Bern Birth Cohort / Trajectory of Microbiota Maturation in Healthy Bern Infants - a Network Approach
Conditions: Maturation of the Healthy Infant Intestinal Microbiota, Microbial Colonization, Nutrition Disorder, Infant, Milk Expression, Breast, Mental Health Disorder
Sponsor: Insel Gruppe AG, University Hospital Bern

 
Bern Infant Microbiota Study, Version 7.0, 20.05.2022 
Universitätsklinik für Viszerale Chirurgie und Medizin 
Klinikdirektoren:  
Prof. Dr. med. Daniel Candinas 
Prof. Dr. med. Andrew J.S. Macpherson 
Prof. Dr. med. Jean-François Dufour 
Project Title 
“Trajectory of microbiota maturation in healthy Bern infants – a network approach” 
 
 
 
Research legislation: 
 Ordinance on human research with the exception of Clinical trials 
(HRO) [1]. 
 
 
Type of Research Project: 
Research project involving human subjects  
 
 
Risk Categorisation:  
Risk Category A 
 
 
Project Leader 
 
 
Prof. Dr. med. Benjamin Misselwitz 
Universitätsklinik für Viszerale Chirurgie und Medizin 
Inselspital Bern, Freiburgstrasse 18, 3010 Bern 
benjamin.misselwitz@insel.ch 
 
 
Co-Investigator Kinderspital  
PD Dr. med. Christiane Sokollik  
Universitätsklinik für Kinderheilkunde, Inselspital Bern  
Freiburgstrasse 15, 3010 Bern 
christiane.sokollik@insel.ch 
 
 
Co-Investigator DBMR 
Prof. Dr. Stephanie Ganal-Vonarburg 
Department for BioMedical Research 
Universitätsklinik für Viszerale Chirurgie und Medizin 
Murtenstrasse 35, 3008 Bern 
Stephanie.ganal@dbmr.unibe.ch 
 
 

 
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TABLE OF CONTENTS 
TABLE OF CONTENTS 
3 
GLOSSARY OF ABBREVATIONS 
4 
1 BACKGROUND AND PROJECT RATIONALE 
5 
2 PROJECT OBJECTIVES AND DESIGN 
5 
2.1 Hypotheses 
7 
2.2 Objectives and Endpoints 
7 
2.3 Project design 
8 
3 PROJECT POPULATION AND STUDY PROCEDURES 
8 
3.1 Project population, inclusion and exclusion criteria 
8 
3.2 Recruitment, screening and informed consent procedure 
9 
3.3 Study procedures 
9 
3.4 Withdrawal and discontinuation 
11 
4. STATISTICS AND METHODOLOGY 
12 
4.1. Statistical analysis plan 
12 
4.2. Handling of missing data 
12 
5 REGULATORY ASPECTS AND SAFETY 
12 
5.1 Local regulations / Declaration of Helsinki 
12 
5.2 Notification of safety and protective measures (HRO Art. 20) 
12 
5.3 Serious events (HRO Art. 21) 
12 
5.4 Radiation 
13 
5.5 Amendments 
13 
5.6 End of project 
13 
5.7 Insurance 
13 
6 FURTHER ASPECTS 
13 
6.1 Overall ethical considerations 
13 
6.2 Risk-Benefit Assessment 
14 
6.3 Rationale for the inclusion of vulnerable participants 
14 
7 QUALITY CONTROL AND DATA PROTECTION 
14 
7.1 Quality measures 
14 
7.2 Data recording and source data 
14 
7.3 Confidentiality and coding 
15 
7.4 Retention and destruction of study data and biological material 
16 
8  FUNDING / PUBLICATION / DECLARATION OF INTEREST 
16 
9 REFERENCES 
16 
 
 

 
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GLOSSARY OF ABBREVATIONS 
 
BASEC 
Business Administration System for Ethical Committees 
CRF 
Case report form 
FOPH 
Federal Office of Public Health 
HRA 
Human Research Act 
HRO  
Ordinance on Human 
 
 
 

