysbiosis) in MOB women, during 
the periconceptional period, pregnancy and delivery as an underlying mechanism of the 
pregnancy and fetal complications.  
Microbial gut symbiosis plays an important role in health (9), whereas dysbiosis is associated 
with diseases as obesity (10) and can be induced by an unhealthy diet, lifestyle, toxins or 
medication (11). Interestingly, also pregnancy (12,13) and pre- or probiotics (14) affect the 
gut microbiome. The microbiome itself can influence many physiological processes, such as 
immune responses (for instance by production of microbial products) (15) and the nutrient 
dependent one-carbon metabolism (supply of substrates and cofactors by the microbiota) 
(16,22). Obesity is associated with gut dysbiosis, also during pregnancy (10). The first 
evidence for a causal link between obesity and dysbiosis was derived from transplanting 
intestinal microbiome from obese mice into germfree mice resulting in replication of the 
disrupted obese metabolism in germfree mice (19). Similar results were obtained by 
transplanting human obese faeces to germfree mice (20). Moreover, faecal microbiota from a 
lean human donor to an obese human host resulted in decreased host insulin sensitivity (21). 
In addition, obesity is associated with derangement of the immune response (f.i. general low-
grade inflammation) and in the one-carbon metabolism (22,23).  
We therefore hypothesize that gut dysbiosis in MOB is an endogenous chronic stressor 
inducing deranged immune responses and one-carbon metabolism. Both processes result in 
excessive oxidative stress, detrimental for cell multiplication, differentiation and epigenetic 
programming of maternal and fetal tissues (24,25). Together, these biological derangements 
contribute to placental and vascular dysfunction resulting in increased risks of preeclampsia 
(26) or GDM (27). Although, gut colonization mainly starts at delivery by horizontal transfer of 

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the maternal vaginal and faecal microbiome to the new-born (28), during pregnancy, 
microbial products are already transferred to the fetus (29). Hence, maternal dysbiosis in 
MOB will be passed to the fetus and new-born, with adverse consequences for the offspring 
gut microbiome. The transfer of dysbiosis, together with adverse fetal and placental 
epigenetic changes may explain the health repercussion of MOB for offspring in later life, i.e., 
obesity “inheritance”. Our hypothesis of the role of gut dysbiosis in MOB is substantiated by 
various studies showing that treatment with prebiotics, such as fructooligosaccharides (FOS) 
and galactooligosaccharides (GOS), or probiotics (Lactobacillus, Bifidobacterium) stimulate 
the restoration of the microbiome resulting in an improved immune responses and one-
carbon metabolism (30) and decreased oxidative stress (31). In addition, these pre-
/probiotics reduce bodyweight and fat mass in obese mice and humans (32), whereas animal 
studies show that these treatments also have positive effects on the offspring (33,34). From 
this background it is our ultimate goal to contribute with this pilot study to better 
understanding, treatment and prevention of MOB-related maternal and fetal complications. 
2. OBJECTIVES 
 
Research question:  
Is maternal microbiome dysbiosis in MOB an underlying mechanism in the pathophysiology 
of adverse maternal pregnancy and offspring outcome? 
 
Objective:  
1) Analyse the differences between the gut microbiome, maternal and fetal immune response 
and one-carbon metabolism in obese vs normal weight pregnant women.  
 
3. STUDY DESIGN 
 
A single center prospective longitudinal observational pilot cohort study, embedded in the 
Rotterdam periconception cohort (Predict study, METC 2004-227) will be performed 
periconceptional/early in the first trimester, during pregnancy and postpartum, at the 
Erasmus MC. The setting will be mainly at the outpatient clinic, partly at the delivery ward 
and partly at home. The study schedule is planned for the period of 48 months.  
 
