Diet Quality of Pregnant Women With a Strict Plant-based Diet Versus an Omnivorous Diet: a Focus on Vitamin B12 Intake and Other Nutritional and Pregnancy-related Outcomes
Conditions: Vitamin B 12 Deficiency, Pregnancy, Nutrient Deficiency, Dietary Exposure
Sponsor: Maastricht University Medical Center

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RESEARCH PROTOCOL (September 2023) 
 
“Diet quality of pregnant women with a strict plant-based diet versus an 
omnivorous diet: a focus on vitamin B12 intake and other nutritional and 
pregnancy-related outcomes.” 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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PROTOCOL TITLE Diet quality of pregnant women with a strict plant-based diet versus an 
omnivorous diet: a focus on vitamin B12 intake and other nutritional and pregnancy-related 
outcomes.  
 
Protocol ID 
NL84796.068.23 
Short title 
Nutrition and pregnancy 
Version 
1.0 
Date 
12.09.2023 
Coordinating 
investigator/project leader 
 
Dr. H.C.J. Scheepers 
Gynecologist-perinatologist 
Dept. Obstetrics and Gynaecology 
Maastricht University Medical Center+ 
+31 (0)433874768 
E: hcj.scheepers@mumc.nl 
Maastricht, The Netherlands 
Principal investigator 
 
 
Dr. H.C.J. Scheepers 
Gynecologist-perinatologist 
Dept. of Obstetrics and Gynaecology 
Maastricht University Medical Center+ 
+31 (0)433874768 
E: hcj.scheepers@mumc.nl 
Maastricht, The Netherlands  
Sponsor  
Maastricht University Medical Center+ 
H.C.J. Scheepers, MD, PhD (PI) 
hcj.scheepers@mumc.nl 
Subsidizing party 
None 
Independent expert  
Dr. Ron van Golde 
Dept. of Obstetrics and Gynaecology, Reproductive 
Medicine 
Maastricht University Medical Center+ 
Ron.van.golde@mumc.nl 
Project team members 
Dr. H.C.J. Scheepers 
Gynecologist-perinatologist 

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Dept. of Obstetrics and Gynaecology 
GROW, School for Oncology and Developmental 
Biology 
Maastricht University Medical Center+ 
 
Prof. dr. Daisy M.A.E. Jonkers 
Associate Professor 
Div. Gastroenterology-Hepatology 
Dept. of Internal Medicine 
NUTRIM, School for Nutrition and Translational 
Research in Metabolism 
Maastricht University, the Netherlands 
 
Dr. Simone J.P.M. Eussen 
Assistant Professor 
Dept. of Epidemiology 
CARIM, School for Cardiovascular Diseases and 
Care 
CAPHRI, School for Care and Public Health 
Research Institute 
Maastricht University, the Netherlands 
 
Dr. Jessica S. Gubbels 
Associate Professor 
Dept. of Health Promotion 
NUTRIM, School of Nutrition and Translational 
Research in Metabolism 
Maastricht University, the Netherlands 
 
Drs. Deidre Meulenbroeks 
Resident Gynaecology 
Dept. of Obstetrics and Gynaecology 
GROW, School for Oncology and Developmental 
Biology 
Maastricht University Medical Center+ 
 

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PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Head of Department: 
Prof. R.F.P.M. Kruitwagen 
Head of the dept. Of Obstetrics and 
Gynaecology 
Maastricht University Medical Center+ 
 
 
 
Principal Investigator 
Dr. H.C.J. Scheepers 
Dept. of Obstetrics and Gynaecology 
Maastricht University Medical Center+ 
 
 
 
 
 

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TABLE OF CONTENTS 
 
1. 
INTRODUCTION AND RATIONALE .............................................................................. 9 
2. 
OBJECTIVES ................................................................................................................12 
3. 
STUDY DESIGN ...........................................................................................................12 
3.1 
General health-related questionnaire .....................................................................13 
3.2 
3-Day food record ..................................................................................................13 
3.3 
Blood sample .........................................................................................................14 
3.4 
Questionnaire pregnancy and post-partum period ..................................................14 
4. 
STUDY POPULATION ..................................................................................................15 
4.1 
Population (base) ...................................................................................................15 
4.2 
Inclusion criteria .....................................................................................................15 
4.3 
Exclusion criteria ....................................................................................................15 
4.4 
Sample size calculation ..........................................................................................15 
5. 
METHODS ....................................................................................................................17 
5.1 
Study parameters/endpoints ...................................................................................17 
5.1.1 
Main study parameter/endpoint .......................................................................17 
5.1.2 
Secondary study parameters/endpoints ..........................................................17 
5.1.3 
Other study parameters ...................................................................................17 
5.2 
Study procedures ...................................................................................................17 
5.3 
Withdrawal of participants ......................................................................................18 
5.4 
Replacement of individual participants after withdrawal ..........................................18 
5.5 
Premature termination of the study .........................................................................18 
6. 
SAFETY REPORTING ..................................................................................................19 
6.1 
Temporary halt for reasons of participant safety .....................................................19 
6.2 
AEs, SAEs and SUSARs ........................................................................................19 
7. 
STATISTICAL ANALYSIS .............................................................................................20 
7.1 
Primary study parameter ........................................................................................20 
7.2 
Secondary study parameters ..................................................................................20 
8. 
ETHICAL CONSIDERATIONS ......................................................................................21 
8.1 
Regulation statement .............................................................................................21 
8.2 
Recruitment and consent........................................................................................21 
8.3 
Benefits and risks assessment, group relatedness .................................................22 
8.4 
Compensation for Injury .........................................................................................22 
8.5 
Incentives ...............................................................................................................22 
9. 
ADMINISTRATIVE ASPECTS, MONITORING, AND PUBLICATION ............................23 
9.1 
Handling and storage of data and documents ........................................................23 
9.2 
Monitoring and Quality Assurance ..........................................................................23 
9.3 
Amendments ..........................................................................................................23 
9.4 
Annual progress report ...........................................................................................23 
9.5 
Temporary halt and (prematurely) end of study report ............................................23 
9.6 
Public disclosure and publication policy ..................................................................24 
10. 
REFERENCES ..........................................................................................................25 

