Elucidating the Dynamics and Impact of the Gut Microbiome on Maternal Nutritional Status During Pregnancy
Conditions: Pregnancy Related, Pregnancy Loss, Pregnancy Complications, Pregnancy; Parasitic Disease, Microbial Colonization, Microbial Disease, Parasitic Disease, Metabolomics, Malnutrition, Malnutrition, Infant, Malnutrition Pregnancy, Breast Feeding
Sponsor: The Hospital for Sick Children

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Elucidating the Dynamics and Impact of the Gut 
Microbiome on Maternal Nutritional Status During 
Pregnancy 
 
 
 
 
 
 
Principal Investigator: Dr. John Parkinson 
 
 
 
 
 
 
 
Funded by: CIHR 
July 19, 2024 

 
 
                       July19, 2024  
 
 
 
1 
Table of Contents 
1 
PROTOCOL SUMMARY .................................................................................................. 3 
1.1 
Synopsis ..................................................................................................................... 3 
1.2 
Schema ...................................................................................................................... 5 
1.3 
Schedule of Activities (SoA) .................................................................................... 6 
2 
INTRODUCTION ............................................................................................................. 13 
2.1 
Study Rationale ....................................................................................................... 13 
2.2 
Background .............................................................................................................. 14 
2.2.1 
Undernutrition Before and During Pregnancy Dramatically Impacts 
Maternal Health, Birth and Life Outcomes....................................................................... 14 
2.2.2 
The Gut Microbiome Impacts Maternal and Infant Nutrition ............ 16 
2.2.1 
Gender Impacts Nutritional Status and the Microbiome .................. 16 
2.2.1 
The Toronto Population ......................................................................... 17 
2.2.2 
Pathogen Exposure Contributes to Environmental Enteric 
Dysfunction 
18 
2.2.3 
Eukaryotic Microbes are a Key Component of the Microbiome ...... 18 
2.2.4 
Marker Gene Surveys Coupled with Advances in Next Generation 
Sequencing Provide Unprecedented Insights into Microbial Communities ................ 18 
2.2.5 
Metagenomics, Metranscriptomics and Metabolomics Deliver 
Readouts of Microbiome Function .................................................................................... 19 
2.3 
Risk and Benefits .................................................................................................... 19 
3 
STUDY OBJECTIVES .................................................................................................... 20 
4 
STUDY DESIGN .............................................................................................................. 24 
4.1 
Study Hypothesis .................................................................................................... 24 
4.2 
Study Design ........................................................................................................... 25 
4.3 
Participating Sites ................................................................................................... 25 
4.4 
End of Study Definition .......................................................................................... 26 
5 
STUDY POPULATION .................................................................................................... 26 
5.1 
Inclusion Criteria ..................................................................................................... 27 
5.2 
Exclusion Criteria .................................................................................................... 27 
5.3 
Strategies for Recruitment and Retention........................................................... 27 
5.3.1 
Recruitment and Obtaining Consent ................................................... 27 
5.4 
Study Honourarium................................................................................................. 29 
6 
DISCONTINUATION AND WITHDRAWAL ................................................................. 29 
6.1 
Lost to Follow-Up .................................................................................................... 30 
6.2 
Removal of Participants ......................................................................................... 30 
7 
STUDY PROCEDURES ................................................................................................. 30 
7.1 
Study Visits .............................................................................................................. 31 
7.2 
Study Assessments and Sample Collection ....................................................... 34 
7.3 
Contact with Study Participants ............................................................................ 39 
7.4 
Safety and Adverse Events ................................................................................... 40 
7.4.1 
Reporting Events to Participants ......................................................... 40 
8 
EMPOWERMENT SUBSTUDY ..................................................................................... 41 
9 
WORKSHOPS .................................................................................................................. 42 
10 
STOOL AND BLOOD SAMPLE ANALYSIS ................................................................ 42 

 
 
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10.1 
Profiling Microbial Community Structure and Function..................................... 42 
10.1.1 
Profiling Microbial Community Structure ............................................ 42 
10.1.2 
Profiling Microbial Community Function ............................................. 44 
10.1.3 
Profiling of Patient and Microbiome Metabolites and Proteomics .. 44 
10.1.4 
Host Markers of Inflammation .............................................................. 44 
10.2 
Potential Problems, Mitigations and Expected Results .................................... 45 
11 
STATISTICAL CONSIDERATIONS .............................................................................. 45 
11.1 
Power Calculation ................................................................................................... 45 
11.2 
SubGroup Analysis ................................................................................................. 45 
11.3 
Statistical Analysis .................................................................................................. 46 
11.4 
Plan For Statistical Analysis .................................................................................. 46 
11.4.1 
Addressing the Primary Objectives ..................................................... 47 
11.4.2 
Addressing the Secondary Objectives ................................................ 48 
11.4.3 
Addressing the Exploratory Objectives ............................................... 49 
12 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS . 49 
12.1 
Regulatory, Ethical, and Study Oversight Considerations ............................... 49 
12.2 
Study Discontinuation and Closure ...................................................................... 50 
12.3 
Confidentiality and Privacy .................................................................................... 50 
12.3.1 
Participant ID System ............................................................................ 50 
12.4 
Future Use of Stored Specimens and Data ........................................................ 51 
12.5 
Protocol Deviations................................................................................................. 54 
12.6 
Conflict of Interest ................................................................................................... 55 
12.7 
Abbreviations ........................................................................................................... 55 
13 
REFERENCES ................................................................................................................. 57 
 
 
 
 
 
 

 
 
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3 
1 
PROTOCOL SUMMARY 
 
1.1 
SYNOPSIS  
 
Title 
Elucidating the Dynamics and Impact of the Gut Microbiome on Maternal Nutritional 
Status during Pregnancy. 
 
Study Short 
Title 
MMIP Study (Microbiome and Malnutrition in Pregnancy Study) 
Principal 
Investigator 
 
Dr. John Parkinson, PhD (Senior Scientist at the Hospital for Sick Children) 
Co-
Investigators 
Dr. Jessie Hulst, MD, PhD (Associate Professor Department of Pediatrics and 
Nutritional Sciences at the University of Toronto; Staff Pediatrician in the Division of 
Gastroenterology, Hepatology and Nutrition at the Hospital for Sick Children)  
Dr. Shazeen Suleman, MD, Msc, MPH (Assistant Professor in the Department of 
Pediatrics at the University of Toronto, and an Associate Scientist at the Li Ka Shing 
Knowledge Institute) 
Dr. Liana Kaufman, MSc. MD (Department of Family and Community Medicine, St. 
Michael’s Hospital) 
Dr. Rachel Spitzer, MD, MPH (Associate Staff, Department of Gynaecology at 
Sickkids and Assistant Professor, University of Toronto) 
Dr. Ashley Vandermorris, MD (Staff Physician, Department of Paediatrics at Sickkids 
Hospital) 
Dr. Nirmala Chandrasekaran, MD (Department of Obstetrics, St. Michael’s Hospital) 
Dr. Zulfiqar Bhutta, PhD, MBBS, FRCPCH, FAAP (Co-Director of Research Centre 
for Global Child Health, Senior Scientist the Hospital for Sick Children) 
Dr. Robert Bandsma, MD, PhD (Pediatric Gastroenterologist, Hepatologist and 
Nutritionist, at the Hospital for Sick Children) 
Dr. Jo-Anna Baxter, PhD, MSc (Centre for Global Child Health, Hospital for Sick 
Children) 
 
Collaborators Linda Moscovitch, RM, Riverdale Community Midwives (Privileges at St. Michael’s 
Hospital) 
Fariba Shodjaie, RM, Community Midwifes of Toronto (Privileges at St. Michael’s 
Hospital) 
Tia Sarkar, RM, Riverdale Community Midwives (Privileges at St. Michael’s 
Hospital) 
 
Dr. Arthur Mortha PhD (Assistant Professor in the Department of Immunology at the 
University of Toronto) 
Dr. Ben Willing, PhD (Associate Professor and Tier 2 Canada Research Chair in 
Microbiology of Nutrigenomics at the University of Alberta)  
Dr. Marie Claire Arrieta Mendez, PhD (Assistant Professor at the University of 
Calgary) 
Dr. Elena Comelli, PhD (Associate Professor and the Lawson Family Chair in 
Microbiome Nutrition Research in the Department of Nutritional Sciences at the 
University of Toronto) 
Dr. Michael Grigg, PhD (Chief of Molecular Parasitology at the NIH)  
Dr. Andrew Roger, PhD (Professor and Canada Research Chair in Comparative 
Genomics at Dalhousie University) 

 
 
                       July19, 2024  
 
 
 
4 
Research  
Coordinator 
Gowshigga Thamotharampillai, (Molecular Medicine at Hospital for Sick Children 
and Department of Pediatrics, Unity Health Toronto, St. Michael’s Hospital) 
Study Design 
Prospective, longitudinal observational study design 
 
Objectives 
 
Primary Objective: 
To assess if alterations of the microbiota in the maternal gut (dysbiosis) is associated 
with maternal gestational weight gain. 
 
To determine the association between maternal microbiome dysbiosis during 
pregnancy and birth outcomes, infant growth, nutritional status and morbidity in the 
first year of life.  
 
Secondary Objectives: 
 
To link the maternal microbiome to dietary intake, with a focus on calories and 
macronutrients. 
 
To integrate maternal anthropometric factors and morbidity, with microbiome data to 
reveal key modulators (microbial taxa and metabolites) of dietary intake during 
pregnancy and the post-partum period. 
 
To determine the impact of the maternal microbiome during pregnancy, including the 
exposure to pathogens and parasites, on the development of the infant microbiome. 
 
To investigate the maternal microbiome’s exposure to pathogens and parasites, and 
the association with intestinal inflammation. 
 
Exploratory Objectives: 
 
To explore the role of the maternal gut microbiome during pregnancy and to identify 
gut community dynamics in pregnant women and how this impacts differences 
between dietary intake and nutritional status. 
 
To investigate socio-economic factors; including gender, poverty, exclusion and 
empowerment, and their influence on the health of a mother’s microbiome (assessed 
by alpha and/or beta diversity, and absence of pathogens). 
 
To explore the role of the human microbiota on nutritional status by performing fecal 
microbiota transplants in germ free mice and sterile piglets. 
Follow-up 
Periods 
Baseline/Initial Recruitment: 8-20 weeks post conception 
2nd time point: 30-34 weeks post conception 
3rd time point: 4 month post-partum  
4th and final time point: 12 months post-partum 
Study 
Population: 
Pregnant women 28 years and younger living in the Toronto and Greater Toronto 
Area and their infants. 
 
No. of 
Subjects 
Total: 800 participants: 
400 women and their infants. 
• 
200 maternal subjects with normal BMI (20-24.9)  
• 
200 maternal subjects with low BMI (less than 20) 
 
Study 
Duration: 
The recruitment target should be met within four years. The study duration should be 
approximately 5 years. 

 
 
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Participant 
Duration: 
• 
Participants will be in the study for approximately 17-19 months.  
• 
Participants will join the study 8-20 weeks post-conception and end their 
participation in the study at 12 months post-partum. 
• 
The infants will be followed for 12 months, from their birth until 12 months of 
age. 
Collaborating 
Institutions:  
 
The Hospital for Sick Children 
 
St. Michael’s Hospital 
 
University of Toronto 
 
St. Michael’s Midwifery Division  
 
Aga Khan University 
 
Dalhousie University 
 
University of Calgary 
 
University of Alberta 
 
The NIH 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.2 
SCHEMA 
Figure 1: Study Flow Diagram 

 
 
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6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
      Infant visit timeline:  
 
 
          Post-Partum Maternal visits: 
         Note maternal and infant visits will take place at the same time as a joint visit.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.3 
SCHEDULE OF ACTIVITIES (SOA) 
 
Table 1: Overview of study visits, visit activities, and data collection  
Baseline (8-20 wks post- conception) 
A stool, blood and rectal swab sample will be obtained. The participant will 
complete 2x dietary recalls, the study questionnaires and have anthropometric 
measurements taken.  
30-34 wks post-conception 
A stool, blood and rectal swab sample will be obtained. The participant will 
complete 2x dietary recalls, the study questionnaires and have anthropometric 
measurements taken.  
Recruitment (8-20 wks post-conception): 
Screening, confirmation of pregnancy and informed consent. 
N=400 
Normal BMI=200  
Low BMI= 200 
Maternal: 4 months post-partum 
A stool and rectal swab sample will be 
obtained. 
The 
participant 
will 
complete the study questionnaires and 
have anthropometric measurements 
taken. A delivery assessment will be 
obtained from chart. 
Empowerment 
Sub-Study 
To be conducted 
during the final 
year of the study 
6-10 months of age 
Maternal participants will receive a 
check-in call to assess health status of 
infant. 
First infant visit: 4 months of age 
A stool and rectal swab sample will be 
collected. The questionnaires and 
anthropometric measurements will be 
obtained. Birth characteristics will be 
obtained from chart. 
 
6-10 months post-partum 
Maternal participants will receive a 
check-in call to assess their health 
status. 
Final visit: 12 months of age 
A stool, blood and rectal swab 
sample will be collected. The 
questionnaires and anthropometric 
measurements will be obtained.  
 