4. STUDY POPULATION 
4.1 Population 
This pilot study is embedded in the Rotterdam Periconceptional Cohort study (Predict 
study), a study that was set up in 2004 and is still ongoing. In total, 2846 women were 

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included up until June 2021 (35). The Predict study population includes preconceptional 
or pregnant women > 18 years and < 45 years old visiting the Erasmus MC and that are 
willing to participate. We will select the participants, that meet our undermentioned 
inclusion criteria, for our study from this cohort: 50 women with a BMI > 30 kg/m2 (cases) 
and 50 women with a BMI ranging 18-25 kg/m2 (controls) and their neonates. 
Given the fact, that the number of inclusions per year in the Predict study is around 235 it 
is likely that we will achieve our sample size. We will longitudinally sample 
first/2nd/3rd/postpartum, 100 women, in addition we will include 10 preconceptional women 
with BMI > 30 kg/m2 and 10 preconceptional women with BMI 18-25 kg/m2, these 
participants do not necessarily have to conceive a pregnancy in order to remain in the 
study (these women are part of the preconceptional Predict population). 
 
4.2 Inclusion criteria 
For eligibility to participate in this study, a subject must meet all of the following criteria: 
 
Participation in Predict study 
 
Preconceptional women who wish to become pregnant or pregnancy <13 weeks 
of gestational age. 
 
BMI > 30 kg/m2 or 18-25 kg/m2 
 
Understanding of Dutch in speaking and reading 
 
Willingness to give written informed consent 
4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from 
participation in this study: 
 
Age < 18 years and > 45 years. 
 
≥13 weeks of gestational age 
 
Multiple pregnancy 
 
Smoking 
 
Gastro-intestinal diseases, heart diseases, liver, pancreas and kidney diseases. 
 
Use of antibiotics < 2 weeks before sampling 
 
Pre-existent diabetes mellitus 
 
Unable or unwilling to give informed consent 
4.4 Sample size calculation 
Since this is a pilot study, exploratory power calculations are not appropriate at this stage. 
We will include 50 (pregnant) women with a BMI > 30 kg/m2 (cases) and 50 (pregnant) 
women with a BMI 18-25 kg/m2 (controls) and their neonates (n =100). And 20 

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preconceptional women with BMI of 18-25 kg/m2 (n=10) and BMI > 30 kg/m2 (n=10). 
Furthermore, performing a power calculation would require prior estimates of all 
parameters in a model explaining the relation between the measurements and the 
outcomes, which is the goal of this study. 
 
5. TREATMENT OF SUBJECTS 
5.1 Investigational product/treatment 
Not applicable. 
5.2 Use of co-intervention  
Not applicable. 
5.3 Escape medication  
Not applicable. 
6. INVESTIGATIONAL PRODUCT  
 

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6.1 Name and description of investigational  product(s) 
Not applicable. 
6.2 Summary of findings from non-clinical studies 
Not applicable. 
6.3 Summary of findings from clinical studies 
Not applicable. 
6.4 Summary of known and potential risks and benefits 
Not applicable. 
6.5 Description and justification of route of administration and dosage 
Not applicable. 
6.6 Dosages, dosage modifications and method of administration 
Not applicable. 
6.7 Preparation and labelling of Investigational Medicinal Product 
Not applicable. 
6.8 Drug accountability 
Not applicable. 
 

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7. NON-INVESTIGATIONAL PRODUCT 
7.1 Name and description of non-investigational product(s) 
Not applicable. 
7.2 Summary of findings from non-clinical studies 
Not applicable. 
7.3 Summary of findings from clinical studies  
Not applicable. 
7.4 Summary of known and potential risks and benefits  
Not applicable. 
7.5 Description and justification of route of administration and dosage 
Not applicable. 
7.6 Dosages, dosage modifications and method of administration 
Not applicable. 
7.7 Preparation and labelling of Non Investigational Medicinal Product 
Not applicable. 
7.8 Drug accountability  
Not applicable. 
 