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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 
AR 
Adverse Reaction 
AVG 
Algemene Verordering Gegevensbescherming 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CV 
Curriculum Vitae 
DSMB 
Data Safety Monitoring Board 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
IC 
Informed Consent 
KNOV 
Koninklijke Nederlandse Organisatie van Verloskundigen 
METC  
Medical research ethics committee (MREC); in Dutch: medisch-ethische 
toetsingscommissie (METC) 
MUMC+ 
Maastricht University Medical Center+ 
RIVM 
Rijksinstituut voor Volksgezondheid en Milieu 
(S)AE 
(Serious) Adverse Event  
Sponsor
The sponsor is the party that commissions the organization or 
performance of the research, for example, a pharmaceutical 
company, academic hospital, scientific organization, or investigator. A 
party that provides funding for a study but does not commission it is not 
regarded as the sponsor but referred to as a subsidizing party. 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 
WHO 
World Health Organization 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 
 
 
 
 

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SUMMARY 
Rationale: The number of people adhering to a strict plant-based diet is increasing globally. 
Adequate maternal nutrition during pregnancy is essential for fetal development, as 
deficiencies in macro- and micronutrients can cause maternal and neonatal complications. 
Nutrition can play a role in developing pre-eclampsia, premature birth, and intra-uterine 
growth restriction. Although there is evidence of nutrient deficiencies in people who follow a 
strict plant-based diet and the significance of maternal diet for both maternal and fetal 
outcomes, there has been limited research on the nutritional intake and outcomes of 
pregnant women on a strict plant-based diet. Additionally, to the best of our knowledge, very 
limited research has been published on the incidence of pregnancy-induced hypertension, 
pre-eclampsia, gestational diabetes, or pre-term birth in women on a strict plant-based diet. 
The relation between these outcomes and nutritional intake/status is also unknown in this 
specific group of women, even though the number of people with a strict plant-based diet is 
increasing. The only two studies that studied pregnancy-related outcomes in women on a 
strict plant-based diet were performed in Israel and were not powered to show statistical 
differences between a strict plant-based diet and obstetrical outcomes. Additionally, the 
dietary intake in Israel differs from other parts of the world. Thus, it is impossible to ascertain 
if the results also apply to a population with a distinct diet. Recently, the Royal Dutch 
Organization of Midwives (Koninklijke Nederlandse Organisatie van Verloskundigen, KNOV) 
published ‘The Guide for Vegetarian and Vegan Diet in Pregnancy.’ They acknowledge the 
limited availability of scientific research concerning the strict plant-based diet in pregnancy 
and the non-existence of data on Dutch pregnant women. They recommend performing 
additional blood tests for vitamin B12 and D in case of possible nutrition deficiencies in this 
group of pregnant women. A significantly lower ferritin level is also described in pregnant 
women on a strict plant-based diet. In conclusion, even though the nutritional intake during 
pregnancy is vital for both mother and child, the nutritional intake amongst Dutch women on 
a strict plant-based diet during pregnancy is unknown. Previously published results from 
Israeli women do not apply to the Dutch population, as nutritional intake differs between 
countries. The recently published KNOV guide suggests studying vitamin blood levels during 
pregnancy. To advise and update guidelines for Dutch women on a strict plant-based diet 
during pregnancy, additional research on nutritional intake and the nutritional status of Dutch 
women on a strict plant-based diet and to compare this with the nutritional status of 
omnivorous pregnant women is crucial. 
Objective:  
The primary objective of the study is to compare vitamin B12 intake between pregnant 
women with a strict plant-based diet and pregnant women with an omnivorous diet. The 
secondary aim of this study is to compare the nutritional intake and status of pregnant 