 
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1 
BACKGROUND AND PROJECT RATIONALE  
The human intestine represents a large interface between the host and the environment, 
mediating absorption of nutrients, minerals and water and defence against pathogens. Intestinal 
functionality is critically shaped by the intestinal microbiota, comprising bacteria, eukaryotes, 
viruses and archaea colonizing the gastrointestinal tract [1, 2]. The intestinal microbiota maintains 
a mutually beneficial relationship with the host which together form a superorganism of which the 
microbiota contributes more than 99% of the combined genomic content [3]. 
Until recently, the complexity and diversity of the microbiota overwhelmed classical culture 
techniques. These limitations have now been surmounted by the extreme analytic depth and high-
throughput of ‘omics techniques (genomics, proteomics, metabolomics). The resultant data attest 
to the instability of the microbiota in early life, its later variability between individuals according to 
lifestyle, nutrition, ethnicity and culture [4] and its association with incidence and severity of 
human disease [5, 6].  
The development of the intestinal microbiota is incompletely understood. However, understanding 
the trajectory of microbiota maturation is likely key to the understanding of the pathogenesis of 
many pathologies in childhood. The intestine is almost sterile in utero. Postnatally mammals 
become colonized with successive waves of microbes seeding from the maternal microbiota. As 
a first step, within the first days after birth, in healthy full-term newborns the intestine is colonized 
by facultative anaerobic bacteria, corresponding to the initial oxygen availability of the newborn 
gut [7, 8]. Relevant species include Enterococcus spp., Escherichia coli, Staphylococcus spp. 
and a-hemolytic streptococci. Oxygen consumption by these bacteria enables further microbiota 
maturation with acquisition of Bacteriodes, Clostridium spp and Bifidobacterium which later 
dominate the healthy infant microbiota [7-9]. The introduction of solid food leads to a progressively 
increased 
diversity 
and 
the 
presence 
of 
Eubacteria, 
Veillonella, 
Fusobacteria, 
Peptidostreptococci and Ruminococci [8].  
The early-life microbiota is unstable as incoming microbes initially populate open ecological 
niches, within a variable environment shaped by nutrition, xenobiotics [10, 11] and protective 
factors [12, 13]. Subsequently, the microbiota matures and reaches far greater stability provided 
the environmental conditions are constant [14].  
Various factors are known to influence the infant microbiota [9, 15]: i) low gestational age leads 
to a dominant colonization with Gram-negative bacilli which in turn predispose infants to infections 
[16]. On the other hand, gestational age also determined the timing of intestinal colonization by 
bifidobacteria [17]. ii) feeding crucially affects microbiota composition with higher levels of 
Bifidobacteria in breast-fed infants and higher levels of bacteroides in formula-fed infants [18]. 
Vice versa, weaning was associated with acquisition of a more adult microbiota [19, 20]. iii) C-
section leads to an infant intestinal microbiota more similar to the maternal cutaneous microbiota 
[21] and differences persist also at 12 months after delivery [20]. iv) the number of siblings also 
influenced the infant microbiota and the presence of older siblings was associated with higher 
bacteria diversity [22]. v) early hospitalization and antimicrobial therapy were shown to affect the 
infant microbiota [9, 15].  
Lactation critically impacts on the health and development of infants. Human milk contains 
oligosaccharides [23] which will reach and shape the infant intestinal microbiota [24], resulting in 
an enrichment of Bifidobacterium species which can represent 70-90% of the faecal community 
[25]. Besides maternal skin bacteria, Bifidobacteria were also identified in breast milk, suggesting 
transfer. Neither the impact of maternal bacteria transferred by breastfeeding nor variations of 
milk constituents on infant intestinal microbiota and well-being have been sufficiently studied [24]. 
It is widely accepted that microbiota composition critically impacts on human health. One 
important benefit of the microbiota is based on the concept of ‘colonisation resistance’: the 