Final visit: 12 months post-partum 
A stool, blood and rectal swab sample 
will be obtained. The participant will 
complete 2x dietary recalls, the study 
questionnaires 
and 
have 
anthropometric measurements taken.  
. 
Workshops 
Development 
and 
Implementation 
To be conducted 
during the final 
year of the study 
 

 
 
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Procedures 
Prior to Initial Baseline 
Visit at ~10-16 Weeks 
Post-Conception 
Enrollment/Baseline 
Visit 1: 8-20 Weeks 
Post-Conception 
Study Visit 2: 30-34 
Weeks Post- 
Conception 
Study Visit 3: 
4 Months  Post-Partum 
Infant Study Visit 3: 4 
Months (same visit as 
mom) 
Maternal Check in Call: 
6-10 Months Post-
Partum 
Infant Check in Call: 6-
10 Months of Age 
Maternal Study Visit 4: 
12-Months Post-Partum 
 
Infant Study Visit 4: 12-
Months (same visit as 
mom) 
Invitation Email and/ or 
Letter 
 
X 
 
 
 
 
 
 
 
 
 
Screening for Eligibility 
and Pregnancy 
Confirmation 
 
X 
 
 
 
 
 
 
 
Informed Consent 
 
X 
 
 
 
 
 
 
 
Baseline demographics  
 
X 
 
 
 
 
 
 
 
Dietary Recall x 2 
(ASA24) 
 
X 
X 
 
 
 
 
X 
 
MDD-W 
Note: this will be 
administered to the 
participant. For the other 
time points the research 
staff will calculate the 
MDD-W from the dietary 
recall data 
 
 
 
X 
 
 
 
 
 
Stool Sample 
 
X 
X 
X 
X 
 
 
X 
X 
Blood Sample 
 
X 
X 
 
 
 
 
X 
X 
Rectal Swab 
 
X 
X 
X 
X 
 
 
X 
X 
Anthropometric 
Measurements 
 
X 
X 
X 
X 
 
 
X 
X 
Empowerment 
Questionnaire  
 
X 
 
X 
 
 
 
 
 
Empowerment Sub-
Study Interview 
This will be for a subset of 
participants during the final 
year of the study. 
 
 
 
X 
 
 
 
 
 
Maternal Health 
Assessment 
 
X 
X 
X 
 
 
X 
 
X 
 
Maternal Birth History 
and Post-Partum 
Questionnaire  
 
 
 
X 
 
 
 
 
 
Birth Assessment 
 
 
 
 
X 
 
 
 
 
Infant Feeding 
Assessment + Minimum 
Dietary Diversity Score  
 
 
 
 
 
X 
 
 
 
X 
Infant Health 
Assessment 
 
 
 
 
X 
 
X 
 
X 
NutricheQ 
questionnaire and food 
insecurity assessment 
 
 
 
 
 
 
 
 
X 
Study Exit  
 
 
 
 
 
 
 
X 
X 
 
*Blue columns: women; grey columns: infant 
Table 2 Overview of the Study Forms 
 

 
 
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Study Form 
Code 
Study Form Name 
Study Visit 
Form Used 
Purpose 
Person 
Responsible 
Consent Forms 
(ICF) 
 
 
 
 
ICF 
Consent form 
Recruitment 
visit 
To obtain 
informed consent 
Research 
Staff  
ICF-2 
Informed consent 
documentation 
Recruitment 
visit 
To document 
informed consent 
discussion 
Research 
Staff 
Invitation/ 
Instructions for 
Participants (I) 
 
 
 
 
I-1 
Invitation email 
 
 
Prior to the 
initial visit, an 
email or letter 
will go out to 
the potential 
participant from 
the clinical 
team. 
 
The email will 
briefly introduce 
the study, and will 
contain the contact 
information for the 
study team. 
N/A- for 
participant’s 
information 
I-2 
Invitation letter 
 
 
Prior to the 
initial visit, a 
letter or email 
will go out to 
the potential 
participant from 
the clinical 
team. 
 
The email will 
briefly introduce 
the study, and will 
contain the contact 
information for the 
study team. 
N/A- for 
participant’s 
information 
I-3 
Instructions for stool 
collection 
 
 
1)Baseline visit 
post-conception 
2)30-34 weeks 
post-conception  
3) 4 months 
post-partum 
4)12-months 
post-partum 
 
The instructions 
will be given to 
each participant 
prior to stool 
collection, to 
advise them on 
how to properly 
collect stool and 
how to return to 
staff. 
N/A- for 
participant’s 
information 
I-4  
Instructions for rectal 
swab collection 
 
 
1)Baseline visit 
post-conception 
2)30-34 weeks 
post-conception  
3) 4 months 
post-partum 
The instructions 
will be given to 
each participant 
prior to rectal 
swab collection, to 
advise them on 
N/A- for 
participant’s 
information 

 
 
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4)12-months 
post-partum 
 
how to properly do 
so. 
Maternal study 
visit forms (M) 
 
 
 
 
M-1 
Baseline eligibility 
screening form 
 
Recruitment 
visit: baseline 8-
20 weeks post-
conception  
 
To confirm 
eligibility 
Research 
Staff 
M-2 
Pregnancy confirmation 
and clinical assessment 
 
 
Baseline post-
conception visit 
 
To confirm 
pregnancy 
Research 
Staff 
M-3  
Baseline visit form 
 
 
Baseline post-
conception visit 
 
To obtain baseline 
participant 
information. 
 
To record maternal 
anthropometric 
measurements. 
 
To assess maternal 
health through 
questions 
regarding 
medication use, 
health seeking 
behavior etc 
Research 
Staff 
M-4  
Empowerment 
questionnaire 
 
 
1)Baseline post-
conception visit 
2)4 months 
post-partum 
visit 
To assess gender 
related variables 
focused on five 
main metrics: 
perceived maternal 
self-efficacy, 
perceived social 
support, decision 
making, perceived 
stress in addition 
to socio-economic 
factors, including 
food and housing 
insecurity, and 
demographic data. 
Research 
Staff 
M-5 
Post ASA24  survey 
 
 
Baseline post-
conception visit 
This will be an 
optional survey for 
participants to 
complete after the 
second ASA24   
Research 
Staff 

 
 
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recall for the 
baseline visit. The 
questionnaire will 
assess their 
experience with 
the ASA24  . 
 
M-6 
Dietary diversity 
questionnaire 
 
 
4 months post-
partum visit 
 
Participants will 
complete a dietary 
diversity score at 
the 3rd visit .  
 
Research 
Staff 
M-7 
Maternal study visit form 
 
 
1)30-34 weeks 
post-conception  
2) 4-months 
post-partum 
3)12-months 
post-partum 
 
To record maternal 
anthropometric 
measurements. 
 
To assess maternal 
health through 
questions 
regarding 
medication use, 
health seeking 
behavior etc. 
Research 
Staff 
M-8 
Maternal and infant check-
in call form 
 
 
6-10 month 
post-delivery 
check-in call 
To assess maternal 
and infant 
morbidity through 
questions 
regarding 
medication use, 
health seeking 
behavior etc. 
 
Research 
Staff 
M-9 
Study Completion/ 
withdrawal form 
 
 
The 12 month 
post-partum 
visit or earlier 
as required. 
To document end 
of study 
participation. 
Research 
Staff 
M-10 
The Edinburgh  
Postnatal Depression Scale 
(EPDS)  
 
This 
questionnaire is 
often 
administered 
clinically; in the 
event that it 
cannot be 
obtained from 
chart review it 
will be 
administered at 
the 3 month 
post partum 
visit. 
Screening 
questions that can 
indicate whether 
the maternal 
participant  has 
symptoms that are 
common in 
women with 
depression and 
anxiety during 
pregnancy and in 
the year following 
the birth of a child 
 
 

 
 
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Maternal Chart 
Review Forms 
(CM) 
 
 
 
 
CM-1 
Chart review maternal 
baseline visit 
 
 
Chart review for 
the baseline 
post-conception 
visit  
 
To obtain baseline 
participant 
information. 
 
To assess maternal 
health  through 
questions 
regarding 
medication use, 
health seeking 
behavior etc 
Research 
Staff 
CM-2 
Chart review maternal 
second visit 
 
Chart review for 
30-34 week 
post-conception 
visit 
To assess maternal 
health through 
questions 
regarding 
medication use, 
health seeking 
behavior etc 
Research 
Staff 
CM-3 
Chart review birth and 
labor form 
 
 
Chart review of 
birth history 
To gather 
information about 
the birth and labor 
history 
Research 
Staff 
CM-4 
Chart review maternal 
post partum visits 
 
 
Chart review for 
the 4 and 12 
month post-
partum visit  
To assess maternal 
health through 
questions 
regarding 
medication use, 
health seeking 
behavior etc 
Research 
Staff 
Infant study 
visit forms (IN) 
 
 
 
 
IN-1 
Infant study visit 1 
 
 
4 months of age 
 
To assess the 
amount and mode 
of feeding, (ie, 
mother’s milk or 
formula; 
complementary 
feeding). 
 
To record infant 
anthropometric 
measurements. 
 
To assess infant 
health through 
questions 
Research 
Staff 

 
 
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12 
regarding 
medication use, 
health seeking 
behavior etc. 
IN-2 
Infant study visit 2 
 
 
12-months of 
age  
 
To assess infant 
health through 
questions 
regarding 
medication use, 
health seeking 
behavior etc. 
 
To assess the 
infants diet, with a 
focus on markers 
of inadequate or 
excessive intake 
and dietary 
imbalances[1], 
minimum dietary 
diversity and to 
assess the annual 
food insecurity. 
 
To record infant 
anthropometric 
measurements. 
 
 
Research 
Staff 
Ages and 
Stages 
Questionnaires 
(ASQ) 
Infant study visit 1 and 2 
Infant follow up phone 
call ~6 months of age  
3 months of age 
12 months of 
age.  
 
Developmental 
screening tool that 
assesses 
developmental 
progress for the 
infant participants. 
Research 
Staff 
Nipissing 
Questionnaire 
Infant study visit 1 and 2 
Infant follow up phone 
call ~6 months of age 
This 
questionnaire is 
often 
administered 
clinically; in the 
event that it 
cannot be 
obtained from 
chart review it 
will be 
administered at 
3, 6 and 12 
month visit. If 
the 
questionnaire is 
administered 
A checklist used to 
monitor infant 
development. 
Research 
Staff 

 
 
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clinically 
throughout the 
first year of the 
infant 
participants life 
it will be 
collected 
through chart 
review. 
Infant chart 
review form 
(CIM) 
 
 
 
 
CIM-1 
Chart review infant visits 
 
 
1) 4 months of 
age 
2) 12 months of 
age 
To assess infant 
morbidity through 
questions 
regarding 
medication use, 
health seeking 
behavior etc 
Research staff 
Adverse Event 
(AE) 
 
 
 
 
AE 
Adverse event form 
As needed. 
To document 
adverse events. 
Research staff 
 
 
 
 
2 
 
INTRODUCTION 
 
Nutritional status during pregnancy plays an important role in maternal health and birth 
outcomes[2, 3]. While few factors impacting nutritional status during pregnancy have been 
identified, studies of undernutrition in children have revealed a key role for the gut 
microbiome[4-10]. Remarkably, studies examining the dynamics of the maternal gut microbiome 
before and during pregnancy and its impact on birth outcomes are limited. Further, relatively 
little is known concerning the influence of enteric eukaryotic microbes, such as parasites, on the 
bacterial microbiome and host nutrition. Here our goal is to define the relationships between host 
nutritional status and microbiome dynamics during pregnancy and how they contribute to birth 
outcomes and growth in infancy.  
 
 
2.1 
STUDY RATIONALE  
 
This project represents the first systematic investigation of the impact of the microbiome on 
nutritional status during pregnancy in young women and directly aligns with global health 
initiatives focused on this vulnerable cohort. Here our goal is to define the relationships between 

 
 
                       July19, 2024  
 
 
 
14 
host nutritional status and microbiome dynamics during pregnancy and how they contribute to 
birth outcomes. The gut microbiome has a profound influence on host nutritional status. 
Dysbiosis (loss of diversity/beneficial microbes and gain of pathobionts) has emerged as a major 
factor in the development of undernutrition. Despite the importance of nutrition during 
pregnancy, few studies have examined the role of the microbiome on maternal health and birth 
outcomes. Further, little is known concerning the influence of enteric eukaryotic microbes, such 
as parasites, on the bacterial microbiome and host nutrition.  
 
At the core of this study are two complementary cohorts of young women that provide an 
exceptional opportunity to obtain longitudinal samples to monitor the dynamic relationships 
between microbiome community structure and function with gut health and host nutritional 
status. This protocol will discuss the Toronto cohort of the study, which will focus on refugee 
and young adult obstetric clinics in Toronto, a population of specific relevance to 
undernutrition[11]. This cohort is expected to yield insights into the influence of eukaryotic 
microbes that are often viewed as asymptomatic. To better define our target demographic, we 
focus on young mothers, 28 years of age and younger, in the Toronto and Greater Toronto Area. 
We will focus on this younger demographic due to our lack of knowledge on the microbiome of 
young women, and their vulnerability to undernutrition[11]. A second complementary cohort 
will be based in the Matiari district of Pakistan. This project will yield unprecedented insights 
into the relationships between prokaryotic and eukaryotic microbes in the gut and their 
associations with maternal health and birth outcomes. 
 