8. METHODS 
8.1 Study parameters/endpoints 
8.1.1 Main study parameter/endpoint 
The composition of the maternal gut microbiome in obese women (BMI >30 kg/m2) 
and non-obese women (BMI 18-25 kg/m2) during the preconceptional period, 
pregnancy and postpartum period. 
8.1.2 Secondary study parameters/endpoints 
The associations between the composition of the microbiome and: 
1) Clinical maternal outcomes (e.g. pregnancy outcome gestational age at delivery, 
preeclampsia, hypertension, gestational diabetes) 
2) Maternal conditions and lifestyle (e.g. diet, medication use, working activities, 
physical activity, intoxications) 

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3) Clinical fetal outcomes (e.g. growth trajectories in first, second and third 
trimester, birthweight) 
4) Maternal (and fetal) immune response and one-carbon metabolism (e.g. 
inflammatory and biomarkers) 
5) Placental function (e.g. placental weight, inflammatory status) 
8.1.3 Other study parameters 
Not applicable 
 
8.2 Randomisation, blinding and treatment allocation 
Not applicable 
 
8.3 Study procedures 
This paragraph describes the investigations and procedures that participants will 
undergo in the detailed scheme followed by an extensive description of procedures. 
8.3.1 
Detailed schedule of visits and assessment of the study: 
 
Preconceptional 1st trimester 
2nd & 3rd 
trimester 
Delivery 
Postpartum 
Optional (n=20) 
 
 
 
 
1 visit 
2-3 visits: 7,9,11th  
week GA 
1 visit: 22-24th and 
30-32th week GA 
 
>8 weeks after 
delivery 
 
 
 
 
 
 
3D Ultrasound scan 
(2x vaginal) 
2D Ultrasound scan 
(2x abdominal) 
 
 
Blood sampling 
Blood sampling (1x) 
Blood sampling 
Blood sampling 
Blood sampling 
Anthropometric 
measurements 
Anthropometric 
measurements (1x) 
 
 
 
Vaginal – rectal 
sampling 
Vaginal – rectal 
sampling (1x) 
Vaginal – rectal 
sampling (2x) 
Vaginal – rectal 
sampling  
Vaginal – rectal 
sampling 
2 questionnaires 
diet and lifestyle 
2 questionnaires 
diet/lifestyle (1x) 
1 questionnaire (1x) 
Placental biopsies 
 
1 questionnaire 
Inclusion in online 
platform 
‘SlimmerZwanger’ 
 
 
Umbilical cord 
blood sampling 
 
 
 
 
Meconium 
sampling 
 
 

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8.3.2 
Extensive description of study procedures 
 
 
Since this study is embedded in the Predict study, below is listed what 
measurements are additional for the PROMOTE study and which measurements 
are already part of the Predict study. 
 
Clinical maternal outcomes (Predict): This information will be obtained via 
questionnaires included in the research module or by electronic medical records. 
 
Maternal conditions and lifestyle (Predict): one/two extensive questionnaires about 
lifestyle, diet and environmental factors must be completed preconceptionally/in the 
first trimester, one general questionnaire in the second trimester at 24 weeks of 
gestation and during postpartum period. These questionnaires are fully digital, by 
using the GemsTracker program.  
 
Anthropometric measurements (Predict): height, weight, chest circumference, waist 
circumference and hip circumference are measured during the intake. 
 
Maternal blood samples (Predict/PROMOTE study): Blood sampling will take place 
at the outpatient clinic after the intake. The laboratory tests described down are in 
addition to the tests of the Predict study (leukocytes differential count and 
Homocysteine). For the PROMOTE study we will assess metabolic indicators such 
as glucose, insulin, cholesterol, HDL, LDL, lipids (standard methods), cytokines 
such as TNF α and IL6 (using Luminex), hsCRP (ELISA), measurements for 
oxidative stress such as total antioxidant capacity, protein oxidation products; 
carbonyl protein and advanced oxidation protein products (AOPP)) and parameters 
of the one-carbon metabolism including folate, homocysteine and vitamin B12. 
Part of the maternal and fetal (cord) blood samples will be prepared for staining in 
Rotterdam and frozen (in DSMO in liquid nitrogen) where after they are transported 
on dry ice to the UMCG in Groningen. Here they will be incubated with various 
panels of antibodies to study monocyte (subsets and activation status), granulocytes 
(activation status) and T cells (subsets and activation status). After antibody 
incubation, cells will be prepared for flow cytometry. 
 