 
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healthy, beneficial intestinal microbiota competes with potentially pathological microbes and thus 
limits vulnerability to intestinal pathogens [26]. Reduced microbiota diversity on the other hand 
decreases resistance against pathogens such as Enterococci [27, 28]. Such microbiota fails to 
provide the beneficial properties of a functional microbiota and may therefore be considered 
‘dysbiotic’. Via transplantation of a healthy microbiota by stool transplantation, this dysbiotic state 
can be revoked and diseases like Clostridium difficile colitis can be cured [29]. Additional 
beneficial microbiota effects include vitamin synthesis, bile salt metabolism and xenobiotic 
detoxification [30]. 
Early microbiota perturbations in infants have been associated with subsequent pathology 
including autoimmune diseases such as type 1 diabetes mellitus [31], childhood atopy and 
asthma [32], childhood obesity [33] and intestinal conditions such as inflammatory bowel diseases 
[34]. The infant microbiota might also impact on the development of motor, social and cognitive 
functions [35] as well as abdominal pain.  
On a global perspective, the most important set of microbiota associated problems for human 
health is in child development. Over 3 million children globally die annually before the age of 5 
years [36]. Further, ~200 million children annually do not reach their developmental potential [37]. 
Malnutrition is especially important in children <2 years [38], and stunts the physical growth and 
cognitive development of survivors; for girls, it also limits the ability to deliver and raise healthy 
children later in life [39]. Childhood malnutrition is to a large degree resistant to simple caloric 
supplementation [24]. Poor health due to low-resources with inadequate nutrition and poor 
hygiene is accompanied by chronic intestinal maladaptive and inflammatory alterations, 
summarized as environmental enteropathy (EE). EE is also characterized by a delayed and 
aberrant maturation of the intestinal microbiota [40, 41], ultimately resulting in dysfunction and 
failure of the microbiota metabolic organ [42].  
Environmental enteropathy is associated with abnormally dense microbial consortia, low-grade 
inflammation, and poor intestinal function [43], which further aggravates infant malnutrition [42, 
44]. Therefore, malnutrition and its consequences of stunted growth and impaired cognitive 
development cannot be attributed solely to food insecurity: early life microbial colonization is 
critical [45, 46] and severe acute malnutrition can be limited and recovery accelerated by 
antibiotics to supplement therapeutic food [47]. Detailed associations with mutualists and 
pathobionts leading to EE are unclear and it is unknown whether these organisms are transmitted 
from the environment or from the mother at birth or during lactation.  
In any case, considering microbiota complexity and functional redundancy, species composition 
alone (on its own) is an insufficient descriptor for metabolic activities [48]. Therefore, recent work 
utilized bacterial full genome metagenomic sequencing to additionally identify bacterial genes 
present and expressed in relevant contexts. In combination with mass spectrometry, the analysis 
can then be constrained to the  actual small molecule metabolites present to even further specify 
relevant mechanisms [49]. Finally, advanced network analysis can subsequently provide a 
comprehensive view of active metabolic pathways [13, 50-52]. Such an approach has not yet 
been applied to the development of the microbiota.  
In summary, there is excellent evidence that microbiota composition is fundamental to human 
health and that the microbiota undergoes critical changes within the first two years of life. We are 
planning to perform a comprehensive analysis using advanced microbiota features and small 
molecule composition of stool samples as well as features of maternal microbiota and maternal 
milk together with clinical characteristics and parameters for infant nutrition and hygiene to 
understand the development of the normal infant microbiota within the first two years of life. Our 
results will allow a better understanding of microbiota pathologies in many conditions including 
environmental enteropathy.  
 

 
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2 
PROJECT OBJECTIVES AND DESIGN  
2.1 
Hypotheses  
Primary hypothesis: Maturation of the intestinal microbiota within the first two years of life is 
characterized by step-wise acquisition of bacteria with an increasing complexity and its trajectory 
can be followed by a comprehensive network analysis of bacteria metabolic activity and 
metabolites.  
Secondary hypothesis:  
1) Infant nutrition and socioeconomic status predict microbiota development in healthy 
children 
2) Maternal microbiota is transferred via maternal milk and/ or skin contact to the newborn 
baby 
3) Infant microbiota maturation characteristics predict:  
a. Growth and weight gain of healthy children  
b. Neurodevelopment of healthy children 
c. Adverse outcomes including obesity, abdominal pain, infectious diseases and 
allergies  
4) In direct comparison with children from the University of Zimbabwe birth cohort, 
trajectories of the microbiota maturation of Swiss infants will resemble healthy 
Zimbabwean children but will strongly differ from African children with malnutrition and 
stunted growth.  
5) Intestinal microbiota composition of the mother determines composition of the breast 
milk regarding its immune-modulatory properties. This affects immune maturation in 
the newborn. 
6) The maternal placenta is expected to be sterile and therefore has no impact on 
intestinal microbiota composition of the newborn. 
2.2 Objectives and Endpoints 
The objectives are explorative in nature and the study should be considered a pilot study. The 
endpoints are not assessed by fixed parameters but are defined by the achievement of said 
objectives. Therefore, objectives and endpoints were combined into one single section.  
 
Primary objective and primary endpoint 
We are aiming for a deep understanding of the maturation of a healthy infant intestinal microbiota 
considering composition, diversity and metabolic activities. We will characterize the composition, 
metabolic potential and activity at various time points by advanced techniques (16S sequencing, 
metagenomics shotgun sequencing and mRNA sequencing) and the metabolites present by mass 
spectrometry. Using this information, we will estimate networks of metabolic activity of the 
microbiota. Network analysis can be informed by information regarding small molecules present. 
The trajectories shared by the microbiota of most healthy infants will be considered normal (for 
details see 3.3 Study procedures). 
 
Secondary objectives and secondary endpoints 
1) To understand the impact of variations of the normal environment in Switzerland on 
microbiota development. To this end we will use parameters for nutrition and 
socioeconomic status, microbiota characteristics,  and metabolomics and correlate 
those with parameters for infant development and immunity. 
2) To understand the transfer of the maternal microbiota to the infant. We will identify 
bacterial species (or operational taxonomic units, OTU) in the maternal microbiota in 
maternal stool, maternal skin, vaginal environment, maternal placenta, as well as 