 
2.2 
BACKGROUND  
 
2.2.1 UNDERNUTRITION BEFORE AND DURING PREGNANCY 
DRAMATICALLY IMPACTS MATERNAL HEALTH, BIRTH AND LIFE 
OUTCOMES 
 
Maternal and child undernutrition, is a tremendous burden on global health. Globally, 
undernutrition contributes to 45% of all deaths in children under 5 years[12]. Deficiencies in 
macro- (e.g. proteins, lipids and carbohydrates) and/or micronutrients (e.g. minerals and 
vitamins) can result in stunting (low height-for-age) and/or wasting (low weight-for-height) or 
thinness in adolescents and adults (low Body Mass Index – BMI). Pregnant women are 
particularly vulnerable due to the high nutrient demands during development of the fetus. 
Consequently undernutrition during pregnancy is associated with increased risk of poor birth 
outcomes, intra-uterine growth restriction of the fetus[13] and can result in complications that 
impact maternal morbidity and mortality[14-18]. In terms of perinatal outcomes, women with 
low BMI are at increased risk for stillbirths and neonatal deaths, preterm birth, low birth weight 
(LBW; <2500g) and delivering babies that are small for gestational age (SGA)[19]. 
Undernutrition prior to or during pregnancy may also have long term impact for the offspring 
(developmental origins of health and disease hypothesis)[20-22]. For example, studies of the 
1944 Dutch winter famine found women exposed to famine during late gestation did not gain 
weight in the third trimester and had babies with reduced birth weights, who were shorter and 
had smaller heads and placentas than unexposed babies[23]. Mothers exposed to famine only in 

 
 
                       July19, 2024  
 
 
 
15 
early -mid gestation however, gained more weight than non-exposed mothers did. The offspring 
of mothers with mid gestation famine exposure were lighter, shorter and had smaller heads, 
whereas the babies that were exposed during early gestations were heavier and longer at birth. 
but had higher rates of obesity[24]. At significant risk are young women (18-28 years of age), 
the target demographic of this study. 
 
The dietary requirements of young women are as high or higher than older age groups[25] and 
adequate nutrition at this key stage of growth is essential as up to 50% of adult weight is gained 
from the ages of 15-19 years. Undernutrition in this demographic has considerable consequences, 
with an increased risk of mortality from pregnancy and childbirth and a higher likelihood of 
LBW infants[13, 26-28]. While the health of young women has traditionally been neglected, they 
are recognized as a critical target for strategic interventions, as global health initiatives seek to 
break the intergenerational cycle of undernutrition and poor health[29-31], A key challenge is 
understanding the factors that predispose young women to nutritional deficits 
(undernutrition) and identifying interventions that improve nutritional status during 
pregnancy.  
 
These findings broadly support the hypothesis that chronic diseases originate through adaptations 
made by the fetus in response to undernutrition. The long-term effects of intrauterine 
undernutrition, however, depend upon its timing during gestation and on the tissues and systems 
undergoing critical periods of development at that time. 
 
A woman’s nutritional status is important both prior to and during pregnancy, as it plays an 
important role in reproductive health and pregnancy outcomes[13, 28]. Prior to pregnancy, 
having sufficient nutrient intake and nutrient stores affect a woman’s ability to maintain 
obligatory physiological functioning, and support fetal development in the case of pregnancy. 
During the periconceptional period and throughout pregnancy, sufficient nutrient intake is 
necessary since different nutrients influence pregnancy outcomes by altering both maternal and 
fetal metabolism via roles in modulating oxidative stress, enzyme function, signal transduction, 
and transcription pathways[32]. Pre-existing micronutrient deficiencies may be exacerbated 
during pregnancy as a result of the increased metabolic requirements[18]. Ultimately, maternal 
undernutrition during pregnancy often leads to fetal growth restriction, which increases the risk 
of neonatal deaths and childhood stunting by 2 years of age[12].  
 
Among young women, and especially among adolescents, ensuring sufficient nutrient intake is 
critical to facilitate the rapid physiological growth and maturation that occurs during the 
transition to adulthood [12]. Nutrition and growth in adolescence is particularly important to 
one’s health and adult stature. Many of the risk factors that impact maternal and newborn health, 
such as nutritional deficiencies, exist from adolescence. Becoming pregnant during this sensitive 
life stage has been found to slow and stunt one’s growth[13].. In some countries, as many as half 
of adolescents are already stunted which further increases the risk of poor perinatal outcomes in 
their off spring[12]. Pregnancies that occur among adolescents are additionally associated with a 
higher risk of complication, maternal and child mortality, and poor birth outcomes than 
pregnancies in older women[33]. Specifically, adolescent pregnancy is associated with a 50% 
increased risk of stillbirths and neonatal deaths, and increased risk of preterm birth, LBW, and 
asphyxia [14, 15, 34]. The prevalence of anemia is suggested to be as high in adolescents as 

 
 
                       July19, 2024  
 
 
 
16 
women 20–28 years of age. Collectively, this makes adolescent nutrition of substantial public 
health importance. 
2.2.2 THE GUT MICROBIOME IMPACTS MATERNAL AND INFANT 
NUTRITION 
 
While both genetic and environmental factors are known to impact BMI[35], our understanding 
on the interactions relating host genetics with the gut microbiome in the context of undernutrition 
remains limited[36, 37]. Indeed, twin based studies have identified few genetic factors that 
predispose to undernutrition[5]. Furthermore, undernutrition is not simply a consequence of food 
insecurity resulting in macro- and micronutrient[12, 38-40]. Instead, the intestinal microbiome 
has emerged as a key factor defining nutritional status, with impaired maturation driving 
undernutrition[4-10]. An immature microbiome may impact the extraction of critical nutrients 
such as vitamins and short chain fatty acids (SCFAs)[41-45], and/or result in the production of 
metabolites that inhibit metabolism or increase host cell turnover[5]. During pregnancy, dramatic 
changes in gut microbiota occur, with a decrease in individual (alpha) diversity but an increase in 
population (beta) diversity[46]. Relative to the 1st trimester, microbiota in the 3rd trimester 
exhibit higher abundances of Proteobacteria, typically associated with obesity in humans, and 
when transplanted to mice, result in increased adiposity and insulin insensitivity. Such 
adaptations may increase energy extraction from the diet to support pregnancy[47], raising 
interest in dietary supplements to improve pregnancy outcomes[48]. While these limited studies 
suggest an important role for the gut microbiome during pregnancy, there is a lack of data 
on gut community dynamics in pregnant women and how this impacts host nutrient 
acquisition. 
  
2.2.1 GENDER IMPACTS NUTRITIONAL STATUS AND THE MICROBIOME 
 
It is increasingly apparent that biological sex impacts the gut microbiome, beginning in 
infancy[49]. While all pregnant individuals have the same biological sex, the experience of being 
a mother is related to gender roles and identity. Intriguingly, recent research is beginning to 
reveal how gender-related variables such as socio-economic status, food security, early marriage 
and maternal empowerment contribute to nutritional status and the development of a healthy 
intestinal microbiome[50-52]. For example, in low-income settings, maternal empowerment may 
play a role in improving breastfeeding, diversity and quality of diet in children, setting up the 
opportunity for a healthy microbiome[53]. Addressing gender roles, specifically with respect 
to maternal empowerment, has the capacity to directly impact intestinal health in infants.  
 

 
 
                       July19, 2024  
 
 
 
17 
 
Figure 2: Conceptual Model of Maternal Socio-Economic Conditions Impact on the Infant 
Microbiome (Adapted from Herd et al, 2018)[54]  
 
2.2.1 THE TORONTO POPULATION 
 
This study will target pregnant women aged 28 years and younger, living in Toronto. Some of 
our potential participants are refugees and newcomers to Canada. In previous studies conducted 
at St. Michael’s Hospital, one of our participating sites, it was found that refugee women 
experienced violence, homelessness, poor health and nutrition during pregnancy [55]. This study 
also found that refugee women were more likely compared to non-refugee controls to give birth 
to low birth weight infants. A scoping review examining maternal health in immigrant and 
refugee women in Canada, found that immigrant women gained less weight during pregnancy 
compared to Canadian born women. In addition, this immigrant population was less likely to 
gain the recommended weight advised by Health Canada during pregnancy [56]. Differences in 
maternal morbidity was also apparent, with immigrants from Sub-Saharan African at a higher 
risk for severe maternal morbidity, with common diagnoses including uterine rupture and 
eclampsia [56]. Together, these findings further highlight the need to examine factors that 
potentially impact maternal weight gain during pregnancy, and the impact on birth 
outcomes, a key outcome of this study.   
 

 
 
                       July19, 2024  
 
 
 
18 
2.2.2 PATHOGEN EXPOSURE CONTRIBUTES TO ENVIRONMENTAL ENTERIC 
DYSFUNCTION 
 
Recently, data from the Malnutrition and Enteric Diseases multisite international study revealed 
that cumulative pathogen exposures confer a high risk for poor growth, with the prokaryotic 
enteroaggregative Escherichia coli and the protozoan Giardia lamblia most commonly 
detected[57, 58]. Exposure to pathogens such as these can contribute to EED, a condition 
characterized by chronic inflammation, poor nutrient absorption and reduced gut epithelial 
barrier function[59-61]. EED is thought to be triggered by dysbiosis (where beneficial microbes 
are replaced by pathobionts with a loss of diversity[62, 63]), initiated by nutrient deficiencies, 
antibiotic treatment and/or pathogen exposure. A ‘healthy’ microbiome normally limits pathogen 
invasion through: antimicrobial compounds; competition for nutrients; and direct stimulation of 
the immune system[64], but dysbiosis may exacerbate pathogen colonization, impair 
development of the mucosal immune system and disrupt, by as yet unknown mechanisms, 
metabolic processes that supply nutrients and energy for normal growth[7]. While maternal 
EED during pregnancy has been shown to adversely impact birth outcomes[65], our 
understanding of the impact of enteropathogens and dysbiosis on maternal gut community 
dynamics and nutritional status is limited. 
 
2.2.3 EUKARYOTIC MICROBES ARE A KEY COMPONENT OF THE 
MICROBIOME 
 
Typically, studies of the microbiome focus on bacterial components and tend to neglect the 
diverse array of eukaryotic microbiota. These include many considered to be parasites such as 
Giardia, Cryptosporidium and Entamoeba, each a significant global healthcare burden[66-69]. 
However not all parasitic infections cause disease; a study of Toronto daycares revealed a ~5% 
incidence of largely asymptomatic Giardia infections[70]. Thus, disease is a consequence of the 
complex interplay between the eukaryotic and bacterial microbiome and the host immune 
system[71, 72]. For example, Prevotella copri, is increased in patients with diarrheagenic E. 
Histolytica infections[69], while indole-producing bacteria such as E. coli CFT073 protect 
against Cryptosporidium[73]. Conversely, eukaryotic microbes may have neutral or even 
beneficial roles; Blastocystis, for example, has been associated with increased microbiome 
diversity[74, 75]. While current knowledge of the interactions between bacteria and eukaryotic 
microbiota and their host is limited, recent advances, discussed below, provide the molecular 
tools required to dissect these interactions. 
 
2.2.4 MARKER GENE SURVEYS COUPLED WITH ADVANCES IN NEXT 
GENERATION SEQUENCING PROVIDE UNPRECEDENTED INSIGHTS 
INTO MICROBIAL COMMUNITIES 
 

 
 
                       July19, 2024  
 
 
 
19 
To date, microbiome studies have largely relied on marker gene surveys (e.g.16S rRNA 
sequences) to profile community structure and dynamics[76-78]. In a typical application, PCR 
reactions are used to amplify marker genes from a DNA 
sample. Sequencing is then performed to yield tens of 
thousands of sequences which are subsequently compared 
against a database of known marker genes to yield readouts 
of the abundance of each taxon in the sample. Key to this 
technology is the identification of a variable genetic region 
that allows unambiguous identification of taxonomy, 
flanked by highly conserved regions that allow universal 
primers to amplify the marker genes. For bacterial surveys, 
hypervariable regions of the 16S rRNA gene are typically 
targeted. Recently, Dr. John Parkinson and Dr. Robert 
Bandsma, targeted 18S and 28S rRNA genes to perform 
equivalent surveys for eukaryotic microbes (Table 3)[79]. In 
combination, 16S, 18S and 28S rRNA markers reveal the 
bacterial and eukaryotic components of the microbiome. 
 
2.2.5 METAGENOMICS, METRANSCRIPTOMICS AND METABOLOMICS 
DELIVER READOUTS OF MICROBIOME FUNCTION 
 
Beyond sequence surveys, whole microbiome DNA and RNA sequencing (metagenomics and 
metatranscriptomics), together with metabolomics, offer more mechanistic insights, detailing the 
biochemical functionalities encoded and expressed by the microbiome[80-89]. For example, Dr. 
Parkinson used metatranscriptomics to reveal how changes in fat absorption in mice impact 
metabolic pathway expression in the gut microbiome[90]. Further, metabolomics has been 
transformed by improved technologies for the sensitive detection of metabolites. For example, 
our group recently revealed significant changes in metabolic pathways involved in lipid and 
protein metabolism in stunted and severely malnourished children relative to controls[91]. 
However, the underlying mechanisms explaining how the microbiome affects intestinal and 
whole body metabolic function is just starting to be elucidated. These tools support the 
functional interrogation of microbiomes to identify microbial pathways that may modulate 
health and disease.  
 
 
2.3 
RISK AND BENEFITS   
 
This is an observational study. Potential risks to participants include the general risks of giving 
blood, including: light-headedness, bruising, minor bleeding where the needle is inserted into the 
arm, pain, and a very low chance of infection.  Participants may feel discomfort providing a stool 
sample and taking a rectal swab, detailed instructions and research staff will be available to 
provide support. If participants are uncomfortable taking a rectal swab, they will have the option 
to opt out of this portion of the study and to still participate. However, the stool samples are an 
essential component of the study, as such it is a required component of participation. Detailed 
Table 3. Protozoa in children with severe 
acute malnutrition from Malawi using 18S 
biomarker technology and clinical 
diagnostics. 

 
 
                       July19, 2024  
 
 
 
20 
instructions will be provided for both the stool and rectal swabs and research staff members will 
be available to answer any questions.  
 