Neonatal cord blood/meconium sampling: the collection of neonatal cord blood will 
be performed at the labour ward, by the obstetrician on duty (as part of the Predict 
study). Measurements that will be assessed are extensively described at the 

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maternal blood samples. Meconium, as part of the PROMOTE study, will also be 
sampled at this time point in the study. 
 
Placental tissue: Placental biopsies will be collected at labour ward and afterwards 
snap frozen. Presence of immune cells in decidua and oxidative stress will be 
characterized by immunohistochemistry. mRNA and protein will be isolated for gene 
(including epigenetics) or protein expression analysis (Dep of Reproduction and 
Development, prof J.Gribnau, Erasmus MC) for instance markers of the one-carbon 
metabolism, immunological markers, vascular markers, cytokines, markers of 
oxidative stress. 
 
Vaginal/Faecal sampling/Microbial sequencing: To analyse the composition and 
differentiation of the maternal microbiome: maternal rectal and vaginal samples will 
be collected by the women during their study visits at the hospital (1st trimester, 2nd 
trimester and third trimester, during delivery and during postpartum checkup). The 
bacteriome profiles will be assessed by 16SrRNA gene amplication sequencing by 
the department of microbiology to analyse the composition of the maternal 
microbiome. Bacterial 16S rRNA gene sequences covering variable regions (V6–V8) 
will be PCR amplified from purified genomic DNA by using the standard primers. 
Sequences will be assigned to OTUs (operational taxonomic units). 
Virome sampling will be performed during clinical visits, and will be stored and 
analysed according to standardized protocols by the department of Viroscience 
Erasmus MC (Prof. Dr. Koopmans). 
8.3.3 
Standard procedure of ultrasound scans 
At 7, 9 and 11 weeks GA the gestational sac, embryo, and placenta are depicted in 
3D ultrasound scans using a GE Voluson E8 or E10 Expert system and 4D View 
software (GE Medical Systems, Zipf, Austria). At 22 weeks and 30 weeks of GA 
standard biometry; Doppler measurements: arteria umbilicalis, arteria media 
cerebralis and arteria uterine will be performed. 
In this study transvaginal scanning time (during first trimester) and transabdominal 
scanning time (second and third trimester) will be kept as short as possible. The 
duration of the scanning time per ultrasound will not exceed 30 minutes in the first 
trimester and 40 minutes in the second and third trimester.  
All scans will be made with standard settings of the ultrasound machine: pulse 
repetition frequency of 0.6 kHZ, gain -2.0, quality “high’’, wall motion filter ‘’low’’.  
The obtained 3D datasets are stored as Cartesian (rectangular) volumes.  

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Pulsed wave Doppler is a standardized additional modality of ultrasound imaging to 
quantify blood flow. Blood flow will be quantified as expressed by the resistance 
index (RI) as well as by the pulsatility index (PI). Hence, the use of pulsed wave 
Doppler signal during ultrasound enables us to perform a non-invasive 
measurement of the blood flow and subsequently to detect changes in flow. All the 
ultrasounds are part of the Predict study. 
8.3.4 
 Sample collection 
Serum, rectal and vaginal samples will be obtained during preconceptional, first 
trimester, second trimester, third trimester, during delivery and after postpartum 
period. All samples will be processed and stored at each setting according to 
standardized protocols. 
 
8.4 Withdrawal of individual subjects 
Subjects may decide to withdraw from 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. 
 
8.4.1 Specific criteria for withdrawal  
Not applicable 
 
8.5 Replacement of individual subjects after withdrawal 
Not applicable 
 
8.6 Follow-up of subjects withdrawn from treatment 
Not applicable 
 
8.7 Premature termination of the study 
Not applicable 
 
 

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9. SAFETY REPORTING 
9.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise subject 
health or safety.  The sponsor 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) 
The interventions performed in this pilot study are blood collection, vaginal and 
rectal swab collection. 
  