 
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maternal milk at various points in time and correlate these parameters to identified 
species/ OTU in the intestinal and skin microbiota of the infant at various points in time.  
3) To understand the impact of the microbiota on child development and health. We will 
correlate infant microbiota characteristics from the primary endpoint with 
a. Parameters for physical development (size, weight, percentiles, head 
circumference, mid upper arm circumference, waist to hip ratio) 
b. Parameters for neurodevelopment and child behaviour  
c. Onset/ occurrence of pathology (obesity, abdominal pain, new onset of 
allergies, asthma, eczema, number of infectious complications, physician 
consultations outside regular preventive medical check-ups).   
4) To understand the impact of low resources with poor nutrition and poor hygiene in 
developing countries on the maturation of the intestinal microbiota. Children from the 
University of Zimbabwe birth cohort were followed in a similar manner as planned for 
the children from the Bern infant microbiota study with the same acquisition of 
biosamples. We will use microbiota characteristics from endpoint 1 to compare 
microbiota maturation in healthy Swiss infants to microbiota maturation in healthy 
Zimbabwean children as well as children with environmental enteropathy and stunted 
growth.  
5) To understand the extent to which the maternal microbiota and maternal diet affects 
the immunomodulatory properties of breast milk and how those properties in turn 
influence immune maturation in the newborn, we will analyse the composition of stool 
and breast milk samples in regard to metabolites, cellular components, cytokines and 
miRNA. We will further use the material  to test the impact of maternal milk in vitro (cell 
culture) and in vivo (mice). Immunomodulatory mechanisms will be identified and 
correlating changes in development and maturation of the immune system of the 
newborn will be analysed.  
2.3 Project design  
We will perform a non-interventional, single center exploratory study. With the help from local 
gynecologists and pediatricians we will enroll mothers from week 20 of pregnancy until birth. 
Participants will be followed as mother-baby pairs until 10 years after birth. Using questionnaires, 
we will assess mother and infant nutrition, clinical parameters, sociodemographic parameters and 
parameters for child development. For clinical outcomes we will mainly rely on the results of 
regular preventive medical checkups performed by pediatricians (“Vorsorgeuntersuchungen”) but 
also acquire anthropometric data (mid-upper arm circumference, MUAC, and waist to hip ratio) 
as well as information regarding mother and infant nutrition using a standard weighted food record 
protocol conducted over 1-3 days as well as validated food frequency questionnaires, infectious 
complications, allergies, abdominal pain and psychological outcomes by questionnaires. 
Biosampling will be performed at enrolment (maternal stool and skin swabs, optional maternal 
vaginal swabs) and during follow-up (infant stool, infant skin swabs, maternal stool, maternal skin 
swabs, maternal milk).  
3 PROJECT POPULATION AND STUDY PROCEDURES 
3.1 Project population, inclusion and exclusion criteria  
We are aiming to recruit 250 pregnant participants which will be followed as mother baby pairs.  
General inclusion criteria: 
• 
Signed informed consent. 
• 
Ability to understand and follow study procedures and understand informed consent 
• 
From week 20 of pregnancy until birth 

 
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• 
General good health, i.e. absence of major severe medical/ surgical/ psychiatric condition 
requiring ongoing management. Minor well controlled conditions (e.g. medically controlled 
arterial hypertension, occupational asthma, gestational diabetes mellitus) may be present.  
• 
Absence of known severe embryonal pathology, expected normal pregnancy (e.g. minor 
conditions including twin/ triplet pregnancy, final pelvic position may be present) 
• 
Age 18-45 years. 
 
General exclusion criteria: 
• 
Participation in another clinical study interfering with study procedures. 
 
3.2 Recruitment, screening and informed consent procedure 
Patients will be recruited by the project team and co-workers in collaboration with pediatricians 
and gynecologists at the Inselspital and in the Bern area. We will preferentially include mothers 
known to local pediatricians during a next pregnancy.  
The patient will be provided with information regarding the study. If the patient shows interest, the 
investigator will explain the study protocol, answer questions, check inclusion and exclusion 
criteria and inquire further about the patient’s current symptoms, medication and medical history. 
In case all criteria are met, patients will be asked to sign the informed consent.   
At the last time point, when the age of the child is 10 years, the study will be explained to the child 
in appropriate terms. If the child does not want to provide information/ biosamples at this point in 
time, this will be respected. Information and biosamples up to this time point will be evaluated and 
statistically analyzed.  
Patients will be compensated for any additional expenses related to the study (e.g. travel). For 
this study, no compensatory fee will be payed.  
3.3 Study procedures 
3.3.1. Visits and procedures 
The project duration for each participant will be from inclusion during pregnancy to 10 years after 
birth. 
 