Participants may also experience psychological and emotional distress when completing 
questionnaires and/ or answering questions about their socio-economic status, their health, and 
their infants’ health. In addition, some participants may go through postpartum issues that may 
impact their mental state during the study period. Participants will have the option leave 
questions blank if they are uncomfortable, and research staff members will be on hand if a 
participant experience’s physiological and/ or emotional distress during a study visit/ study 
procedures. If this occurs the study staff members will bring in members of the clinical team to 
assist as necessary. 
 
Participants will also have the option to consent to future genetic testing of their blood samples. 
Genetic information can never be full de-identified. Although procedures will be put in place to 
de-identify genetic information, there is a risk that the information gained from genetic research 
can be linked to participants. While this is very unlikely at this time, rapid scientific advances 
mean that re-identification may be more likely in the future. There is also a risk of unintentional 
release of information that could lead to loss of privacy and to possible future discrimination 
against participants and/ or their biological relatives. The potential future use of genetic 
information is unknown and therefore not all potential future risks are known.  The potential 
psychological and social risk of participating and receiving genomic information is not fully 
known at this time. It may be upsetting to learn about genetic causes and medically actionable 
findings.  
 
There are no direct benefits to participants in the study. The societal benefits of the proposed 
study are that it is expected to yield insights into the relationships between prokaryotic and 
eukaryotic microbes in the gut and their association with maternal health and birth outcomes. 
The study results are expected to have an impact beyond the study setting. The actual risk of 
harm caused by a non- interventional trial is expected to be low, and the potential benefits in 
terms of knowledge generation outweigh the theoretical risks. 
 
 
3 
STUDY OBJECTIVES 
 
Table 4: Study Objectives 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
To assess if alterations of the 
microbiota in the maternal gut 
(dysbiosis) are associated with 
maternal gestational weight gain.   
The primary endpoint will be 
maternal gestational weight gain 
(GWG) during pregnancy, measured 
between the first (8-20 weeks post-
conception) and second time point 
(30-34 weeks post conception).  
 
Weight gain is a key 
predictor of adverse 
birth outcomes[92, 93]. 
 
 

 
 
                       July19, 2024  
 
 
 
21 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
To monitor microbiome dynamics, 
16S and 18S rDNA surveys will be 
applied to the maternal stool samples 
to monitor the bacterial and 
eukaryotic components of the 
microbiome. 
 
To determine the association between 
maternal microbiome dysbiosis during 
pregnancy and birth outcomes, infant 
growth, nutritional status and 
morbidity in the first year of life. 
The specific birth outcomes evaluated 
will include preterm birth, birth 
weight, SGA, LGA, head 
circumference at birth, and morbidity.  
 
The specific infant outcomes 
evaluated will include the following 
growth and nutritional status 
parameters; WHO z-scores for 
weight, length, head circumference 
and MUAC during the first year. 
 
Birth characteristics will be collected, 
including information on vaginal or 
cesarean birth (elective, emergency), 
placental insufficiency, APGAR 
score, and antibiotic usage.  
 
The infant’s health status and 
morbidity will be assessed at follow-
up visits, with questions regarding 
care-seeking, hospitalizations and 
treatments for any morbidity; 
including antibiotic usage. 
 
For the infants an assessment of 
amount and mode of feeding will be 
obtained at 4-months and 12-months. 
 
To monitor microbiome dynamics, 
16S and 18S rDNA surveys will be 
applied to the maternal stool samples 
to monitor the bacterial and 
eukaryotic components of the 
microbiome. 
This will allow for an 
examination of the 
association between the 
maternal microbiome 
and birth outcomes.  
Secondary 
 
 
To link the maternal microbiome to 
dietary intake, with a focus on calories 
and macronutrients. 
The maternal participants will 
complete a 24 hour dietary recall at 
both time points during pregnancy 
and at 1-year post partum.  
 
Quantitative measures 
of nutritional status will 
provide parameters to 
better inform 
microbiome analyses. 

 
 
                       July19, 2024  
 
 
 
22 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
To better relate to individual nutrient 
intake, recalls will be implemented on 
two separate days (separated by 3-10 
days) for each time point. 
 
Food insecurity will be assessed 
through the use of a food insecurity 
questionnaire administered at time of 
recruitment and 4 months post-
partum. 
 
The quality of the diet will be 
assessed through dietary diversity 
scores. 
 
To integrate maternal anthropometric 
factors and morbidity, with 
microbiome data to reveal key 
modulators (microbial taxa and 
metabolites) of dietary intake during 
pregnancy and the post-partum period.   
Maternal morbidity will be assessed 
at follow-up visits, with questions 
asked about illness or complications 
during pregnancy, childbirth and the 
post-partum period, care-seeking, 
hospitalizations and treatments for 
any morbidity; including antibiotic 
usage. 
 
Maternal anthropometric 
measurements will include maternal 
height, weight, MUAC, and SFT.  
 
Integration with clinical 
information will allow 
for the analysis of 
microbiome data in the 
context of patient 
nutritional status and 
health outcomes. 
To determine the impact of the 
maternal microbiome during 
pregnancy, including the exposure to 
pathogens and parasites, on the 
development of the infant 
microbiome. 
Maternal and child stool and blood 
samples will be collected and 
analyzed for microbiome 
composition, micronutrients and 
macronutrients. 
These analyses will 
inform on the role of 
the maternal 
microbiome during 
pregnancy to impact 
the developing child’s 
microbiome. Our 
hypothesis is that 
exposure to pathogens 
and/or parasites during 
pregnancy will 
negatively impact the 
development of the 
child’s microbiome 
with downstream 
consequences for 
nutrient uptake. 
 
To investigate the maternal 
microbiome’s exposure to pathogens 
The maternal stool samples will be 
examined for markers of intestinal 
These markers will 
reflect gut health and 
pathogen challenge. 

 
 
                       July19, 2024  
 
 
 
23 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
and parasites, and the association with 
intestinal inflammation. 
mass, inflammation, and gut 
permeability. 
 
To monitor the bacterial and 
eukaryotic components of the 
microbiome, 16S and 18S rDNA 
surveys will be conducted on the stool 
samples. 
 
Exploratory  
 
 
To explore the role of the maternal gut 
microbiome during pregnancy and to 
identify gut community dynamics in 
pregnant women and how this impacts 
differences between dietary intake and 
nutritional status. 
 
 
To gain more mechanistic insights 
into the relationship between 
microbiome function and maternal 
health and birth outcomes, 
metatranscriptomics, metabolomics 
and markers of inflammation will be 
selectively deployed on the stool 
samples.  
 
The output of these 
analyses are 
concentrations of 
metabolites that are 
expected to correlate 
with pathway 
expression data; linked 
to readouts of microbial 
gene expression 
detailing biochemical 
activity and the taxa 
responsible. 
 
To investigate socio-economic factors; 
including gender, poverty, exclusion 
and empowerment, and their influence 
on the health of a mother’s 
microbiome (assessed by alpha and/or 
beta diversity, and absence of 
pathogens). 
Participants will answer 
questionnaires at two time points: 
baseline and 4-months post-partum. 
The questionnaire will assess gender 
related variables focused on five main 
metrics: perceived maternal self-
efficacy, perceived social support, 
decision making, perceived stress in 
addition to socio-economic factors, 
including food and housing 
insecurity, and demographic data. 
 
 
 
The use of previously 
validated 
questionnaires within 
this population, will 
allow for comparison 
between the Pakistan 
and Toronto cohorts, to 
account for gender 
related variables that 
impact maternal and 
infant gut health. 

 
 
                       July19, 2024  
 
 
 
24 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
To explore the role of the human 
microbiota on nutritional status by 
performing fecal microbiota 
transplants in germ free mice and 
sterile piglets. 
The pregnancy rates, birth outcomes, 
microbiome dynamics, health 
outcomes, host nutritional status, 
inflammation and barrier integrity 
will be collected and assessed.  
 
 
 
Exploiting animal 
models will allow us to 
define causal 
interactions between 
diet, microbiome, 
pathogen exposure and 
nutritional status during 
pregnancy. Through 
recapitulating patient 
phenotypes with fecal 
microbiome 
transplants, our animal 
studies will set the 
stage for developing 
new therapeutic 
strategies that promote 
gut health. 
 
 
 
 
4 
STUDY DESIGN  
 
4.1 
STUDY HYPOTHESIS  
 
The central hypothesis of the study is that alterations of the microbiota in the maternal gut 
(dysbiosis) exacerbated by nutritional status or pathogen exposure during pregnancy, impacts 
weight gain because of impaired nutrient absorption, leading to corresponding negative birth 
outcomes. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3: Project Hypothesis. During the course of pregnancy a shift to an ‘unhealthy’ microbiome, perhaps 
triggered by exposure to pathogens, antibiotics or poor diet, results in impaired nutrient absorption, low weight 
gain and poor birth outcomes.  

 
 
                       July19, 2024  
 
 
 
25 
4.2 
STUDY DESIGN 
 
The study will be a prospective, longitudinal, observational study to investigate the impact and 
relationship between prokaryotic and eukaryotic microbes in the gut and their association with 
maternal health and birth outcomes among young women, 28 years of age and younger in the 
Toronto and Greater Toronto Area. The study will aim to recruit 400 women into two groups 
based on BMI at time of recruitment (Normal BMI will be defined as between 20 and 24.9 kg/m2 
and Low BMI will be defined as less than 20 kg/m2). With a goal of having 200 participants 
within the normal BMI group and 200 participants within the low BMI group. Although this our 
recruitment aim, in the event that we are unable to recruit 200 women with a low BMI, we will 
recruit more women that fall within the normal BMI range.  The study will follow women and 
their infants over the course of their pregnancy and for a year post-partum, collecting stool and 
blood samples, nutritional information, heath assessments, anthropometric measurements and 
empowerment metrics at different time points. 
 
4.3 
PARTICIPATING SITES 
 
For this study, we will recruit from the St. Michael’s Hospital Family Medicine Obstetrics 
Department, the St. Michael’s Hospital Obstetrics and Gynecology Department and St. 
Michael’s Hospital Midwifery Clinics. The Department of Family and Community Medicine at 
St. Michael’s Hospital, provides care to over 45,000 individuals annually, including prenatal 
care, with a focus on underserved inner-city populations. We expect to recruit around 75 women 
per year from this population. We also have a partnership with Dr. Rachel Spitzer, the Medical 
Director of the Young Prenatal Program (YPP) at SickKids and Dr. Ashley Vandermorris from 
the SickKids Young Families Clinic, we will be recruiting participants from the YPP. From the 
YPP we expect to recruit around 15-20 women per year. We therefore expect to meet our 
recruitment target within the first four years of the project.  
 
See the figure below for the anticipated participant numbers at each site, and participant’s 
potential paths through the health care system from pre-pregnancy to a year after child birth. 

 
 
                       July19, 2024  
 
 
 
26 
 
Figure 4: Pathways to Care for Pregnant Women and Their Infants in the Pregnancy and Microbiome 
Study 
4.4 
END OF STUDY DEFINITION 
 
A participant is considered to have completed the study if she has completed all phases of the 
study including the last visit or the last scheduled procedure shown in Section 1.3, Schedule of 
Activities (SoA). The duration of participation for each individual participant who completes all 
study visits will be approximately 17-19 months in duration, from 8-20 weeks into their 
pregnancy, until around one year post-partum.  
 
It is estimated that it will take 4 years for the study to reach its recruitment target, and 
approximately 5.5 years from when the study opens to the final participant study visit. 
 
5 
STUDY POPULATION 
 
The study population will consist of pregnant women, 28 years of age and younger, living in the 
Toronto and Greater Toronto Area. We will focus on younger women, due to our lack of 
knowledge about their microbiome and their vulnerability to undernutrition[11].  Many of the 
participants in our target population do not speak English; the main languages we anticipate are: 
Spanish, Arabic, Cantonese, Bengali, Tamil and French. During our study visits/ check-in call 
we will have a translator present, either, in person or over the phone. In addition, we will also 
have our study consent documents translated into the six languages indicated above. Once our 
initial consent form is approved by REB, we will have the consent forms translated by 
a certified translator. The translated versions will be back-translated into English and compared 

 
 
                       July19, 2024  
 
 
 
27 
with the original to ensure accuracy. These documents will then be submitted to the REB as an 
amendment and will include the translator’s seal/attestation/certificate with the translations. 
 
In addition, we anticipate that many individuals in our target population may have low literacy 
rates. Our study forms and documents will be written in easy to understand language, and we 
will have study staff members available to assist study participants with any questions they may 
have while filling out the forms.  
 
5.1 
INCLUSION CRITERIA 
 
In order to be eligible to participate in this study, an individual must meet all of the following 
criteria: 
1. Consent provided 
2. Participant is between 8-20 weeks post-conception 
3. Female aged 28 years of age and younger 
4. Confirmation of pregnancy 
5. Intend to comply with study procedures and follow up 
 
5.2 
EXCLUSION CRITERIA 
 
An individual who meets any of the following criteria will be excluded from participation in this 
study: 
 
1. Women who do not meet the enrollment age criteria 
2. Women who are 20 + weeks post-conception  
3. Women who have taken antibiotics within the past 3 months 
Note: it is common practice to give the mother penicillin in perinatal period if they are GBS 
positive; because this is standardized across the board it would not act as an exclusion 
factor. 
 
 
 
5.3 
STRATEGIES FOR RECRUITMENT AND RETENTION 
 
5.3.1 RECRUITMENT AND OBTAINING CONSENT  
 
Recruitment may take place in person, or virtually via Microsoft Teams, Zoom Healthcare or if 
preferred by phone. Our study will employ recruitment posters in the waiting rooms of 
participating clinics at SickKids, St. Michael’s Hospital and at participating midwifery clinics. In 
addition, prior to the clinic visit, participants will be sent an email or a letter introducing them to 
the study. In the event that a participant is not sent the recruitment letter/ email ahead of their 
clinic visit, they can still be approached by a member of the study team, as long as they agree to 
be approached. 
 