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women following a strict plant-based diet with those following an omnivorous diet. 
Additionally, the maternal and fetal outcomes of pregnant women on a strict plant-based diet 
will be compared to those on an omnivorous diet. 
Study design: The study comprises an observational non-inferiority design. 
Study population: Pregnant women with a strict plant-based diet (n=128) and pregnant 
women with an omnivorous diet (n=128) will be recruited to participate in this study.  
Main study parameters/endpoints: The main study parameter is the vitamin B12 intake 
(mcg/day). Additional parameters are the daily intake of other macronutrients and 
micronutrients and the adherence to Dutch dietary guidelines, plasma vitamin D, plasma 
vitamin B12, plasma hemoglobin, and plasma ferritin. Other parameters are maternal and 
fetal outcomes (e.g., gestational age at delivery, gestational diabetes, pregnancy-induced 
hypertension, pre-eclampsia, weight gain during pregnancy, birth weight, and breastfeeding 
initiation).  
Nature and extent of the burden and risks associated with participation, benefit, and 
group relatedness: Participants may encounter a minor burden during this study. The 
participant will complete a health-related questionnaire. The estimated time required to 
complete the questionnaire is approximately 10 minutes. Additionally, between 27 and 30 
weeks of pregnancy, participants will fill out a three-day food diary. It is estimated that the 
questionnaire will take approximately 30 minutes to complete each day. Finally, the 
participants must complete a questionnaire following the baby's delivery. This questionnaire 
will cover various aspects, such as the pregnancy, delivery, and blood results obtained 
during pregnancy. The estimated time required to complete the questionnaire is 
approximately 15 minutes. 
During the blood sampling, 10 ml of blood will be collected using an evacuated tube system 
through venepuncture. This may cause minor discomfort and potentially result in a small, 
temporary hematoma at the puncture site. Taking blood samples is a routine part of clinical 
practice, not solely for this study. Pregnant women typically undergo venipuncture between 
27 and 30 weeks as part of regular obstetrical care. Participants can have their blood 
sampled at a nearby laboratory without traveling for the study. 
The study's results will provide valuable information about the nutritional intake and status of 
pregnant women who follow a strict plant-based diet compared to those who follow an 
omnivorous diet during pregnancy. It will also examine the impact of these diets on the health 
of both the mother and fetus. This information can be used in developing and improving 
guidelines on diet in pregnancy and may contribute to the health of pregnant women and 
their newborn children in the future.  
 
 

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1. INTRODUCTION AND RATIONALE 
The number of people adhering to a strict plant-based diet, which consists of fruits, 
vegetables, grains, legumes, nuts, seeds, herbs, and spices, and excludes all animal 
products (often also referred to as a vegan diet), is increasing globally. The prevalence is 
estimated to be 1.5% of the Dutch population, and the number of vegans in Great Britain 
quadrupled between 2014 and 2019 to 1.21% of the population.1 
 
Adequate maternal nutrition during pregnancy is essential for fetal development, as 
deficiencies in macro- and micronutrients can cause maternal and neonatal complications. 
Nutrition can play a role in developing pre-eclampsia, premature birth, and intra-uterine 
growth restriction. Vitamin B12, folate, iodine, calcium, zinc, selenium, magnesium, and iron 
are essential elements in maternal and fetal health. 2 In a strict plant-based diet, sufficient 
intake of vitamin B12 can only be achieved by using supplements since vitamin B12 is 
naturally not present in plant-based foods unless being fortified. Maternal iron deficiency, 
linked to inadequate ingestion of iron, but also of, e.g., vitamin B12 or folate, in the first and 
second trimester, can lead to premature birth or low birth weight and jeopardize the 
newborn's health.3,4  Iron deficiency is associated with neuronal changes and problems in 
myelinization, neuronal transmission, and frontal cortex and basal ganglia development.5 
Anaemia is related to a higher maternal mortality rate and can lead to miscarriage during the 
first trimester.5,6 Maternal iodine deficiency is associated with aberrations in the neuronal 
development of children. It can cause irreversible damage to the central nervous system, 
instilling permanent mental retardation.7 Calcium deficiency increases the risk of maternal 
pre-eclampsia and is medically induced prematurely.8 Additionally, it can contribute to 
spontaneous prematurity and intra-uterine growth restriction, and it implies a variety of 
physical and cognitive consequences in the long term, including increased atrophy.9 
Selenium deficiency can cause alterations in the lipid profile, mental and psychomotor delay, 
oxidative stress in the mother and the fetus, premature birth, miscarriage, and problems in 
the neural tube of the newborn.2 Selenium acquired during pregnancy is maintained in 
newborns' reserves; the build-up of this mineral in the uterus occurs in the third trimester.2 
When in low concentrations in newborns, especially prematurely, they are at higher risk for 
developing retinopathy, bronchopulmonary dysplasia, and other lung disorders. However, 
excess selenium can trigger cardiovascular problems, dyslipidaemias, and insulin 
resistance.2 Zinc deficiency during pregnancy can cause premature birth, complications in 
childbirth, and neurological deficits. It can also cause a reduced natural skin and mucosa 
barrier function and deleterious effects on the immune system.2 Magnesium deficiency can 
lead to severe pre-eclampsia, premature birth, and hypoxic-ischemic encephalopathy.10,11 
Maternal intake of macronutrients is also of importance for both mother and fetus. Maternal 
energy intake is needed to provide energy for the fetus for the synthesis of new tissue; 