Table 1: Timeline of study procedures  
Time 
± tolerance 
interval  
Enrollment 
Birth 
Days after birth 
Weeks after birth 
 
Years 
after 
birth 
0 
0-3  
±3 
10  
±5 
 
6 
±2 
10 
±4 
14 
±4 
24 
±6 
36 
±8 
48 
±8  
96 
±12 
5 
±1 
10 
±1 
Information about 
the study 
+ 
 
 
 
 
 
 
 
 
 
 
 
 
Check inclusion/ 
exclusion criteria 
+ 
 
 
 
 
 
 
 
 
 
 
 
 
Signing of written 
informed consent 
+ 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
+ 
 
 
 
 
 
 
 
 
 
 
 
 
Baseline 
questionnaire 
+ 
 
 
 
 
 
 
 
 
 
 
 
 
In-depth dietary 
questionnaires of 
mothers 
 
 
 
 
+ 
 
+ 
+ 
 
 
 
 
 

 
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(optional) – 1-3 
days weighed 
food record 
food frequency 
questionnaire  of 
the mother 
+ 
 
 
 
+ 
 
+ 
+ 
 
 
 
 
 
In-depth dietary 
questionnaires of 
infant (optional) – 
1-3 days weighed 
food record 
 
 
 
 
 
 
 
 
 
 + 
+ 
+ 
+ 
food frequency 
questionnaire of 
the infant 
 
 
 
 
 
 
 
 
 
+ 
+ 
+ 
+ 
Anthropometric 
measures 
 
 
 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
Follow-up 
questionnaire 
 
 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
Maternal stool 
samples 
+ 
 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
Maternal skin 
swab 
+ 
 
 
 
+ 
 
 
+ 
 
+ 
+ 
+ 
+ 
Maternal vaginal 
samples 
+ 
 
 
 
 
 
 
 
 
 
 
 
 
Maternal placenta 
sample 
 
+ 
 
 
 
 
 
 
 
 
 
 
 
Maternal milk 
samples, if 
nursing 
 
 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
 
 
Infant stool 
samples 
 
 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
Infant skin swabs 
 
 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
Child Behaviour 
Check list (CBCL) 
 
 
 
 
 
 
 
 
 
+ 
 
+ 
+ 
 
3.3.2. Material sampling 
Sampling will be performed/ assisted by the study team.  
- 
For maternal stool samples, a sampling kit will be provided to the participant. 
- 
If specific consent is given, vaginal swabs will be retrieved. 
- 
At birth, small samples will be taken from the placenta. 
- 
Infant stool samples will be acquired from infant diapers. In case sampling is not possible 
during the visit, a sampling kit with instructions for sampling will be provided. 
- 
Maternal and infant skin swabs will be acquired in the inguinal region 
- 
For acquisition of milk samples, the breast will first be cleaned followed by acquisition of 
1-2ml of milk by squeezing of the breast. The first drops of acquired milk will be discarded.  
 
3.3.3. Methods for sample analysis: 
For the primary objective and outcome/ endpoint of our study, bacterial content of infant stool 
will be analyzed by:  
- 
Mass spectrometry to assess intestinal content (metabolome). Techniques have been 
established in the laboratory of Prof. U. Sauer who already collaborated with our group in 
previous studies.  
- 
16S rRNA sequencing for bacterial species composition as well as microbial diversity. 
- 
Bacterial full genome metagenomics shotgun sequencing to identify bacterial genes 
present (metabolic potential of the microbiota).  
- 
Bacterial mRNA sequencing to assess transcription and a functional role of the microbiota 
(metabolic activity of microbiota).  
- 
Analysis of the intestinal virome and eukaryotic intestinal populations by appropriate 
sequencing or culturing techniques. 

 
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- 
Analysis of IgA antibodies in human milk and stool and the interaction of antibodies with 
intestinal bacteria. 
 
- 
For the secondary endpoints identical analyses will be performed in skin swabs, maternal 
milk, maternal vaginal swabs, maternal placenta probes and maternal stool. Parameters 
for infant growth, neurodevelopment, immune maturation and potential occurrence of 
pathology will be assessed at every visit. Maternal and infant nutrition, hygiene, 
socioeconomic status and clinical history will be assessed by questionnaires at every visit. 
All methods described above have already been used for microbiota analyses in human 
and murine samples for previous unrelated questions [13, 50-52]. Milk samples will further 
be analyzed for their cellular contents by flow cytometry and single cell RNA-sequencing, 
as well as for cytokines and exosome-based miRNAs. All biosamples will be analyzed by 
mass spectrometry to assess impact of the environment on infant metabolism and 
physiology. 
 
 
Further follow-up experiments with the acquired samples are possible. Specifically, individual 
bacteria strains can be isolated and cultured in vitro and also tested alone or in combination in 
experimental animals.  
 