In person Recruitment: 

 
 
                       July19, 2024  
 
 
 
28 
When the potential participant arrives for their clinic visit, someone within the participant’s 
circle of care will ask the participant if the research team can approach them about the study. If 
the potential participant agrees, a member of the research team will approach the patient to 
explain the study and screen the participant for eligibility. If the participant is eligible and agrees 
to participate, the research team member will obtain informed consent. As part of the eligibility 
screening, the research staff member will obtain a confirmation of pregnancy and the participants 
BMI (based on their height and weight) from the clinical team and/ or the participant’s chart. 
  
Remote Recruitment:  
 
When the participant arrives for their clinic visit (either remote or in-person), the clinical team 
will ask the participant if they can be approached by someone from the research team. If the 
participant agrees, their contact information will be sent to the research team. The research team 
will reach out to the participant to set up a time for the video call by Zoom Healthcare or 
Microsoft Teams, or if the participant prefers, the research member will conduct the visit over 
the phone. During the remote recruitment visit the research member will confirm the identity of 
the potential participant and first briefly explain the study, if the participant agrees the research 
member will then confirm participant eligibility and go through the consent document with the 
participant. No audio or video recording will be done. If the participant is eligible and agrees to 
participate, the participant will be able to either send in their signed consent document via email 
or regular mail.  
 
The consent document will be written in simplified, easy-to-understand language to ensure 
participant understanding. Many of our potential participants have low literacy rates and/ or will  
not speak English. The main languages we anticipate our participants to speak are: Spanish, 
Arabic, Cantonese, Bengali, Tamil and French. If a participant does not speak English, a 
translator will be present either in person or via a phone call translation service. In addition, the 
consent documents will be translated into the six main languages encountered. Participants will 
sign the consent document in English as well as the translated version. The participant will be 
provided with a signed copy of both the English and translated version for their records.  
 
In a systematic review conducted to investigate potential tools to enhance strategies for 
comprehension in the informed consent process, it was found that extended discussion with 
participants had the most significant impact on a participant’s understanding[94]. To ensure 
informed consent and participant understanding, the research team member obtaining informed 
consent will engage in extended verbal discussion of the study procedures and the informed 
consent form. The participants understanding will be probed and documented, by having the 
participant answering the following questions in their own words: 
o What is the purpose of the study? 
o What samples will we be collecting during the study? 
o How long is your participation in the study? 
The participant’s responses to these questions, as well as any questions the participant raises, will 
be documented. If an interpreter is present for the informed consent discussion, the research 
member will document their presence as well as their name on the informed consent documents.  
 
The informed consent form will contain three additional sections for the participant to either 
provide consent for or to opt out of. 

 
 
                       July19, 2024  
 
 
 
29 
1. Empowerment sub-study, the participant will be asked to indicate whether or not they 
consent to be approached/ to participate in an empowerment sub-study. A subset of 
participants (around 20-30) who consent to this additional sub-study will be selected to 
participate using non-random purposive sampling (more information on this sub-study 
can be found in section 8). The empowerment sub-study will run during the final year of 
the study, only participants involved in the final year of the study will be approached for 
the sub-study. This section will be marked as ‘non-applicable’ for participants recruited 
in previous years. 
2. Workshops, a final section of the consent form will ask for participants consent to be 
approached for future workshops that will be designed to provide knowledge translation 
on some of the key findings of the study (more information on the workshops can be 
found in section 9).  
3. Genetic testing: Participants will also have the option to sign an additional section of the 
consent form for future genetic testing of their blood samples. With an additional option 
for their infant’s blood samples. Participants will have the option to opt out of this future 
testing for themselves and their infant. If they sign this section of the consent form, the 
samples will be stored to further dissect patterns of microbiome genotype associated 
traits. (More information about genetic testing can be found in section 12.4.1) 
 
The study staff will keep a record of all the study recruitment attempts, including participants 
who decline. The study recruitment file will be stored on the St. Michael’s shared drive for St. 
Michael’s participants, and on Onedrive for SickKids participants in a password protected excel 
file. 
 
5.4 
STUDY HONOURARIUM   
 
As a token of appreciation for the time and effort spent on the study, participants will receive a 
grocery gift card after each study visit. The grocery gift cards will be given either as physical gift 
cards or as electronic gift cards (sent via email); this will be dependent on the participant’s 
preference. Participants will receive $20 for the first and final study visits, as these visits will be 
longer in duration. For the 2 visits in between, participants will receive $10 grocery gift cards. 
 
6 
DISCONTINUATION AND WITHDRAWAL 
 
Participants are free to withdraw from participation in the study at any time upon request. 
An Investigator may discontinue or withdraw a participant from the study for the following 
reasons: 
 
• Withdrawal of informed consent (participant can withdraw for any reason) 
• If any clinical Adverse Event (AE), laboratory abnormality, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best 
interest of the participant 
• Significant study non-compliance  
• If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 

 
 
                       July19, 2024  
 
 
 
30 
The reason for participant discontinuation or withdrawal from the study will be recorded in the 
study file Form M-9 Study Completion/ Withdrawal and Exit Form. The data from participants 
who are withdrawn or discontinued from the study will be used in the analysis unless the 
participant requests otherwise. 
 
6.1 
LOST TO FOLLOW-UP 
 
A participant will be considered lost to follow-up if they are unable to be contacted by the study 
site staff.  If a participant misses one of the scheduled visits, they will still be included/ contacted 
for future study visits.  
 
The following actions must be taken if a participant fails to return to the clinic for a required 
study visit: 
• The site will attempt to contact the participant and reschedule the missed visit and 
counsel the participant on the importance of maintaining the assigned visit schedule and 
ascertain if the participant wishes to and/or should continue in the study. 
• Before a participant is deemed lost to follow-up, the Principal Investigator or designee 
will make every effort to regain contact with the participant, where possible, 3 contact 
attempts, through telephone calls, emails and a voicemail. These contact attempts will be 
documented in the participant’s study file.  
• Should the participant continue to be unreachable, they will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
 
6.2 
REMOVAL OF PARTICIPANTS 
 
Efforts will be made to obtain complete follow-up data from enrolled participants. If a 
participant experiences a serious adverse event, follow-up and scheduled data collection will 
continue to the extent that is possible. However, study activities will be stopped for a participant 
when any one of the following events occurs: 
• Consent for follow-up is withdrawn. 
• Participant moves residences and is lost to follow-up. Loss to follow-up will be 
considered to have occurred if study personnel receive information that the participant 
has moved away and will not return to follow-up at any time during the remainder of the 
study. 
7 
STUDY PROCEDURES 

 
 
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31 
 
 
Figure 5: Overview of Study Visits Including Sample Collection, Measurements and Surveys 
 
7.1 
STUDY VISITS  

 
 
                       July19, 2024  
 
 
 
32 
 
Study participants will be involved in four main study visits. We aim to conduct these four visits 
at: 8-20 weeks post conception, 30-34 weeks post-conception, 4 months post-partum and  12 
months post-partum. However, in the event that participants miss their study visits, we will still 
conduct visits and collect participant samples/data outside of these windows. We will also have a 
check-in call that will fall between 6-10 months post-partum. 
7.1.1.1 
BASELINE ASSESSMENT: 8- 20 WEEKS POSTCONCEPTION 
 
A member of the clinical team will ask the participant if they can be approached for a research 
study. If participants are interested, the research staff member will approach the participant to 
explain the study, screen for eligibility/ confirm pregnancy and obtain informed consent (see 
section 5.3 for more information). Following consent, research staff will administer the baseline 
enrolment form, the pregnancy confirmation form, the first ASA24  dietary recall and the 
empowerment questionnaire. The research staff and/ or the healthcare staff will obtain 
anthropometric measurements, a blood sample and give participants the stool collection kit. The 
participant will collect a rectal swab during this visit after detailed instructions and will return it 
to the research staff.  
 
Study staff will follow-up with participants over the phone 3-10 days after this baseline visit to 
administer the second ASA24  dietary recall. After completing this recall, the research staff 
member will ask the participant if they are willing to answer some questions about their 
experience with the ASA24  . The questions will revolve around the participant’s experience 
with the ASA24  ; were they able to find all of the food items they looked for, how long it took, 
was the recall easy/ difficult to fill out etc. The survey questions will be pilot tested with a 
convenience sample, and based on participant responses to the questionnaire, we may revise the 
questions, and submit the revised questions as an amendment.  Participants will have the option 
to decline answering these questions. 
 
The mother’s chart will be reviewed for pregnancy details and a health and medication 
assessment using the chart review form CM-1 Chart Review Maternal Baseline Visit. The 
purpose of the chart review is to minimize participant burden during the visit in order to keep the 
visit as short and convenient for the participant as possible. 
7.1.1.2 
STUDY VISIT 2: 30-34 WEEKS POSTCONCEPTION 
 
During this visit, research staff will administer the first ASA24 dietary recall and the study visit 
form. The research staff and/or the health care staff will obtain anthropometric measurements, a 
blood sample and give participants the stool collection kit. The participant will collect a rectal 
swab during this visit and will return it to the research staff. Study staff will follow-up over the 
phone 3-10 days after this visit to administer the second ASA24  dietary recall for this time 
point.  
 
The mother’s chart will be reviewed for pregnancy details and a health and medication 
assessment using the chart review form CM-2 Chart Review Maternal 2nd visit.  
 

 
 
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33 
7.1.1.3 
STUDY VISIT 3: 4 MONTHS POST PARTUM 
 
This will be the third maternal time point and the first infant time point. The research staff will 
aim to conduct the visit at around 4 months post-partum, however this time point will be flexible 
to allow participants to come in at a time convenient to them that aligns with a clinic visit i.e. 
“well-baby visit”. This visit will be conducted within 6 wks-4 months post-partum. The research 
staff will administer the maternal study visit form and the infant study visit form, the 
empowerment questionnaire, and the dietary diversity questionnaire. The research staff and/ or 
the health care staff will obtain anthropometric measurements from the infant and mother. If the 
infant arrives in a freshly soiled diaper and/ or they soil their diaper during the visit, then stool 
will be taken from the freshly soiled diaper. However, if the infant does not have a freshly soiled 
diaper, a rectal swab will be taken and the participant will be sent home with a stool collection 
kit. The mother will also take a rectal swab and will be sent home with a stool collection kit. 
 
Note at this visit, participants will not provide a blood  sample or complete the 2 ASA24  
recalls. 
 
Delivery details, a birth assessment and infant and maternal health details will be obtained from 
the mother and infant’s charts using the chart review forms, CM- Birth and Labor History, CIM-
1 Chart Review Infant Visits and CM-4 Chart Review Maternal Post-Partum Visits. In the event 
that a mother gave birth outside of SickKids and/ or St. Michael’s Hospital she will be asked to 
bring in her discharge summary to collect birth and labor details. The Edinburgh Postnatal 
Depression Scale (EPDS)[95], Ages and Stages Questionnaire [96],  and Nipissing Development 
Questionnaire [97] will be obtained from the participant’s chart, if the questionnaires are 
unavailable from the chart, they will be administered to the participant. 
7.1.1.4 
CHECK IN CALL: 6-10 MONTHS POST-PARTUM 
 
There is a large gap between the 3rd study visit and the final study visit, the purpose of this call is 
to have a check-in midway through, to check in with the participant and to keep them engaged 
with the study. The call will take place between 6-10 months post-partum; with the timing of the 
call dependent on when the third visit took place. For instance, if the third visit took place at 6 
weeks post-partum, we will aim to call earlier around 4 months, alternatively if the 3rd visit took 
place later around 4 months, then we would call later into the post-partum period around 10 
months. The research staff member will call the participant to check in and administer the check-
in call form with questions addressed to both the mother and infant. 
7.1.1.5 
STUDY VISIT 4: 12 MONTHS POST PARTUM 
 
In order to coincide with the participant’s clinic visits, this visit can be scheduled within 11-13 
months post-partum. This will be the final time point for both the mother and infant. The 
research staff will administer the study visit forms, and the first ASA24  recall for this time 
point. The research staff and/ or the health care staff will obtain anthropometric measurements 
and blood samples from the infant and mother. If the infant arrives in a freshly soiled diaper and/ 
or they soil their diaper during the visit, then stool will be taken from the freshly soiled diaper. 
However, if the infant does not have a freshly soiled diaper, a rectal swab will be taken and the 

 
 
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34 
participant will be sent home with a stool collection kit. The mother will also take a rectal swab 
and will be sent home with a stool kit. The research staff will follow-up 3-10 days after this visit 
to administer the second ASA24  dietary recall over the phone. The research staff will fill out 
the study completion form at this time point as well. 
 
The mother’s chart will be reviewed for a post-partum health and medication assessment using 
the chart review form CM-4 Chart Review Post-Partum Visit and the infant’s chart will be 
reviewed for health and medication use using the chart review form CIM-1 Chart Review Infant 
Visits. The Ages and Stages Questionnaire [96],  and Nipissing Development Questionnaire [97] 
will be obtained from the participant’s chart, if the questionnaires are unavailable from the chart, 
they will be administered to the participant. 
 
 
7.2 
STUDY ASSESSMENTS AND SAMPLE COLLECTION 
7.2.1.1 
BLOOD SAMPLE COLLECTION 
 
For Maternal Participants: 
A venous blood sample of 5 mL will be collected; 0.6 ml in SST Serum, 4.4 ml in EDTA tubes 
at enrolment, 30-34 weeks post conception and 12-months post-partum. When possible the 
bloodwork will take place at the same time as the clinical bloodwork to avoid the participant 
requiring an additional poke. 
 