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protein is involved in both structural and functional biological roles and influences birthweight; 
and fatty acids impact the development of the brain and retina in the fetus and the risk of 
preeclampsia.12 Although evidence is limited, a low glycaemic index, low glycaemic load, and 
a high fiber diet may benefit preconception or early pregnancy for women at risk of 
developing gestational diabetes mellitus or preeclampsia or having a large for gestational 
age infant.12 
In a systematic review by Bakalouhi et al., including 48 studies (12 prospective 
cohorts and 36 cross-sectional studies), differences in the nutrient intake and nutrient status 
of non-pregnant people on a strict plant-based diet compared to World Health Organization 
recommendations were summarised.13 Sample sizes differed between 6 and 2596 
participants. The general conclusions of the review were that people on a strict plant-based 
diet generally have a higher fiber intake and a total energy intake from fat comparable to an 
omnivorous diet. However, deficiencies or low intake of various nutrients were also 
described. Recommended levels of protein intake (i.e., 15% of daily energy intake, 0.66 g/kg 
daily) were not always reached. The vitamin B12 intake was significantly lower, and a serum 
deficiency of vitamin B12 was frequently observed. Even though the iron intake was higher 
among people on a strict plant-based diet compared to individuals who followed other dietary 
patterns, this did not always translate into higher ferritin levels due to the lower bioavailability 
of non-haem iron from plant-based foods. However, overall, the risk of iron deficiency among 
vegan women was not higher than in omnivorous women. Vitamin D intake was also found to 
be lower than the WHO limit, but deficiencies were only sometimes observed. Calcium intake 
was low due to the exclusion of dairy products and the lower bioavailability of calcium in 
plant-based foods, but still above the WHO limit of 525 mg/day. Zinc intake was reduced 
among people on a strict plant-based diet, and a risk of deficiency was frequently observed. 
Intake of iodine below the limit was reported for most people on a strict plant-based diet in 
several studies. And lastly, there seemed to be a decreased selenium intake. Importantly, in 
the last ten years, more and more meat substitutes and other plant-based products have 
become available on the market. Therefore, the current dietary intake will differ from the 
nutritional intake 20 or 30 years ago. 
Despite the observed findings on deficiencies in nutrient intake in the general 
population on a strict plant-based diet and the importance of maternal diet for maternal and 
fetal outcomes, limited research has been performed on the nutritional intake and the 
maternal and fetal outcomes of pregnant women on a strict plant-based diet. Additionally, to 
the best of our knowledge, very limited research has been published on the incidence of 
pregnancy-induced hypertension, pre-eclampsia, gestational diabetes, or pre-term birth in 
women on a strict plant-based diet. The relation between these outcomes and nutritional 
intake/status is also unknown in this specific group of women, even though their number is 

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increasing. The only two studies that researched pregnancy-related outcomes in women on a 
strict plant-based diet were performed in Israel and were not powered to show statistical 
differences between a strict plant-based diet and obstetrical outcomes. The prospective 
observational study by Avnon et al. among 60 pregnant women on a strict plant-based diet 
compared to other diets showed an increased risk of small-for-gestational-age newborns 
compared to omnivores (RR 5.9, 95% CI 1.2-21.8) with a lower birthweight of newborns from 
mothers with a strict plant-based diet compared to omnivores (3015 gram ± 420 g versus 
3328 gram ± 495 g, p<0.001).14 The most recent study by Kesary et al. included 234 women 
on a strict plant-based diet in a retrospective, web-based study and compared them with 
vegetarians and omnivores.15 After adjusting for BMI, they concluded that there was no 
association with lower birth weight for offspring of women on a plant-based diet during 
pregnancy compared to omnivores, with an adjusted odds ratio for small for gestational age 
of 1.59 (95% CI 0.95 – 2,65). There was a decreased risk for gestational diabetes, although 
this association did not reach significance (OR = 0.54 95% CI 0.28-1.03). A strict plant-based 
diet had no significant association with the risk of preterm birth. The dietary intake in Israel, 
where both studies were performed, differs from other parts of the world.16 It is impossible to 
determine whether the results apply to a population with a different diet.  
Recently, the Royal Dutch Organization of Midwives (Koninklijke Nederlandse 
Organisatie van Verloskundigen, KNOV) published ‘the guide for vegetarian and vegan diet 
in pregnancy.’19 They acknowledge the limited availability of scientific research concerning 
the strict plant-based diet in pregnancy and the non-existence of data on Dutch pregnant 
women. Therefore, they recommend additional blood tests for vitamin B12 and D in case of 
possible nutrition deficiencies in this group of pregnant women. A significantly lower ferritin 
level is also described in pregnant women on a strict plant-based diet.   
In conclusion, even though the nutritional intake during pregnancy is vital for both 
mother and child, the nutritional intake amongst Dutch women on a strict plant-based diet 
during pregnancy is unknown. Previously published results from Israeli women do not apply 
to the Dutch population as nutritional intake differs between countries. The recently published 
KNOV guide suggests checking vitamin blood levels during pregnancy. To advise and 
update guidelines for Dutch women on a strict plant-based diet during pregnancy, additional 
research is crucial on nutritional intake and the nutritional status of Dutch women on a strict 
plant-based diet.  
This study will primarily evaluate nutritional intake of vitamin B12 of pregnant women 
on a strict plant-based diet, as this nutrient needs to be supplemented for these women to 
have a sufficient vitamin B12 intake. Secondly, the study will assess the nutritional intake of 
other relevant micronutrients and macronutrients, the nutritional status (vitamin B12, D, 
ferritin, and hemoglobin), and their maternal and fetal outcomes.   