Bacterial sequencing by the methods described above will also inevitably identify maternal or 
infant DNA sequences, since metagenomic shotgun sequencing cannot differentiate between 
bacterial and human DNA. These human DNA sequences will not be analyzed within the scope 
of this project. However, these sequences might be the subject of future studies. Study 
participants will therefore be asked for permission to analyze human DNA from mother and/ or 
child at the page for “further analyses” within the consent form. An option to “opt out” for human 
DNA analysis will be provided and refusal will not lead to exclusion from the study.  
Any findings of clear relevance to the health of the participant (i.e. mother or child) will be reported 
to the participant in collaboration with their treating pediatrician. Participants will need to inform 
the study team if they do not wish to be informed.    
3.4 Withdrawal and discontinuation 
If an individual withdraws informed consent during the investigation he/she will be withdrawn from 
the project. Additional reasons for withdrawal include clear cases of non-compliance with study 
procedures where the integrity of the samples/questionnaires is clearly in question.  
Samples that belong to patients that discontinue/ drop-out during the study (e.g. attend to only to 
initial visit but fail to attend some of the following visits) or withdraw informed consent will still be 
considered for study purposes and analyzed accordingly. This approach allows the utilization of 
an ‘intention to treat’ analysis. By using all collected samples, we aim to reduce confounding 
factors which would otherwise critically compromise the validity of the generated data considering 
the small number of participants.  
Further, considering the length of the project it is also very likely that at the time point of withdrawal 
generated data from the first study period have already been analyzed and/ or published which 
cannot be undone. 
Subjects who discontinue from the study or withdraw consent should always be asked about the 
reason(s) for their discontinuation and about the presence of any adverse events. Adverse events 
should be followed up.  
At 10 years of age the child will be informed about study participation in appropriate terms. If the 
child expresses unwillingness to continue the last time point of the study this will be respected. If 
the child expresses the wish for withdrawal of the informed consent provided by the mother, this 
will also be respected.  

 
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4. STATISTICS AND METHODOLOGY 
4.1. Statistical analysis plan 
Only very limited knowledge exists regarding networks of bacterium-metabolite interactions in the 
small intestine. Therefore, a formal power analysis cannot be performed and the current study 
should be considered a pilot study. The number of study participants has been chosen for 
practical reasons for this pilot study, also considering capacities of our lab for the expected 
analyses. 
 
Data from the current study will enable to build hypothesis and perform power analyses for future 
trials. Observed effects and interactions will be statistically substantiated by parametric and non-
parametric tests and/ or multivariate analyses. A p-value <0.05 will be considered significant. 
Infant intestinal microbiota will be compared to maternal microbiota in stool samples, milk 
samples, skin swabs, vaginal swabs and maternal placenta probes. Therefore, a microbiota 
clustering analysis based on a Bray-Curtis dissimilarity principal coordinate analysis (PCoA) will 
be performed.  
A pipeline for detailed assessment of sequence data (QIIME v1.9.1 and several R packages 
including phyloseq, ggplot2, shape, ape, vegan and Maaslin) and further measures including a-, 
b-diversity and Bray-Curtis dissimilarity principal coordinate analysis (PCoA) has been 
established at DBMR, University of Bern and will be available for this project. 
4.2. Handling of missing data  
The nature of this exploratory study allows for some tolerance regarding missing data. Patients 
with a limited number of missing samples will not be replaced as long as the missing data does 
not compromise assessment of microbiota characteristics. Due to the explorative nature of our 
study, a specific threshold for participant exclusion/ replacement cannot be provided. Patients 
with an insufficient number of biosamples can be replaced by newly recruited individuals. 
Dropouts within the first 2 years can also be replaced by newly recruited individuals, if feasible 
until the final number of individuals indicated at 3.1 will be reached. 
5 
REGULATORY ASPECTS AND SAFETY 
5.1 Local regulations / Declaration of Helsinki 
This research project will be conducted in accordance with the protocol, the Declaration of 
Helsinki [3], the principles of Good Clinical Practice, the Human Research Act (HRA) and the 
Human Research Ordinance (HRO) [1] as well as other locally relevant regulations. The Project 
Leader acknowledges his responsibilities as both the Project Leader and the Sponsor. 
5.2 Notification of safety and protective measures (HRO Art. 20) 
The project leader/ sponsor is promptly notified (within 24 hours) if immediate safety and 
protective measures have to be taken during the conduct of the research project. The Ethics 
Committee will be notified via BASEC of these measures and of the circumstances necessitating 
them within 7 days. 
5.3 Serious events (HRO Art. 21) 