For Infant Participants: 
A venous blood sample of 3 mL; 0.6 ml in SST Serum and 2.4 ml in EDTA tubes at 12 months. 
When possible the bloodwork will take place at the same time as the clinical bloodwork to avoid 
the participant requiring an additional poke. 
 
All blood samples will be labelled with the participant’s name, DOB, the study time point, and 
the date and time of collection. This, and other identifiable data of participants from both sites 
will be shared with SickKids hospital along with the samples for processing. Once the blood 
samples are collected 1.1 ml (0.5 ml in EDTA tube and 0.6  ml in SST serum) will be analyzed 
for CBC with a focus on HB + MCV, ferritin, and CRP. Bloodwork will be analyzed at SickKids 
laboratory or St. Michael's Laboratory depending on the timing and logistics of the visit. The 
remaining sample will then be transported to the Parkinson lab where they will be flash frozen 
and stored at -80oC. Samples will be brought to the lab by the research team member, if the team 
member is not present at the visit, the blood samples will be transported via Canada Post pre-
labelled envelopes or through the KJV courier service. Upon arrival in the Parkinson lab, the 
sample will be de-identified to include only the participant ID, study visit time point (Baseline 
will be marked as visit 1, 30-34 weeks post-conception will be marked as visit 2, and 12 months 
post-partum will be marked as visit 4*) and the date and time of collection. Blood samples from 
individuals who do not complete the study will be processed and their data analyzed, as possible, 
for selected outcomes. 
 
*Note there is no blood sample collection at the third visit, 4 months post-partum. 
See section 11 for more information on blood sample analysis. 

 
 
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35 
7.2.1.2 
STOOL SAMPLE COLLECTION 
 
 
Participants will receive a stool sample collection kit with illustrated pictorial and written 
instructions that explain the procedure during each study visit. The samples will be collected 
using the OMNiGene fecal collection tube. The materials will be provided to the participant by 
the research team during their scheduled research visits, these supplies can also be sent to 
participants by mail. Upon collection of the stool samples, participants will place the sealed tube 
containing the stool sample in a Ziploc bag, inside the biohazard bag, and mail it to the 
Parkinson lab using the pre-labelled FEDex envelope or send it in via KJV, a prepaid courier 
service, whichever is their preference. For follow-up visits participants will be provided with 
stool kits ahead of time and will be instructed to bring their stool sample in with them to the 
study visit. If preferred these samples can also be sent in via prepaid courier or pre-labelled 
FEDex envelope. Participants will receive detailed instructions for the collection and shipment of 
their stool samples.  
 
A subset of participants will split their stool sample into two containers; the first is the container 
indicated above (the OMNiGene kit which allows for DNA/ RNA analysis) and a second: the 
OMNImetGUT for Metabolomics kit. As our study is focused on recruiting 200 participants with 
a low BMI and 200 participants with a normal BMI. Our approach will be to have the first 50 
participants with a BMI below 19.5 and the first 50 participants with a BMI above 19.5 collect 
stool using both kits and then for all participants after that to collect their stool samples using 
only the DNA/ RNA OMNiGene kit.  This would allow us to conduct the more advanced 
analysis on a subset of participants, while remaining within our budget. 
 
If the infant arrives to the study visit with a soiled diaper, or they soil their diaper during the 
study visit, a study team member will collect stool from the diaper into a labelled stool collection 
tube. The stool collection tube will be transported by the study team member to the Parkinson 
Lab, in the event that a study team member is not present at the study visit, then a health care 
staff member or the mother will collect stool from the diaper into the labelled stool collection 
tube, and the sample will be shipped to the Parkinson Lab through a pre-labelled FEDEx 
envelope or through the KJV courier service.  
 
The stool sample will be labelled with the participant’s name, study ID, DOB, the visit number 
and the date and time of collection. Upon arrival at the lab the sample will be de-identified, to 
only include the study ID, the visit time point and the date and time of collection. The sample 
will then be stored in the Parkinson lab -80 Celsius freezer, until sample analysis. Stool samples 
from individuals who do not complete the study will be processed and their data analyzed, as 
possible, for selected outcomes. 
 
See section 11 for more information on stool sample analysis. 
7.2.1.3 
RECTAL SWABS 
 
We anticipate that some participants may not return their stool samples, so in addition to stool 
samples, maternal participants will also provide a rectal swab at baseline, 30-34 weeks post-
conception, at 4 month’s post-partum and at 12 months post-partum. If infants do not have a 

 
 
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36 
soiled diaper available, they will also have a rectal swab taken at 4 months and 12 months. 
However, if participants are uncomfortable with the collection of the rectal swabs, they can opt 
out of having them taken. 
 
Maternal participants will receive detailed verbal and written instructions on how to safely and 
properly collect the rectal swab. Maternal participants will receive all of the necessary 
equipment, including; biohazard bags, plastic Ziploc bags, paper towel and the COPNAN 
Floqswab rectal swab kit transported in eNAT medium.. Maternal participants will collect the 
rectal swab on their own, once they collect the rectal swab, they will place the swab in a pre-
labelled tube. The tube will be labelled with the participant’s full name, date of birth, study ID, 
visit time point, and the date and time of collection. The tube will be placed in the Ziploc bag, 
which will be placed in the biohazard bag. If a study team member is present at the study visit, 
they will transport the rectal swab to the Parkinson lab. If a study team member is not present at 
the study visit, the study team member will call the KJV courier service to pick up the rectal 
swab, where it will be transported to the Parkinson lab. 
 
Infant participants will have a rectal swab taken if they do not have a soiled diaper available, the 
swab will be taken by a health care provider or by a trained research member. The rectal swab 
will be taken according to the study rectal swab SOP. Once the swab is taken it will be placed in 
a pre-labelled tube. The tube will be labelled with the participant’s full name, date of birth, study 
ID, and the date and time of collection. The tube will be placed in the Ziploc bag, which will be 
placed in the biohazard bag. If a study team member is present at the study visit, they will 
transport the rectal swab to the Parkinson lab. If a study team member is not present at the study 
visit, the study team member will call the KJV courier service to pick up the rectal swab, where 
it will be transported to the Parkinson lab. 
 
We will be following the SOP developed by the CHAIN network, Chain Rectal Collection SOP 
v1.04[98].  
 
See section 11 for more information on stool sample analysis. 
7.2.1.4 
ANTHROPOMETRIC DATA COLLECTION 
 
Maternal height and weight will be measured using a digital floor scale and stadiometer; MUAC 
will be determined using a measuring tape. A triceps skinfold thickness (SFT) measurement will 
be taken using a skinfold caliper. All measurements will be collected by the study staff member 
and/ or the health care provider, using standardized procedures.  
 
Infant anthropometry will include measurement of weight, length, MUAC, SFT, and head 
circumference; these measurements will be conducted using standardized procedures: a digital 
weight scale, infantometer, and measuring tape and skinfold caliper. All infant measurements 
will be collected by study personnel and/ or health care providers, using standardized procedures. 
 
The staff member taking the anthropometric measurements will take one measurement for 
weight, length, height, MUAC and head circumference. The staff member will take 3 
measurements for SFT, and will take the average of the three measurements.  
 

 
 
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37 
Note if visits are conducted remotely, available measurements will be obtained from the chart, 
the rest will be marked on the study form as unavailable. 
7.2.1.5 
STUDY FORMS 
 
The information collected will depend on the visit, for the maternal participant, the forms 
collected will include; a pregnancy confirmation and clinical assessment, a baseline and 
enrolment questionnaire, a dietary diversity questionnaire, an empowerment questionnaire (see 
section 8 for more information), a health/ medication assessment questionnaire and a birth and 
labor history form. A summary of all the study questionnaires and visit-specific forms that will 
be used throughout the study is provided in Table 2. 
 
The baseline study visit form will collect participant information regarding the participant’s age, 
gender, sex, number of pregnancies, number of children, marital status, the language spoken in 
the home, fluency of English and/ or French, race/ ethnicity, country of origin, immigration 
status, income, # of people this income supports, education and housing. The maternal study visit 
form will collect anthropometric measurements (described in the previous section) as well as a 
health assessment to document morbidity and medication usage. This form will also document 
the first three digits of the participants postal code. The postal code will be used to conduct GIS 
mapping, in order to gain a better understanding of the environmental impacts of the microbiome 
and  pregnancy and birth outcomes 
 
 
The birth and labor history form will gather information through chart review of both maternal 
and infant charts including: delivery characteristics (vaginal/cesarean section), gestational age, 
birthweight and other measurements at birth (head circumference and length), placental 
insufficiency, APGAR score and antibiotic use. 
 
At 30-34 weeks post-conception, 4 months post-partum and 12-months post-partum, a health 
assessment of the mother and child will be administered. The assessment will include questions 
related to the use of medications, including antibiotics, and the presence of illnesses and other 
health problems leading to doctor’s visits/hospitalizations.  
 
For the infant an anthropometric, feeding and health assessment will be completed at both visits 
and during the phone follow-up. The form will collect the infant anthropometric measurements 
(described in the previous section), a breastfeeding assessment, and an infant feeding assessment 
as well as a health assessment to assess infant health status, morbidity and medication usage. At 
12 months the Nutricheq form will also be administered, this form assesses potential dietary risk 
factors in children aged 1-3 years of age[1]. The feeding assessment is discussed further in the 
section below.  
 
The participants charts will be reviewed for information on participant’s health, medication use 
and birth and labor details. The chart review forms detail the exact information that will be 
obtained from the chart. For SickKids participant’s EPIC will be reviewed and for SMH 
participant’s both the family medicine EMR and the hospital EMR will be reviewed to ensure the 
most robust data. Participant’s that deliver outside of SickKids and St. Michael’s hospital, will 

 
 
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38 
be asked to bring in their discharge summary. The Edinburgh Postnatal Depression Scale 
(EPDS)[95], Ages and Stages Questionnaire [96],  and Nipissing Development Questionnaire 
[97] will be obtained from the participant’s chart, if the questionnaires are unavailable from the 
chart, they will be administered to the participant. Note if the ASQ questionnaire and Nipissing 
Development Questionnaire are administered clinically throughout the first year of the infant 
participant’s life it will be collected through chart review, even if administered outside the 
research visit windows. 
7.2.1.6 
DIETARY RECALLS AND FEEDING ASSESSMENT 
 
To link the microbiome to nutritional status and nutritional intake, with a focus on calories and 
macronutrients, we will use the Automated Self-Administered 24-Hour Dietary Assessment Tool 
(ASA24-Canada2018)[99]. The ASA24 has been used in multiple cohort studies in Canada and 
includes details of all food items available in Canada; moreover, it is also found to be useful among 
low-income females[100, 101] and pregnant women[102]. Participants will complete the recall, 
with assistance from a research staff member, at both time points during pregnancy and at 1 year 
post-partum. To better capture reliable individual nutrient intake, recalls will be completed on two 
separate days (separated by 3-10 days). The first recall for each time point will be completed in 
person, or virtually over Microsoft Teams or Zoom Healthcare and assisted by the research team. 
The second recall will take place either virtually through Zoom HealthCare or Microsoft teams. If 
the participant prefers they can also complete their visits via phone call. If required, an interpreter 
will also be present on the video call / phone call. Each participant’s usual intake will be obtained 
through averaging the two 24-hr dietary intakes per time point. The ASA24 is linked to the 
Canadian Nutrient File and provides a detailed analysis of diet[99]. 
 
The participants will receive the links to complete the ASA recalls via email. The recall works on 
all web browsers, and can be used on smartphones, tablets, laptops and desktops. ASA24 is linked 
to the Canadian Nutrient File and provides a detailed analysis of diet (macronutrients and 
micronutrients)[99]. Dietary data is saved on the National Institute of Health (NIH) website, the 
NIH has no access to them.  
 
At the 3rd visit, which will take place around 4 months post-partum, participants will complete a 
dietary diversity questionnaire using the Minimum Dietary Diversity Score for Women (MDD-
W). The MDD-W is a population level indicator for dietary diversity for women aged 15-49, based 
on 10 food groups[103]. The MDD-W will reflect what they have eaten over the previous 24 hours, 
and they will be asked at the end of the questionnaire whether this reflects their diet over the 
previous 3 months. The research team will calculate the MDD-W from the ASA recalls completed 
at baseline, 30-34 weeks post-conception and at 12 months. 
 
For the infant, feeding assessments will take place at the 4 month and 12 month visits. At 4 month’s 
the form will include an assessment of feeding initiation, and the amount and mode of 
breastfeeding/formula feeding and dietary supplements. At 12 month’s this form will also include 
an assessment of young child feeding, with questions to address the introduction of complementary 
foods (solid, semi-solid and soft foods), meal frequency and the minimum dietary diversity score 
(MDD, see appendix X) will be assessed to estimate quality of food intake. Overall, the 
questionnaire will be designed based on the guidance developed by the WHO in 2010, in the 
“Indicators for assessing infant and young child feeding practices (Part 2 Measurement)”[104] 

 
 
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At the 12 month visit, the research staff will also administer the NutricheQ questionnaire, a tool 
designed for toddlers aged 1 to 3 years of age, with a focus on markers for inadequate or excessive 
intake and dietary imbalances[1]. Also, household annual food insecurity will be assessed based 
on a validated 2 question score[105].  
 