Adverse events are defined as any undesirable experience occurring to a subject 
during the study, whether or not considered related to an intervention. All adverse 
events reported spontaneously by the subject or observed by the investigator or his 
staff will be recorded. 
9.2.2 Serious adverse events (SAEs) 
Not applicable. 
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 
Not applicable. 
 
9.5 Data Safety Monitoring Board (DSMB) / Safety Committee 
Not applicable. 

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10. STATISTICAL ANALYSIS 
10.1 Primary study parameter(s) 
Since this study is a pilot, including 60 obese and 60 non-obese women, exploratory 
power calculations are not performed at this stage. Furthermore, performing a power 
calculation would require prior estimates of all parameters in a model explaining the 
relation between the measurements and the outcomes, which is the goal of this study. 
 
Descriptive statistics will be presented for maternal and lifestyle conditions and such as 
but not limited to; medication use, intoxications, infections, physical activity, working 
activities, blood pressure, nutrition, smoking, alcohol, and folic acid supplement use to 
give an overview of the data. We will use mean and standard deviation for variables that 
are approximately normally distributed, median and interquartile rand for variables that do 
not appear to have a normal distribution. 
 
We will investigate the microbiome by using different analyses. The alpha-diversity 
(microbiome within a sample) will be described using the Shannon-index. We will use 
univariate analysis to describe and compare alpha-diversity per person and per analysis 
group (normal BMI and BMI >30) by chi-square test. These descriptions will be presented 
and compared at different time points (preconceptional, 1st trimester, 2nd trimester, 3rd 
trimester, postpartum). 
 
The alpha diversity will further be analysed using a marginal model for repeated 
measurements (fitted using generalized least squares). BMI-group, time and their 
interaction will be included as covariates as well as the potential confounders ('parity, age, 
mode of conception, infection, and fetal gender'). Several common variance-covariance 
structures will be used and the one that achieves the best fit according to Akaikes 
information criterion will be used for further analyses. A likelihood ratio test will be used to 
test the hypothesis that the evolution of the alpha diversity differs between the BMI 
groups. 
 
Beta diversity (variation of microbial communities between the samples) will be examined 
per time unit (possibly preconceptional, 1st trimester, 2nd trimester, 3rd trimester, 
postpartum), per person and between the 2 groups of persons (normal BMI and BMI > 30) 
at the different time units (1st trimester, 2nd trimester, 3rd trimester, postpartum). The 
possible changes in microbiome over time in the different BMI groups will be analysed 
with a confidence interval based on bootstrapping. Potential confounders (parity, age, 

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mode of conception, infection, and fetal gender) will be also corrected for. Missing data 
will be handled by multiple imputation. A p-value <0.05 will be considered statistically 
significant unless otherwise stated.  
 
10.2 Secondary study parameter(s)  
We will investigate the association between the composition of the microbiome and the 
maternal, fetal and pregnancy outcomes by using a multivariate linear or logistic 
regression model. A multivariable linear regression is used for the association between 
the microbiome (in alpha and beta diversity) and the continuous outcomes (birth weight, 
placenta weight, immune response and one-carbon metabolism (markers with continuous 
outcome). The multivariable logistic regression model is used for the association between 
the microbiome and the dichotomous outcome measures: (hypertension yes/no, pre-
eclampsia yes/no, gestational diabetes yes/no).  
The association between the composition of the microbiome (alpha and beta-diversity) at 
different timepoints (preconceptional, 1st trimester, 2nd trimester, 3rd trimester and 
postpartum) and fetal growth (at 1st, 2nd and 3rd trimester), will be analyzed by a linear 
mixed effect model (repeated measurements in the same person). We stratify for normal 
BMI and BMI>30 and also include BMI among the potential confounders (parity, age, 
mode of conception, infection and fetal sex). 
Continuous, normally distributed variables will be presented as mean with standard 
deviation, and variables with a skewed distribution as median with the range. Categorical 
variables will be presented as count and proportions. The independent samples t-test and 
Mann-Whitney U test will be used for continuous data. Two-sided p-values less than 0.05 
will be considered statistically significant. 
 