 
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If a serious event occurs, the research project will be interrupted and the Ethics Committee 
notified on the circumstances via BASEC within 7 days according to HRO Art. 211. 
5.4 Radiation 
Not applicable (patients will not be exposed to radiation for this study). 
5.5 Amendments 
Substantial changes to the project set-up, the protocol and relevant project documents will be 
submitted to the Ethics Committee for approval according to HRO Art. 18 before implementation. 
Exceptions are measures that have to be taken immediately in order to protect the participants.  
5.6 End of project 
All encrypted biological materials will be stored in a biobank at Inselspital Bern for 10 years under 
the responsibility of the sponsor. Clinical data (questionnaires and digitalized material) will be 
stored by Prof. Dr. med. Benjamin Misselwitz for 10 years after end of study or publication of data 
analysis (whatever comes last). 
5.7 Insurance 
In the event of project-related damage or injuries, the liability of Inselspital Bern provides 
compensation, except for claims that arise from misconduct or gross negligence. 
6 
FURTHER ASPECTS 
6.1 Overall ethical considerations 
The intestinal microbiota critically impacts on utilization of nutrients, host metabolism, immunity 
and defense. The importance of the host-microbiota interaction is increasingly recognized. The 
maturation of the intestinal microbiota is insufficiently understood and has not been sufficiently 
investigated with state-of-the-art analysis techniques and network analyses. However, microbiota 
maturation likely critically impacts on infant development, health and subsequent diseases (see 
introduction). Insights gained during this study might provide a solid framework for further 
investigations and development of future treatment and prevention strategies for many diseases 
related to the intestinal microbiota.  
 
This observational study involves only minor burden to the participants, but does not require any 
modification of clinical care and does not pose any risk to participants (see 6.2., risk-benefit 
assessment). The study requires handling of their own stool by participants, which might be 
considered inconvenient or unpleasant by some participants. The study requires several study 
visits, which poses an inconvenience for the participants. During these visits, participants 
(mothers) will fill in questionnaires and non-invasive sampling of biomaterials will be performed. 
All procedures are not associated with risks to patients (see 6.2 risk-benefit assessment). The 
extensive sampling during this study is crucial for answering the primary and all secondary 
endpoints.  
 
Individuals will be informed about any incidental finding with clear implications regarding the 
health of an individual. However, given the pilot character of the current study and the current 
 
1 A serious event is defined as any adverse event where it cannot be excluded, that the event is attributable to the 
sampling of biological material or the collection of health-related personal data, and which: 
a. requires inpatient treatment not envisaged in the protocol or extends a current hospital stay; 
b. results in permanent or significant incapacity or disability; or 
c. is life-threatening or results in death. 

 
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understanding on the topic of study the microbiota information obtained during this study is 
unlikely to have an impact on individual patient health. 
 
Overall, the very low risk of the study, only minor inconveniences associated with the study and 
the chance of helping to improve medical care for infants ensure an overall fair balance for each 
study participant.  
6.2 Risk-Benefit Assessment 
None of the study procedures is associated with risks for relevant complications.  
Stool sampling: Handling of one’s own stool (for mothers) is not associated with any risk. 
Similarly, infant stool is not considered infectious and handling infant’s stool (i.e. changing 
diapers) is part of daily routine baby care anyways.  
Skin swabs: are considered to be entirely safe 
Sampling of maternal milk: is also considered entirely safe 
Vaginal swabs: are also considered to be entirely safe 
Placenta probes: the collection of tissue samples from an expelled placenta is also considered 
risk-free 
For these reasons (lack of risks, potential benefits for future patients) the potential benefit 
outweighs the potential risks in this study. 
6.3 Rationale for the inclusion of vulnerable participants  
This study will be observing healthy pregnant women and healthy infants; information regarding 
healthy infant and maternal microbiota cannot be obtained by other means. When informed 
consent is obtained, we will carefully explain the right to refuse and to withdraw informed consent. 
7 
QUALITY CONTROL AND DATA PROTECTION 
7.1 Quality measures  
All individuals involved in the project will be trained regarding study procedures. The sponsor 
(Prof. Dr. B. Misselwitz) will continuously monitor the appropriate handling of data and samples.  
In case the sponsor leaves the Inselspital Bern before completion of the project, the sponsorship 
will be transferred preferably to a member of the study team. If this should not be possible, the 
sponsorship will be transferred to a long-term employee of the Universitätsklinik für Viscerale 
Chirurgie und Medizin or the Universitätsklinik für Kinderheilkunde of the Inselspital Bern. 
However, as all participants of the study team are long-term employees of the Inselspital Bern, 
we are not expecting any change of sponsorship. 
For quality assurance the Ethics Committee may visit the research sites. Direct access to the 
source data and all project related files and documents must be granted on such occasions. 
7.2 Data recording and source data 
Study data is recorded in the worldwide used research electronic data capture application 
REDCapTM. This study database is run by the Clinical Trial Unit (CTU) of the University of Bern. 
Data is physically stored on servers of the University of Bern that are located in Bern, Switzerland. 
 
Source data include the medical history of the patient, questionnaires filled out by the patient and 
our structured study documentation. Source data will be collected during screening and follow-
up, coded and entered in the electronic database (electronic CRF, eCRF). During and at the end 

 
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of the project period data will be transferred into the electronic database. After the end of data 
collection the database will be frozen. 
7.3 Confidentiality and coding 
Project data will be handled with uttermost discretion and is only accessible to authorized 
personnel who require the data to fulfil their duties within the scope of the research project. On 
the CRFs and other project specific documents, participants are only identified by a unique 
participant number.  
 