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2. OBJECTIVES 
 
2.1 Primary Objective  
In this observational non-inferiority study, we aim to compare vitamin B12 intake in pregnant 
women with a strict plant-based diet and pregnant women with an omnivorous diet. We 
hypothesize that the vitamin B12 intake amongst women with a strict plant-based diet is non-
inferior to pregnant women with an omnivorous diet. 
 
2.2 Secondary Objectives:  
The secondary aim of this study is to compare pregnant women with a strict plant-based diet 
and pregnant women with an omnivorous diet concerning their: 
- Intake of micronutrients and macronutrients 
- Plasma vitamin B12, vitamin D, ferritin, and hemoglobin  
- Maternal and fetal outcomes, including gestational age at delivery, gestational diabetes, 
pregnancy-induced hypertension, pre-eclampsia, weight gain during pregnancy, and fetal 
birth weight. 
 
 
3. STUDY DESIGN 
 
In this observational non-inferiority study, a total of 256 pregnant women will participate, with 
n=128 pregnant women with a strict plant-based diet and n=128 pregnant women with an 
omnivorous diet.  
Participant recruitment will start after the Medical Research Ethics Committee (METC) of 
Maastricht University Medical Center+ (MUMC+) has approved this study according to the 
revised version of the Guidelines of the Declaration of Helsinki (64th WMA General Assembly, 
Fortaleza, Brazil, October 2013).  
Participants will be recruited via their obstetrician (midwife or gynecologist). They will provide 
a brief explanation to potentially eligible pregnant women regarding the study. Furthermore, 
participants will be recruited through nutrition and pregnancy-related social media 
advertisements and other online platforms (website/newsletter Nederlandse Vereniging voor 
Veganisme, nutrition and/or pregnancy-related magazines). 
If a woman shows interest, she will be directed to the study website 
(www.voedingsonderzoek.nl), which contains concise information about the study (see 
“E3_Advertentieteksten_NL…._Versie1.0_19-07-2023”). Potential participants can contact 
the researchers at the Maastricht University Medical Center+ via e-mail. In addition, we will 
be contacting women who participated in another previously conducted study that has been 

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finished already by our research group titled "Raising Children on a Vegan Diet: A Qualitative 
Exploration of Feeding Practices and Determinants of Mothers Following a Vegan Diet." 
These women have given their consent to be approached for future research and will be 
contacted via email (see “E3_WervingstekstInterviewstudie_NL…_V1.0_19.07.2019”). The 
researcher will contact those who respond and invite them to fill out a screening 
questionnaire about their nutritional diet, pregnancy, and medical history (see 
“F1_vragenlijstscreening_NL..._Versie1.0_19-07-2023”). The researcher will also send the 
participant information brochure (PIF) through email or regular mail.  
The PIF and the informed consent form are enclosed in this study protocol (“see 
E1+E2_Proefpersoneninformatie_NL……Versie1.0_datum”). When considered eligible to 
participate in the study, the participant will be asked to sign informed consent. 
 
Women are given at least three days to decide if they want to participate in the study. If the 
woman is interested and willing to participate, an informed consent form will be signed (co-
signed by the investigator). The signing of the informed consent form needs to be finished 
before 27 weeks of pregnancy for a participant to join the study. After signing informed 
consent, the total duration of the study will be from 27 weeks of pregnancy until the post-
partum questionnaire is received. A total of 256 pregnant women will be included in the 
study. 
 
The study consists of the following: 
1. General health-related questionnaire 
2. 3-Day food record 
3. Blood sample 
4. Questionnaire regarding pregnancy, delivery, and post-partum period 
These steps are briefly described below; more elaborate information can be found in 
paragraph 5.2.  
 
3.1 General health-related questionnaire 
Once the participant has provided their informed consent, they will receive an email 
containing a link to a general health-related questionnaire which they will need to complete 
(see “F2_vragenlijstgezondheid_NL..._Versie1.0_19.07.2023”). This email will be sent via 
Castor.  
3.2 3-Day food record 
As part of standard care in the Netherlands, pregnant women have their blood sampled 
between 27-30 weeks. For this study, participants will be asked to keep track of their 

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nutritional intake for three days, including two weekdays and one weekend day, in the two 
weeks leading up to their blood test. This will help establish a connection between their blood 
results and dietary habits. To log their daily intake, participants will be directed to download 
the 'Traqq' app (see “F3_traqqapp_NL..._Versie1.0_04.08.2023”) 
3.3 Blood sample 
The participant will be asked to provide their obstetrician at a regular consultation before 27 
weeks of pregnancy with an information document about the blood sampling (see 
“E4_overigvoorlichtingsmateriaal_NL…_Versie1.0_21-07-2023”). In this document, the 
obstetrician is requested to measure hemoglobin, ferritin, vitamin B12, and vitamin D in the 
blood sample taken from the participant as regular pregnancy care between 27-30 weeks of 
pregnancy. The obstetrician most likely already conduct these blood tests as part of their 
routine care. If this is the case, there's no need for any extra tests to be done. The 
obstetrician can proceed with their usual actions of care based on the study results; no 
additional study-related actions are required. 
 