10.3 Other study parameters 
Not applicable. 
 
10.4 Interim analysis 
Not applicable. 

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11.  ETHICAL CONSIDERATIONS 
 
11.1 Regulation statement 
The study will be conducted according to the principles of the Declaration of Helsinki 
(version October 2013) and in accordance with relevant national guidelines, regulations 
and Acts (e.g., for Erasmus MC the Medical Research Involving Human Subjects Act 
(WMO). 
 
11.2 Recruitment and consent 
For recruitment of high risk groups of participants and their (unborn) children we will use 
the infrastructure of the preconception, early pregnancy and reproductive life planning 
clinics of Erasmus MC. Participants can enter the study during the preconception period 
and/or at <13 weeks GA.  
Women contemplating pregnancy or being pregnant and visiting the preconception and 
reproductive life planning clinics at Erasmus MC will be informed by their clinician and 
invited to participate in the cohort. Participation is fully voluntary. Participants will be 
informed about the voluntary nature of their participation by their clinician as well as by 
means of the information leaflet and informed consent form. Full comprehensive 
information will be given to all participants according to the regulations of the authorities 
that need to approve the clinical studies. Subjects that will be addressed are: 
- research objectives 
- the nature, extent and duration of the procedures 
- details of the additional risk and burden caused by the research project and the 
protective measures to prevent or address the potential problems 
- possible adverse events 
- voluntary participation 
- the opportunity to ask questions and receive understandable answers before making a 
decision 
- who will benefit from participation 
- the procedures that will be implemented in case of incidental findings 
- how their data will be collected, protected during the project and either destroyed or 
reused at the end of the research 
- being able to withdraw themselves and their data from the project at any time 
- any potential commercial exploitation of the research 
 

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Informed consent: 
The study will be performed according to written informed consent procedures and the 
study protocol approved by the METC Erasmus MC. The informed consent procedures 
ensure personal data protection and confidentiality. Data will be coded, i.e. information will 
not be directly traceable to an individual person. 
Written information will be presented clearly, using short sentences and either avoiding or 
explaining all technical terms. Information sheets will be provided in the participants’ own 
languages. Participants will be informed about the study according to their understanding 
and need to provide written consent to participate, only after a full explanation has been 
given and understood, an information leaflet offered and time allowed for consideration. In 
case of inclusion during pregnancy, additional signed consent regarding the use of the 
child’s data is requested from both parents (i.e. the legal representatives of the child). The 
right for the participant (and/or the legal father) to refuse to participate without giving 
reasons will be respected, they are free to withdraw at any time from the protocol 
treatment without giving reasons and without prejudging further treatment. 
 
11.3 Objection by minors or incapacitated subjects 
Not applicable 
 
11.4 Benefits and risks assessment, group relatedness 
Information gained by ultrasound will provide doctors and parents with information. It is 
also hoped that the information gained from this study will be valuable to future parents to 
assess the influence of obesity on the pregnancy and outcomes.  
Risks: Worldwide ultrasounds are routine antenatal care. Following specifications as set 
by ISUOG, the assumed risks with respect to ultrasound examinations and phlebotomy 
are very minimal. 
 
For all participants the risks involve primarily the burden of participating in a study, which 
means additional hospital visits and additional assessments. All efforts will be taken to 
limit the number of visits and assessments and to harmonize the visits and assessments 
with usual clinical care. The risks of participation are considered to be minor and the 
potential benefit outweighs the risks. A detailed risk assessment will be included in the 
study protocol, informed consent documents and other study documents delivered to the 
ethic committees. From the above, it is clear that there are no obvious risks associated 

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with participation in the study and that participants included will benefit from receiving 
more clinical care. 
11.5 Compensation for injury 
The Erasmus MC has a liability insurance which is in accordance with article 7 of the 
WMO. 
The Erasmus MC (also) has an insurance which is in accordance with the legal 
requirements in the Netherlands (Article 7 WMO). This insurance provides cover for 
damage to research subjects through injury or death caused by the study. The insurance 
applies to the damage that becomes apparent during the study or within 4 years after the 
end of the study. However, as participation in this study involves, at most, a negligible risk 
for injury the METC Erasmus MC imparted a waiver for this insurance.  
 