Access to source documents will be permitted for purposes of monitoring and audits. All involved 
parties must keep the participant data strictly confidential. Data protection will be based on local 
and national guidelines and applicable local and national law. The anonymity of the participants 
will be guaranteed when presenting the data at scientific meetings or publishing them in scientific 
journals. Individual subject medical information obtained as a result of this study is considered 
confidential and disclosure to third parties is prohibited. 
 
All data entered into the eCRF are transferred to the database using Secure Sockets Layer (SSL) 
encryption. Each data point has attributes attached to it identifying the user who entered it with 
the exact time and date. Retrospective alterations of data in the database are recorded in an audit 
table. Time, table, data field and altered value, and the person are recorded (audit trail).  
 
The server hosting the EDC system and the database is kept in a locked server-room. Only the 
system administrators have direct access to the server and back-up tapes. A role concept with 
personal passwords (site investigator, statistician, monitor, administrator etc.) regulates 
permission for each user to use the system and database as he/she requires. 
 
A multi-level back-up system is implemented. Back-ups of the whole system including the 
database are run internally several times per day and externally backed up once a day.  
 
At interim or final analyses, data files will be extracted from the database into statistical packages 
to be analyzed. The status of the database at this time is recorded in special archive tables. The 
study database with all archive tables will be securely stored by CTU Bern for at least 10 years. 
The sponsor also keeps the Trial Master File and interim and final reports both in electronic and 
in hard copy form for at least 10 years. 
 
The investigator will create and Trial Master File and is responsible to keep this Investigator Site 
File updated and available for review by authorized persons. All paper documents will be stored 
at the investigator’s sites. All documents pertaining to the conduct of the study must be kept by 
the investigator for a period of 10 years. 
 
The investigator is responsible for treating patients’ study information as confidential. The 
investigator ensures that identifying patient data (name, date of birth, initials) will not be made 
publicly available. All patient records are identified by the patient identification number (that must 
not contain patient’s data of birth or initials) to maintain patient’s confidentiality. Identification code 
lists that link the subjects’ names to the subjects’ identification number must be stored in the 
Investigator Site File. 
 
Biological material in this project is not identified by participant name but by a unique study ID. 
Biological material is appropriately stored in a restricted area only accessible to authorized 
personnel. 
The samples will be stored at the Mucosal Immunology Laboratory, Gastroenterology Group from 
Benjamin Misselwitz, Stephanie Ganal-Vonarburg and Andrew Macpherson, in Bern and the PI, 
Benjamin Misselwitz and collaborators from the lab who are actively involved in the study will 
have access to them. 

 
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All freezers have temperature control implemented (temperature is continuously monitored) and 
are alarm protected. Most analyses will be performed in the facilities of Bern University. Mass 
spectrometry will be performed in the laboratory of Prof. U. Sauer, Institute for Molecular Systems 
Biology, ETH Zürich. Otto-Stern-Weg 3, 8093 Zürich. Appropriate storage of samples and 
participant confidentiality is also guaranteed by our collaborators. 
7.4 Retention and destruction of study data and biological material 
The data and samples collected during this study are stored for 10 years after the end of the 
study. It is possible that the data and samples will be reused at a later date for other examinations 
(projects), or later be sent to a different database/biobank in Switzerland or abroad for further 
studies not yet defined (reuse). This other database/biobank must meet the same standards as 
the database/biobank for this study. 
After termination of the study, samples will be autoclaved and put to the waste which will 
subsequently be burned. These procedures will be documented in our study documentation.  
 
7.5 Infrastructure 
The necessary infrastructure for this project including clinical care of participants, equipment for 
sequencing, bacterial culturing and further sample analysis is available at the Department of 
Visceral Surgery and Medicine and the Department of Biomedical Research, Inselspital Bern.  
8  FUNDING / PUBLICATION / DECLARATION OF INTEREST 
This study will be funded by Prof. Dr. med. Benjamin Misselwitz at Inselspital Bern and Prof. Dr. 
S. Ganal-Vonarburg, DBMR. Prof. Misselwitz and Prof. Ganal-Vonarburg will apply for additional 
funding for this project and follow-up projects (for instance at the Schweizerischer Nationalfonds 
(SNF)). Additional costs will be covered by the Department of biomedical research (DBMR) of the 
University of Bern.  
There are no conflicts of interest from the sponsor and the other investigators of this study. 
We are aiming to publish data from this project in a peer-reviewed journal. Authorships of all 
publications will reflect the relative contribution of a given investigator to this project and agreed 
on by mutual consent. Following current standard for scientific publishing, we will aim to make 
raw-data of this project available as electronic supplemental material to a publication. 
9 
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