3.4 Questionnaire pregnancy and post-partum period 
Six weeks after the participants’ due date, they will be asked to complete the questionnaire 
regarding their pregnancy, delivery, and postpartum period (see 
“F4_vragenlijstpstpartum_NL..._Versie1.0_19.07.2023”). They will receive the link to this 
questionnaire by email, sent via Castor.  
 
 

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4. STUDY POPULATION 
 
4.1 Population (base)  
Pregnant women with a strict plant-based diet from the Netherlands and pregnant women 
with an omnivorous diet will be recruited in the Netherlands.  
4.2 Inclusion criteria 
To be eligible to participate in this study, a participant must meet all of the following criteria. 
- 
Before 27 weeks of pregnancy 
- 
Age >18 years 
- 
Strict plant-based diet (vegan diet), defined as a diet without ingestion of animal 
products (i.e., intake less than once a month during) the entire pregnancy, or 
omnivorous diet, defined as a diet that includes meat 
- 
Willing and able to give written informed consent and to understand, participate and 
comply with the research project requirements 
 
4.3 Exclusion criteria 
A potential participant who meets any of the following criteria will be excluded from 
participation in this study: 
- 
Food allergy that influences the nutritional intake, e.g., milk allergy, celiac disease, 
and lactose intolerance 
- 
Medical history of gastrointestinal diseases such as Crohn’s disease and other 
diseases associated with vitamin B12 deficiencies such as (pre-existing) diabetes. 
- 
Medical history of abdominal surgery interfering with nutrient uptake, such as gastric 
bypass and intestinal resections 
- 
Pre-existing cardio-vascular diseases or auto-immune diseases 
- 
Insufficient understanding of the Dutch language  
 
4.4 Sample size calculation 
Our calculation for the sample size is based on the primary outcome measure, which is the 
intake of vitamin B12. Pregnant women in the Netherlands are advised to consume 3.3 mcg 
of vitamin B12 daily. According to the 'Voedselconsumptiepeiling' of the RIVM (Rijksinstituut 
voor Volksgezondheid en Milieu) conducted between 2012-2016, 63.2% of Dutch women 
(without considering their diet) had a sufficient intake.17 The significance level (alpha) and, 
thus, the chance of a false positive result will be 0.05. To obtain a power (1-beta) of 80%, 
with a percentage of ‘success’ of 63.2% in the group of women with a strict plant-based diet 

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and the group of women with the omnivorous diet, and a non-inferiority limit of 15%, a 
sample size of 128 participants per group is required. So, a total sample size of 256 
participants is required.  
There will only be recruitment for additional participants if the primary outcome (vitamin B12 
intake) is missing. Sample size calculation was conducted on the statistical power analysis 
website https://www.sealedenvelope.com/power/binary-noninferior/.  
 
 

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5. METHODS 
5.1 Study parameters/endpoints 
5.1.1 Main study parameter/endpoint 
The primary outcome is vitamin B12 intake in mcg/day.  
5.1.2 Secondary study parameters/endpoints  
1. Nutritional intake (micronutrients and macronutrients) 
2. Plasma vitamin B12, vitamin D, ferritin, and hemoglobin  
3. Maternal and fetal outcomes: gestational age at delivery, gestational diabetes, 
pregnancy-induced hypertension, pre-eclampsia, gestational weight gain, 
birthweight 
5.1.3 Other study parameters  
Age, parity, Body Mass Index before pregnancy (BMI) and after pregnancy, 
smoking, alcohol use, drug use, educational level, employment, medical history, 
obstetrical history, family history (e.g. (gestational) diabetes, pregnancy-induced 
hypertension, pre-eclampsia), medication use, supplement use, when they started 
being on the strict plant-based diet 
5.2 Study procedures 
Questionnaires 
All questionnaires will be completed in a secured, electronic environment (Castor) 
that can be accessed via the internet by electronic devices (telephone, tablet, or 
computer).  
 
Participants will be asked to fill out the following questionnaires 
 
Health-related questionnaire 
See “F2…Naam_studienummer_Versie1.0_datum” 
This questionnaire comprises 30 questions covering various topics and takes 
about 15 minutes to complete. The participants are requested to provide their 
birth details, educational qualifications, employment status, and dietary habits 
during pregnancy. In addition, they are inquired about their pregnancy, 
obstetric record, present height, and weight, smoking habits, alcohol 
consumption, drug usage, medication intake, and supplement usage. 
 