7.3 
CONTACT WITH STUDY PARTICIPANTS 
 
Study participants will be recruited at around 8-20 week’s post-conception, with our aim to 
recruit participants around 12-16 weeks post-conception. Participants will be followed from 
recruitment, until 12 months post-partum. Study staff will aim to schedule research visits at the 
same time as clinic visits and infant check-ups to avoid having the participant come in for 
additional visits. Study participants can contact the Pregnancy and Microbiome study coordinator 
and research team for any question or concerns they might have. The study team will 
communicate with participants via email and/ or phone for study visit/ procedure reminders, to 
follow-up about outstanding study tasks, to send study information (like the links for the ASA 
dietary recalls) and to answer any participant questions. The expected number and duration of 
visits is detailed in table 7 below. 
 
All participant contact, including follow-up calls/ emails will be documented in a study contact 
log. The study contact log will include the participant ID, the participant contact information and 
a log of all the participant contact. The contact log will be stored on the St. Michael’s shared 
drive for St. Michael’s participants and on Onedrive for SickKids participants, in password 
protected excel files.  
 
Table 7: Overview of Study Visit Duration 
Visit  
Visit Duration 
Enrolment and baseline 
~1.5-2 hours 
2nd baseline dietary recall  
30-40 minutes 
30-34 weeks post conception 
1-1.5 hours 
2nd 30-34 week dietary recall 
30-40 minutes 
Empowerment sub-study  
4 months post-partum 
20-30 minutes 
Maternal visit 4 months post-
partum 
1-1.5 hours (This visit will be 
completed at the same time as 
the infant visit) 
Infant visit within 4 months 
of birth 
1-1.5 hours (This visit will be 
completed at the same time as 
the maternal visit) 
6-10 month check- in call 
10-20 minutes. 
Maternal visit 12-months 
post-partum 
1-1.5 hours (This visit will be 
completed at the same time as 
the infant visit) 
Infant visit 12 months post-
partum 
1-1.5 hours (This visit will be 
completed at the same time as 
the maternal visit) 

 
 
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40 
2nd 12 month post-partum 
dietary recall 
30-40 minutes 
 
 
 
7.4 
SAFETY AND ADVERSE EVENTS 
 
Although study-related adverse events are not anticipated, the participants may encounter 
pregnancy-related or other unrelated adverse events, including, but not limited to, miscarriages, 
abortions, stillbirths and maternal and newborn deaths. These events will be recorded (AE – 
Adverse event form). Women who experience clinical events during study participation will be 
referred to the appropriate health facility. Serious adverse events will be reported to the Hospital 
for Sick Children and St. Michael’s REB according to their respective institutional requirements.  
 
Serious adverse events include the following events, irrespective of their association with study 
procedures: 
• Death 
• Life-threatening complication, such that death was averted by medical or surgical 
interventions (the term "life-threatening" in this context refers to an event in which the 
patient was at risk of death at the time of the event; it does not refer to an event, which 
hypothetically might have cause death, had it been more severe) 
• Inpatient hospitalization or prolongation of an existing hospitalization 
• Persistent or significant disability/incapacity following the resolution of the acute event 
• Reporting of certain events (e.g., suspected child abuse) is mandatory because of the 
study population  
 
7.4.1 REPORTING EVENTS TO PARTICIPANTS  
 
Participants will be informed in a timely manner of any new information, including safety 
information, that is relevant to that participant’s willingness to continue participation. The 
communication of this information will be documented through a revised REB approved 
Informed Consent Form, where possible, based on the timeliness of the information. 
 
In the event that a study procedure detects a new clinically important secondary 
finding/incidental finding, the qualified physician will notify the Most Responsible Physician 
(MRP) at St. Michael’s Hospital or The Hospital for Sick Children or request the participant’s 
family doctor’s name and contact information in order to arrange medical follow-up to interpret 
the significance of the findings. 
 
In the event of discovering a medically actionable incidental finding, or if any new clinically 
important information about the participant’s health is obtained as a result of participation in the 
study and where a second sample is already available, the test result will be validated clinically 
in an accredited laboratory. For this study, the presence of a pathogen might be identified, in the 
event of this finding, the follow up would include clinical microbiology. The qualified physician 

 
 
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41 
will work with the participant, their family physician and MRP at St. Michael’s Hospital or The 
Hospital for Sick Children to arrange referral to the appropriate specialist as needed.  
 
8 
EMPOWERMENT SUBSTUDY 
 
Gender-related variables that may influence how mothers understand, promote and maintain 
intestinal microbiome health will be explored. While there may be sex- specific interactions 
between the microbiome and hormones, here we focus explicitly on gender. Validated 
modules will be administered by a trained member of the research team to explore maternal 
education status[106], household food insecurity[107], generalized self-efficacy[108] and 
decision making[106], with modifications made and beta- tested for internal validity. Other 
gender-related variables including self-identified race and ethnicity, religion, primary 
language, immigration status and income will be captured. Validated modules that have been 
used in the MaPPS cohort will allow for comparison between the cohort based in Pakistan and 
the Toronto cohorts, to account for cultural factors that further intersect with gender norms in 
their specific context.  
 
Participants will fill out an empowerment questionnaire at baseline and 4-months post-partum. 
The five main metrics will be: perceived maternal self-efficacy, perceived social support, 
decision making, perceived stress in addition to socio-economic factors, including food and 
housing insecurity, and demographic data. These metrics align with major social constructs 
contributing to infant feeding practices and suspected microbiome health (Figure 4). These 
constructs will be measured using validated questionnaires that have been previously translated 
and utilized with this study population. Questions pertaining to perceived decision-making, and 
demographic data including maternal education, income, housing and gender norms are from the 
Pakistan Demographic and Health Survey (PDHS)[106]; food insecurity will be assessed using 
the Household Food Insecurity Access Scale (HFIAS)[107]. Self-efficacy will be measured using 
the Generalized Self-Efficacy scale, developed by Schwarzer and Jerusalem[108]. Perceived 
social support will be measured using the Multi-dimensional Scale of Perceived Social Support 
(MSPSS), developed by Zimet and colleagues[109]. Perceived parental stress will be measured 
using the Perceived Stress Scale (PSS-10)[110].  
 
A sub-set of 20-30 participants will be invited to participate in qualitative in-depth interviews. 
Participants will be approached for this sub-study around 4 months post-partum. In order to get a 
broader perspective of empowerment, this sub-study will also be open to participant’s who are 
not involved in the main study. These participants will be selected using non-random purposive 
sampling in line with recommended sampling strategies. Using grounded theory 
methodology[111], we will conduct in-depth interviews with mothers in the Toronto cohort to 
further explore knowledge and attitudes toward intestinal gut health. Interviews will be recorded 
and transcribed and coded for themes using an inductive coding strategy. Thematic analysis will 
yield opportunities for knowledge translation to empower young mothers to improve gut health. 
The visit will take around 20-30 minutes, these interviews will be conducted in person and / or 
over the phone. This sub-study will take place during the final year of the study, the interview 
guides for the sub-study will be submitted as a study amendment. 
 

 
 
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42 
We expect that the impact of gender-related variables may vary by cohort as cultural 
expectations of gender roles widely differ. We anticipate our qualitative investigation may 
identify that young mothers lack knowledge concerning gut health and may benefit from targeted 
education opportunities early in pregnancy.  
 
9 
WORKSHOPS 
 
We will work with our partner clinics in Toronto as well as our participants, to identify gaps in 
knowledge and to integrate findings from our gender focused investigations into accessible 
material that can be delivered as workshops to promote the importance of a healthy gut 
microbiome during pregnancy and beyond. We will develop two kinds of workshops: 
1. The first workshop will be directed at clinicians and healthcare providers. It will deliver 
tangible material on how to improve patients gut health in general. The content of the 
workshop will be designed based on participant and clinician feedback. These workshops 
will be delivered during the final year of the study, once the clinicians and healthcare 
providers have completed their participation in the study.  
2. The other workshop will be directed at participants who have completed their 
participation in the study. These workshops will be designed/ delivered during the final 
year of the study, and will be developed based on participant identified gaps in 
knowledge. Potential topics may include how to improve gut health, how to develop a 
healthy gut microbiome in toddlers, etc. 
 
These workshops will be centered on patient experiences and knowledge translation, once we 
design the workshop content, we will submit an amendment application to our REB. We will 
only deliver the workshops once we have approval from the REB. 
 
 
10 STOOL AND BLOOD SAMPLE ANALYSIS  
 
10.1 PROFILING MICROBIAL COMMUNITY STRUCTURE AND FUNCTION 
 
To assess nutritional status, metabolomics targeting minerals and general metabolites will be 
applied to the blood samples. To monitor microbiome dynamics, 16S and 18S rDNA surveys will 
be applied to the maternal and infant stool samples. To gain more mechanistic insights into the 
relationship between microbiome function and maternal health and birth outcomes, we will deploy 
metatranscriptomics, metabolomics, proteomics and markers of inflammation.  
 
10.1.1 PROFILING MICROBIAL COMMUNITYSTRUCTURE  
 
Microbial communities will be analyzed through 16S and 18S rDNA surveys using established 
methods that target the V4 region of the 16S rRNA gene to capture bacterial taxa [112-114] and 
the V4V5 region of the 18S rRNA gene to capture eukaryotic taxa. DNA library preps will 
include error-correcting barcodes[115] for multiplexing of samples. Sequencing will be 
performed to generate ~50,000 2x300bp paired end reads generated per sample. To define 
taxonomic diversity, species profiles from 16S and 18S rDNA data will be clustered to identify 
differences in community structure across samples.  

 
 
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Prior to implementing these lab analyses, a pilot experiment will be performed using 10 
previously sequenced DNA samples derived from human stool at the Integrated Microbiome 
Resource (IMR) facility, which applies a modified approach to preparation of DNA sequencing 
libraries compared to that used in our previous studies, to ensure a comparable quality of output.  
The pilot experiment will involve PCR amplification of the 18S rRNA variable regions 4 and 5 
using primers V4-1 and V4-4[130,79], which will be fused to Illumina adapter sequences unlike 
in previously published analyses. Sequencing will be performed using Illumina MiSeq v3 
chemistry, 300 cycles, as previously, and the quality and number of sequenced reads as well as 
success of detecting protozoa will be compared to outputs from the published data. As the DNA 
extraction procedure in the current study differs from that of our previous studies, we will 
include 5 archived DNA samples from each of two different cohort studies (NCT00705445 and 
NCT02308917), where DNA samples were extracted using two different kits to minimize 
extraction bias. We will also submit the same 10 DNA samples for 16S rRNA variable region 4 
sequencing to IMR, which uses slightly modified versions of EMP primers we previously used 
(515FB instead of 515F, and 806RB instead of 806R) to ensure reproducibility of results.  
A waiver of consent is being requested for the secondary use of the 5 samples from the study 
(cRCT NCT00705445, AKU (752-Peds/ERC-07)), that were previously approved by the 
research ethics board (REB) at SickKids (REB No. 1000054244) for a use in a retrospective 
analysis (NCT00705445). Currently, these 5 samples are stored in Dr. Bandsma’s lab. 
Informed consent was obtained from the participants in the study (NCT02308917, REB 
1000039604) to use their samples for future research. Participants in this study are allowed to 
withdraw their specimens. At the governance level, a sample can be withdrawn as long as it 
remains within the research network and has not been utilized. If participants choose to withdraw 
their specimen, they should contact the research coordinator of the study, who will ensure the 
specimen will no longer be used for research activities. However, there are some limitations. If 
the sample has been published as a part of grouping with other samples, or if it has been sent for 
third party analysis based on an agreement, and the participant has consented to future research 
activities, the sample cannot be withdrawn.  
In this pilot experiment, only deidentified samples will be used for sequencing and analyzing the 
data, meaning all the personal information linking the samples to specific participants will be 
removed. DNA extracted from stool samples sent to IMR will be assigned a study ID number, 
ensuring that no patient identifying information will be shared with the IMR facility. The 
information that links the study ID to the participant’s personal information will be stored in a 
separate, encrypted file in a password protected computer which can only be accessed by the 
principal investigator of the original study. This process ensures the privacy and confidentiality 
for all participants. The secure transfer process will involve packaging the deidentified samples 
in compliance with IATA P.I. 650 shipping standards, ensuring safe and secure transport. 
Additionally, a material transfer agreement (MTA) with IMR will be fully executed prior to 
transfer of de-identified samples, further ensuring that all legal and ethical guidelines for 
handling the de-identified samples are met. 
At the end of the pilot testing, any unused samples will be destroyed at the IMR facility. The 
microbiome sequencing processing takes approximately 6-8 weeks, after which the unused 

 
 
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44 
samples will be destroyed. After a successful pilot, the stool samples from the MMIP study will 
be sent to IMR for 16s and 18s sequencing.  
We will utilize the QIIME2 platform[116], MOTHUR[117], multivariate approaches such as 
Permutation Multivariate Analysis of Variance (i.e. PERMANOVA-S a method that can 
associate microbiome changes with outcome measures while accounting for confounders[118]) 
among other state of the art tools. Differences between groups in microbiome community 
structure will be tested by analysis of similarities (ANOSIM) and co-occurrence analysis[119]. 
To better define bacterial pathogen burden, we will apply TaqMan array card technology for the 
simultaneous detection of 19 common enteropathogens[120]. 
 
10.1.2 PROFILING MICROBIAL COMMUNITY FUNCTION 
 
After total RNA extraction and rRNA depletion (RiboZero Gold Kit, Illumina, San Diego, Ca, or 
equivalent), libraries will be constructed and Illumina-based sequencing will be performed to 
generate ~30 million 2x150bp paired end reads per sample (our rarefaction analyses have 
previously shown such sequencing depth is sufficient to identify the vast majority of species and 
enzymes present in the samples[90]). Reads will be processed for quality and contaminants using 
established pipelines (e.g. [66, 68, 104]). Reads will be assembled using appropriate state of the 
art tools such as SPAdes[105] and subsequently annotated with taxonomic and functional 
assignments Expression will be normalized to Reads per Kilobase of transcript per million 
mapped reads. Annotations are mapped onto biochemical pathways and complexes such as those 
defined by the Kyoto Encyclopedia of Genes and Genomes [121]. The output of these analyses 
are readouts of microbial gene expression detailing biochemical activities as well as the taxa 
responsible. 
 