11.6 Incentives  
Study participants will receive 2D and 3D ultrasound examinations and pictures, if 
possible, as compensation for their participation. 
 
12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1 Handling and storage of data and documents 
 
Individually identifiable private information 
Individually identifiable private information will only be accessible to clinicians involved in 
the clinical care of the participant, the research team, the study monitor, and IGJ 
(Inspectie Gezondheidszorg en Jeugd). Importantly, no contact details will be included in 
datasets that are obtained from each participant and no identifying details will leave the 
participating centre. 
 
Protection of data 
The Erasmus MC will provide data in a format using a common data model that will be 
defined upfront. Data will be de-identified (pseudo-anonymised) in order to guarantee the 
privacy of the participants, by assigning a study-ID. We will keep a (strictly confidential) 
mapping from their local patient ID to the study-ID. 
 
Safety of assessments 
The as-low-as-reasonable achievable (ALARA) principal has been recommended in 
statements of the Food and Drugs Administration (FDA), American Institute of Ultrasound 
in Medicine (AIUM), World Federation of Ultrasound in Medicine and Biology (WFUBM), 

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International Society of Ultrasound in Obstetrics and Gynaecology (ISUOG), European 
Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) and Australian 
Society for Ultrasound in Medicine (ASUM) regarding medical use of ultrasound. 3D 
ultrasound imaging is not associated with higher risks than two-dimensional (2D) B-mode 
ultrasound, as 3D is a post-processing procedure using captured fields. Off-line 
evaluation of stored 3D images furthermore reduces the duration of exposure time in 
comparison to the use of real time 2D. At present Power Doppler ultrasound is not used 
routinely in clinical practice. When performing Doppler ultrasound in the first trimester of 
pregnancy, the displayed TI should be ≤ 1.0 and exposure time should be kept as short 
as possible (usually no longer than 5 -10 min) and should not exceed 60 min. The 
proposed study will comply with above mentioned recommendations. 
 
Security of personal data 
The study-consortium considers the safety and security of the personal and individual 
data obtained and stored in the study-database hosted by participant Erasmus MC to be 
most important. All data will be collected, stored, processed and disclosed in compliance 
with national regulations. Data transfers within the EU/EEA are not subject to specific 
requirements (i.e. specific authorizations or other restrictions) when complied with the 
general requirements of Directive 95/46/EC. All national laws and regulations (e.g. Code 
of conduct on health research and Personal Data Protection Act) on data protection will be 
respected. 
 
Storage of data 
All data entered by the participants will be stored in the database hosted by Erasmus MC. 
All imaging data will be stored and handled in the same way and according to the same 
rules and regulations as other patient related imaging data at Erasmus MC. It will be 
stored as files on a separate research storage platform. Access will be limited to 
authorized medical personnel. Should data transfer to other parties within the study-
consortium be necessary for testing or validation purposes, we will code the datasets and 
remove all personal data before the transfer. 
 
Privacy of data 
The data will always be handled confidentially and coded using a random identification 
code. The key to this code will be kept separately from the dataset at all times. If datasets 
are to be transferred to other locations this will only be performed after stripping them of 
all identifiers, leaving only the subject identification code. The key will remain on site with 
the local research team. 