Three-day food records 
see “F3_traqqapp_NL..._Versie1.0_04.08.2023” 

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All participants will receive instructions to use the Traqq app (developed by 
Wageningen University) to record their food intake for three days. This will 
include two regular days and one weekend day. The food records will contain 
information about the time of day when the food was consumed and the type 
and amount of food eaten. Participants will receive instructions on estimating 
serving sizes and recording recipes in the app. The food record must be 
completed within the two weeks before the blood sample collection between 
27-30 weeks of pregnancy.  
 
Maternal and fetal outcomes questionnaire 
See “F4…Naam_studienummer_Versie1.0_datum” 
This questionnaire comprises a maximum of 15 questions covering various 
topics and takes about 10 minutes to complete. Participants are asked about, 
e.g., their blood sample results, the occurrence of complications related to 
pregnancy, the delivery, and the newborn. Participants will have access to the 
questionnaire six weeks after their anticipated due date, and must be 
completed within 4 weeks.  
 
Blood sampling 
The local laboratory will collect blood samples from an antecubital vein in the forearm, 
which may cause minor discomfort and result in small and temporary hematoma or 
bruises. However, it's important to note that obtaining blood samples from pregnant 
women for hemoglobin testing is a routine part of clinical practice between 27 and 30 
weeks of pregnancy, and not specifically for this study. For study purposes, 10mL of 
additional blood will be sampled per participant during this blood withdrawal.  
 
5.3 Withdrawal of participants 
Participants can leave the study at any time for any reason if they wish to do so without any 
consequences. 
 
5.4 Replacement of individual participants after withdrawal 
Participants will not be replaced after signing informed consent. However, if the primary 
outcome (vitamin B12 intake in mcg/day) is missing, an additional participant will be recruited 
to meet the calculated sample size.  
 
5.5 Premature termination of the study 
There are no foreseeable reasons for the study to end prematurely. 
 

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6. SAFETY REPORTING 
6.1 Temporary halt for reasons of participant safety 
In accordance with section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardize the 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.  
6.2 AEs, SAEs and SUSARs  
The study makes use of questionnaires and food diaries. Blood sampling is part of current 
and daily clinical routine; for this study, additional blood will be sampled (10mL). 
Therefore, study-related adverse events (AEs), serious adverse events (SAEs), and 
suspected unexpected serious adverse reactions (SUSARs) are not applicable.  

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7. STATISTICAL ANALYSIS  
- 
All data will be presented quantitatively. Means and standard deviations or medians 
with inter quartile ranges depending on variable distributions of maternal and fetal 
outcomes will be given for continuous variables (such as gestational weight gain and 
birthweight), and frequencies and percentages will be presented for categorical 
variables (such as gestational age at delivery, gestational diabetes, pregnancy-
induced hypertension, pre-eclampsia, pre-term birth).  
- 
Continuous (such as (micro)nutrient intake and blood levels, age, BMI) will be 
presented as means with standard deviations (normal distribution) or median with 
interquartile range (non-normal distribution) and categorical (such as smoking, alcohol 
use, drug use, education, employment, obstetrical history, obstetrical family history, 
medication use, supplement use) will be presented as percentages. 
- 
Comparison between women on a strict plant-based diet and the omnivorous group 
will be presented with the independent t-tests (normal distribution) or Mann-Whitney U 
test (non-normal distribution), or chi-square test (for categorical variables (with Fisher 
exact when needed)). Additionally, a multivariable linear regression analysis will 
assess the association between maternal and fetal outcomes with vitamin B12 intake, 
adjusted for potential confounders (such as BMI). 
 
7.1 Primary study parameter 
The main study parameter is vitamin B12 intake (mcg/day).  
 
7.2 Secondary study parameters  
Secondary study parameters are: 
- Other micro- and macronutrient intake 
- Plasma vitamin B12, vitamin D, ferritin, and hemoglobin 
- Gestational age at delivery, gestational diabetes, pregnancy-induced hypertension, pre-
eclampsia, gestational weight gain, birthweight, initiation of breastfeeding 
 

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8. ETHICAL CONSIDERATIONS 
8.1 Regulation statement 
This study has to be approved by the Medical Ethical Committee of Maastricht University 
(METC). The study will be conducted according to the Declaration of Helsinki (8th revision, 
2013) and in accordance with the Medical Research Involving Human Subjects Act (WMO).  
 
8.2 Recruitment and consent 
After approval of the study by the METC of MUMC+, participants will be recruited via their 
obstetrical healthcare giver (midwife or Gynaecologist). The obstetrician will provide pregnant 
women with a brief introduction to the study. If the woman expresses interest, she can be 
asked if the researcher may contact her to give more information about the study orally. The 
obstetrician will then share the woman's contact details with the researcher. The woman can 
visit the study website (www.voedingzwangerschap.nl) to find brief information about the 
study and email the researchers at Maastricht University Medical Center+. 
 
Furthermore, participants will be recruited through nutrition and pregnancy-related social 
media advertisements and other online platforms (website/newsletter Nederlandse 
Vereniging voor Veganisme, nutrition and/or pregnancy-related magazines).  
Lastly, women who participated in the study ‘Raising children on a vegan diet, a qualitative 
exploration of feeding practices and determinants of mothers following a vegan diet’ and who 
gave consent to be approached for further research will be contacted by email or letter (see 
“E3_WervingstekstInterviewstudie_NL…_V1.0_19.07.2019”). Women will be referred to the 
study website (www.voedingzwangerschap.nl), where brief information about the study can 
be found and where she can contact the researchers at the Maastricht University Medical 
Center+ via e-mail. 
 
If a woman is interested in participating in this study, she can contact the research team by 
e-mail or telephone, as shown on the website. Women can always contact the researcher or 
independent medical doctor if any question arises and will be referred to the VWO brochure 
“General information for research participants,” which contains general information about 
medical-scientific research. The researcher will contact the women interested in the study by 
e-mail or telephone and send them the PIF via e-mail or general mail (“see 
E1+E2_Proefpersoneninformatie_NL……Versie1.0_datum”). Participation will be voluntary. 
Three days (minimum) until 27 weeks of pregnancy (maximum) are provided to decide 
whether the woman would like to participate. The woman is asked to contact the investigator 
by email or telephone if she wants to participate. However, if the participant does not contact 

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the researcher, the researcher will contact the participant (at the earliest three days after 
sending the written information brochure). Permission to contact the patient will be logged in 
the subject screening and enrolment log. If the woman is interested and willing to participate, 
she will be asked to sign a digital informed consent form that will be co-signed by the 
investigator, both in duplicate. The informed consent form has to be filled out completely. The 
date of the assignment is necessary.  
 
The participants' privacy will be protected, meaning the study results will be related to an 
anonymous identification code. Codes and related participant information will not be 
available to individuals other than the principal investigator and a researcher (Drs. D. 
Meulenbroeks). They will be stored in a password-protected file. Participants can appeal to 
the independent doctor (Dr. R. van Golde) appointed to this study at any time. Participants 
will be informed that their decision to participate is voluntary, and they can withdraw at any 
time without giving a reason. Participants can be informed about the group results at the end 
of the study. 
 
8.3 Benefits and risks assessment, group relatedness 
Participants will not receive any direct benefits from participating in this study. The results of 
this study will be used to gain more knowledge about nutritional intake by pregnant women in 
the Netherlands with a strict plant-based diet and an omnivorous diet, which can affect the 
health of the mother and the unborn baby. There are no specific risks that need to be 
considered.  
 
8.4 Compensation for Injury 
We will apply for a waiver for the insurance because of the nature of this study. Liability 
insurance is available for current study.  
 
 
8.5 Incentives  
To thank the participants for participating, a pregnancy or newborn photoshoot will be made 
available to win for every 50 participants who join the study.  

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9. ADMINISTRATIVE ASPECTS, MONITORING, AND PUBLICATION 
9.1 Handling and storage of data and documents 
Data will be handled confidentially (coded), and the privacy of the participants will be 
guaranteed, according to the Dutch Personal Data Protection Act (“Algemene Verordering 
Gegevensbescherming” (AVG)). All eCRFs (in Castor) will be coded so that no personal 
information about the participant can be derived.  
The project team members, assigned monitors from the CTCM, IGZ, and members of the 
medical ethical committee will have access to the research data. All primary documents 
and data shall be kept for 15 years after the end of the experimental phase of the study 
for possible inspection. The electronic CRF, electronic diary, and electronic 
questionnaires (using Castor) will be developed, considering current regulations.  
 
9.2 Monitoring and Quality Assurance  
The Clinical Trial Centre Maastricht will perform monitoring and quality assurance. 
 
9.3 Amendments  
Amendments are changes made to the research after a favorable opinion by the 
accredited METC has been given. All amendments will be notified to the METC that gave 
a favorable opinion.  
 
9.4 Annual progress report 
The sponsor/investigator will submit a summary of the trial's progress to the accredited 
METC once a year. Information will be provided on the date of inclusion of the first 
participant, the number of participants included, and number of participants that have 
completed the trial, problems, and amendments.  
 
9.5 Temporary halt and (prematurely) end of study report 
The investigator/sponsor will notify the accredited METC of the end of the study within 
eight weeks. The end of the study is defined as the last patient’s post-partum information 
received or six months after the last participant gave birth.  
The sponsor will notify the METC immediately of a temporary halt of the study, including 
the reason for such an action. If the study ends prematurely, the sponsor will notify the 
accredited METC within 15 days, including the grounds for the premature termination. 
Within one year after the end of the study, the investigator/sponsor will submit a final 

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study report with the study's results, including any publications/abstracts, to the 
accredited METC.  
 
9.6 Public disclosure and publication policy 
Publication policy is in agreement with the Centrale Commissie Mensgebonden 
Onderzoek (CCMO-statement publicatie beleid). The results of the scientific research will 
be published in peer-reviewed scientific journals. Both positive and negative results of the 
study will be disclosed. The article's authorship shall be determined in appropriate 
consultation based on a considerable contribution to the set-up and execution of the study 
and active participation in publication. 

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