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Study-database and disclosure of data 
With respect to the existing clinical observational datasets, all research data will be 
retained and stored in the study-database at the research institutions where data were 
primarily collected. Ownership of the data remains with the institutions that collected the 
data. Any data transferred from one centre to another will be stripped from all identifying 
variables, i.e. completely anonymized. If for verification purposes it is not possible to 
completely anonymize, files will be coded using a random identifier number. The key to 
this identifier number will at all times remain with the local research team at the institution 
where the data was collected. Any data transferred or combined by one of the consortium 
partners will be stored at the secured server of the partner’s institute during the running of 
the project. It will remain there for at least 15 years after completion of the project. 
Due to the sensitive nature of the participant’s data, datasets will never be made publicly 
available. Data will only be stored at secured servers of the participating research centres. 
All researchers who are not seated in the primary consortium and who are given access to 
the confidential information that has been pseudo-anonymised must provide a signed 
statement that they will maintain confidentiality. 
The Principal Investigator agrees to archive the study documents for 15 years from the 
study end. 
 
Data management 
Any data storage and handling process will ensure data protection and confidentiality. The 
data entered in the study-database will be coded using a study-ID. Results from 
ultrasound examinations will be supplied in a file. Standardized electronic files will be used 
that supply output with a unique personal code without information that is directly 
traceable to the person. For extraction of data for research purposes by external parties, 
the study-ID will be removed. In the absence of this ID, researchers cannot identify the 
individual subjects. All data will be stored on hospital computer systems. All project data 
will be back-upped automatically via the O:disk and copies will be kept in two separate 
locations. 
 
Disclosure of data 
The personal identification data will not be made available to persons outside the local 
research team, including the other researchers in the study-consortium. Results of 
research will be published without any reference to individual participants of the study. 
 
Accountability 

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The study-consortium is responsible for the data collection, secure storage and disclosure 
of data. Any misconduct can be directed to the coordinating investigator of the study-
consortium. 
 
12.2 Monitoring and Quality Assurance  
The risk of this study is qualified based on the guideline by the NFU (Dutch Federation of 
University Medical Centers) about quality insurance in human research 
(“Kwaliteitsborging van mensgebonden onderzoek”). The NFU guideline states that the 
extent of risk has to be estimated by considering the additional risk of an intervention 
compared to standard treatment. The risk of this study is qualified as ‘Negligible’.  
 
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 that gave 
a favourable opinion.  
 
12.4 Annual progress report 
The sponsor/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, numbers of subjects included and numbers 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/sponsor will notify the accredited METC of the end of the study within a 
period of 8 weeks. The end of the study is defined as the last participant’s last visit.  
The sponsor will 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 sponsor 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/sponsor will 
submit a final study report with the results of the study, including any 
publications/abstracts of the study, to the accredited METC.  
 

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12.6 Public disclosure and publication policy 
Project results will be disseminated to healthcare professionals and to scientific and 
industrial peers through events, professional organisations and publications in peer-
reviewed journals as well as presentations at scientific congresses. 
The project will be communicated to the general public via a press release at the start of 
the project and by using the internal and external communication means of the partners, 
such as institutional websites, magazines and the yearly news letter from the Predict 
study. All data will be available for verification and re-use for at least 15 years. Full 
permission to access the data will be granted only to the principal investigator at the 
location where data are stored. In case of an additional research question, this research 
can only be done with permission of all members of the Leadership Team. Before access 
is granted to any person outside of the consortium, a signed statement ensuring 
confidentiality must be obtained. 

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13. STRUCTURED RISK ANALYSIS  
 
13.1 Potential issues of concern 
a. Level of knowledge about mechanism of action 
Not applicable. 
 
b. Previous exposure of human beings with the test product(s) and/or products with a 
similar biological mechanism 
Not applicable. 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo 
human cell material? 
Not applicable. 
 
d. Selectivity of the mechanism to target tissue in animals and/or human beings 
Not applicable. 
 
e. Analysis of potential effect 
Not applicable. 
 
f. Pharmacokinetic considerations 
Not applicable. 
 
g. Study population 
Not applicable. 
 
h. Interaction with other products 
Not applicable. 
 
i. Predictability of effect 
Not applicable. 
 
j. Can effects be managed? 
Not applicable. 
 

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13.2 Synthesis 
Not applicable.

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