10.1.3 PROFILING OF PATIENT AND MICROBIOME METABOLITES AND 
PROTEOMICS  
 
To generate read outs of stool and blood metabolites, samples will be sent on dry ice to the 
Metabolomics Innovation Centre. Stool and blood samples will be analyzed for general 
metabolites (e.g. SCFAs, amino acids, intermediates in glycolysis and nucleotide metabolism, 
among other metabolites, using the TMIC Prime Metabolomics platform[122]). Blood samples 
will additionally be analyzed for vitamins and minerals through the TMIC center. The output of 
these analyses will be concentrations of metabolites that are expected to correlate with pathway 
expression data. 
 
Blood proteomics will allow for a deeper investigation into the relationship between the maternal 
microbiome and maternal weight gain and the effects on offspring. Proteomics will be conducted 
at the Sickkids SPARC (Sickkids Proteomics, Analytics, Robotics and Chemical Biology 
Centre)[123] 
 
10.1.4 HOST MARKERS OF INFLAMMATION  
 
In addition to microbiome function, we will also obtain host readouts that reflect gut health and 
pathogen challenge. Here we will examine markers in the stool for intestinal mass, inflammation, 
and gut permeability and circulating lipopolysaccharide, among other markers. 

 
 
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45 
 
10.2 POTENTIAL PROBLEMS, MITIGATIONS AND EXPECTED RESULTS 
 
In the unlikely event we find RNA yields are poor, we will revert to performing whole shotgun 
DNA sequencing (metagenomics), which also has the capacity to provide functional insights.  
 
Our expectation is that: 1) study participants exhibiting positive maternal health and birth 
outcomes will be associated with more diverse communities than those exhibiting poor maternal 
health and/or birth outcomes; 2) pregnancies associated with poor nutritional status at the start of 
pregnancy, will exhibit a greater incidence of dysbiosis (e.g. loss of microbial diversity and 
decrease in otherwise dominant stool taxa e.g. Firmicutes and/or Bacteroidetes) throughout 
pregnancy; 3) pregnancies associated with pathogen exposure will result in dysbiosis; and 4) 
poor nutritional health will correlate with altered fecal metabolite profiles and changes in the 
expression of metabolic pathway enzymes encoded by the microbiome. Since analysis of 
microbiome data is a rapidly evolving field, to ensure data analysis is in line with best practices, 
we will adopt current state of the art bioinformatics tools for sequence processing, assembly, 
annotation and analysis. In case of delays or other issues that may be experienced at the 
Metabolomics Innovation Centre, we may rely on the SPARC Biocentre, hosted by the Hospital 
for Sick Children, to perform equivalent analyses 
 
As previous[46], we may expect specific taxa (prokaryotic or eukaryotic) will directly correlate 
with nutritional status during pregnancy and birth outcomes. The core deliverables will be an 
unprecedented view of dynamic changes in microbial communities that contribute to nutritional 
status during pregnancy, together with the prediction of biochemical pathways and taxa 
responsible. 
 
11 STATISTICAL CONSIDERATIONS  
 
11.1 POWER CALCULATION 
 
The adequacy of the sample size was verified using the ‘pwr’ package (version 1.2-2) in R 
(version 3.6.1). Calculations were based on the correlation between α-diversity (Shannon index) 
and weight gain during pregnancy. Assuming 400 participants are recruited into the study, and a 
type I error rate of 0.05, there will be 80% power to detect a correlation coefficient (r) >0.14. 
This is conventionally considered a small effect size[124]. Thus, we expect to be powered to 
reveal significant association between weight gain during pregnancy and diversity. Further, a 
study of 123 lean and 24 obese individuals, revealed 96% power to detect differences in 
Bacteroidetes abundance and 80% power for Firmicutes[125], while a cohort of 44 Malawian 
malnourished children was sufficient to detect significant differences in bacterial α-diversity in 
the presence and absence of Blastocystis.  
 
11.2 SUBGROUP ANALYSIS 
 
To reduce study complexity and to avoid confounding effects due to existing micronutrient 
deficiencies, we will focus specifically on interactions between macronutrient deficiencies with 

 
 
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46 
the host microbiome and health. Given the high prevalence of micronutrient deficiencies 
experienced within the study population, the effect of micronutrient deficiencies of public health 
importance (ie. iron deficiency anemia, vitamin A deficiency and vitamin D deficiency) will be 
explored within a subgroup analysis. We will achieve this by monitoring for anemia as well as 
serum mineral content. Patients exhibiting deficiencies will still be included in the study, but will 
be analyzed separately.  
 
In addition, we may conduct further sub- group analysis for participants with documented 
substance use, this sub-group analysis will be dependent on the prevalence of substance use 
among our study population.  
 
11.3 STATISTICAL ANALYSIS 
 
We will work with the research core in Calgary to apply multivariate analyses and other 
integrative methods to analyze microbiome data in the context of patient nutritional status and 
health outcomes. Our initial strategy is to apply the Similarity Network Fusion framework[126] 
to combine the various datatypes (i.e. metabolomics, microbiome, and clinical). In this approach 
networks are first constructed from each datatype, with nodes representing patients and links 
representing similarities between participants (e.g. based on correlations between microbiome or 
metabolite profiles). Networks are then fused using a method based on message-passing theory 
to identify links shared between patients supported by multiple datatypes, eliminating poorly 
supported links and strengthening links supported by multiple datatypes. This allows the 
integration of all available datasets to uncover their global substructures that can be associated to 
relevant outcomes. In an alternative approach, we will also employ Random Forests to identify 
combinations of predictors (microbial and/or metabolic factors) that best correlate with our 
clinical outcomes. Beyond integrative methods we will also apply simple statistical tests (e.g. 
Wilcoxon, t-test, Spearman’s rank correlation, PERMANOVA) to examine pairwise interactions 
(e.g. presence of pathogens vs. bacterial diversity and/or abundance of specific taxa; metabolic 
pathway expression vs. inflammation; and stool metabolites vs. blood metabolites). Further, we 
will also attempt to include additional quantitative measures of nutritional status as well as 
gender-related variables from the empowerment questionnaires, as covariates where appropriate. 
These analyses will reveal relationships between taxonomic composition, microbiome diversity 
(both prokaryotic and eukaryotic), metabolomics and the primary and secondary outcome 
measures gathered. 
 
11.4 PLAN FOR STATISTICAL ANALYSIS 
The following section details the plans for evaluating the aims of this study. Table 8 lists the 
variables to be considered in the analyses. 
Table 8. List of variables collected in the study to be utilized in analysis 
Clinical Variables 
1. 
Maternal BMI, MUAC and SFT (continuous variables) 
2. 
Maternal gestational weight gain (continuous) 
3. 
Maternal markers of inflammation (continuous) 
4. 
Infant sex 

 
 
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5. 
Infant morbidity  
6. 
Infant weight, height, head circumference z-scores (continuous) 
7. 
Infant gestational age  
8. 
Infant birth weight z-scores (continuous) 
9. 
Breastfeeding duration (continuous) 
10. 
Maternal age years (continuous) 
11. 
Maternal use of antibiotics 
12. 
Infant use of antibiotics 
13. 
Maternal calorie and macronutrient intake 
14. 
Maternal incidence of pathobionts 
15. 
Infant incidence of pathobionts 
16. 
Infant and maternal diet diversity 
Microbiome Variables 
1. 
Maternal gut bacteria profile 
2. 
Maternal eukaryotic microbe profile 
3. 
Infant gut bacteria profile 
4. 
Infant eukaryotic microbe profile 
5. 
Maternal metabolomic profile of stool 
6. 
Maternal metabolic pathway expression profile 
7. 
Maternal bacterial gene expression profile 
 
Gender-related Variables 
1. 
Food insecurity 
2. 
Self efficacy 
3. 
Perceived decision making 
4. 
Perceived social support 
5. 
Perceived parental stress 
 
 
 
 
11.4.1 ADDRESSING THE PRIMARY OBJECTIVES 
This study has two primary objectives: 1) To assess if alterations of the microbiota in the 
maternal gut (dysbiosis) is associated with maternal gestational weight gain; and 2) To determine 
the association between maternal microbiome dysbiosis during pregnancy and birth outcomes, 
infant growth, nutritional status and morbidity in the first year of life.The first objective will be 
addressed through the following approaches. First, maternal gut bacteria and eukaryotic profiles 
will be used to calculate Bray-Curtis dissimilarity metrics between individual samples which will 
be leveraged in principal co-ordinate analyses to determine the extent samples collected at the 
first or third trimester, exhibiting similar gestational weight gains, co-cluster. Permutational 
multivariate analysis of variance (PERMANOVA) tests will assess the degree of overlap 
between samples exhibiting low gestational weight gain versus samples exhibiting high 
gestational weight gain. A lack of clustering in the first trimester, followed by clustering of 
samples on the basis of similar gestational weight gain would indicate that alterations in the 

 
 
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48 
maternal microbiota during the course of pregnancy, impacts gestational weight gain. Next, we 
will attempt to correlate changes in the alpha diversity (as measured by the Shannon and 
Simpson indices) of the gut microbiome samples between the first and third trimester, with 
gestational weight gain. A decrease in alpha diversity (indicative of dysbiosis) associated with 
low gestational weight gain, would again indicate that dysbiosis is associated with maternal 
weight gain. To examine the influence of individual taxa on gestational weight gain, we will 
perform bivariate analyses that examine the correlation (Pearson, Spearman) of each taxon with 
gestational weight gain. Correlations will examine taxon abundance in first and third trimester, in 
addition to the change in abundance between the two trimesters. The Benjamini-Hochberg 
procedure will be applied to correct p-values while controlling for false-discovery rates. 
Equivalent approaches will be used to investigate other clinical outcomes in the first year of life. 
To complement these analyses we will also undertake an integrative modeling strategy based on 
Similarity Network Fusion framework[127] to analyze the contribution of each variable (clinical, 
microbiome and gender-related) on gestational weight gain. In this approach we construct a 
series of networks (one for each variable) in which nodes represent patients and links represent 
similarities between patients (e.g. based on correlations between e.g. microbiome or metabolite 
profiles). Networks are then fused using a method based on message-passing theory to identify 
links shared between patients supported by multiple datatypes, eliminating poorly supported 
links and strengthening links supported by multiple datatypes. This allows the integration of all 
available datasets to uncover their global substructures that can be associated with gestational 
weight gain. In an alternative approach, we will also employ Random Forests to identify 
combinations of variables (clinical, microbiome and gender-based; Table 8) that correlate with 
gestational weight gain.  
 
11.4.2 ADDRESSING THE SECONDARY OBJECTIVES 
This study has the following secondary objectives: 1) To link the maternal microbiome to dietary 
intake, with a focus on calories and macronutrients; 2) To integrate maternal anthropometric 
factors, morbidity with microbiome data to reveal key modulators (microbial taxa and 
metabolites) of dietary intake during pregnancy and the post-partum period; 3) To determine the 
impact of the maternal microbiome during pregnancy, including the exposure to pathogens and 
parasites, on the development of the infant microbiome; and 4) To investigate the maternal 
microbiome’s exposure to pathogens and parasites, and the association with intestinal 
inflammation. 
To assess these secondary objectives, we will employ general linear models. In a first approach 
we will perform PERMANOVA and probe the association of microbial community structure 
with the clinical variables. Pair-wise differences in microbiome structure between patient 
samples at similar time-points are defined by abundance-weighted pair-wise differences using 
the Bray-Curtis dissimilarity metric. DESeq2, a method for differential analysis of count data, 
will subsequently be applied to investigate both associations of specific taxa with the clinical 
variables, together with the strength of those associations. DESeq2 will utilize default settings, 
and q-values calculated with the Benjamini-Hochberg procedure to correct p-values while 
controlling for false-discovery rates. These analyses will reveal which clinical variables 

 
 
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49 
(including exposure to pathogens and parasites) correlate with the maternal microbiome from a 
taxonomic perspective. 
To probe the association of clinical variables with the function of the microbiome, 
PERMANOVA and DESeq2 analyses will be performed replacing microbial community 
structure metrics with microbial community function metrics, specifically: 1) metatranscriptomic 
profiles; and 2) stool metabolomic profiles. Profile differences will be defined through Spearman 
rank differences for metatranscriptomic data and Euclidean distance metrics for metabolomic 
profiles. These analyses will reveal microbial genes, metabolites and pathways that correlate 
with clinical variables. 
 
11.4.3 ADDRESSING THE EXPLORATORY OBJECTIVES 
This study has the following exploratory objectives: 1) To explore the role of the maternal gut 
microbiome during pregnancy and to identify gut community dynamics in pregnant women and 
how this impacts differences between dietary intake and nutritional status; 2) To investigate 
socio-economic factors; including gender, poverty, exclusion and empowerment, and their 
influence on the health of a mother’s microbiome (assessed by alpha and/or beta diversity, and 
absence of pathogens); 3) To explore the role of the human microbiota on nutritional status by 
performing fecal microbiota transplants in germ free mice and sterile piglets. 
For the first exploratory objective, we will apply the same statistical framework as we utilized 
for the secondary objectives, here however, we are concerned with changes in the microbiome 
within a patient from the first to third trimester. Consequently, microbiome structural and 
functional profiles will be generated from the difference in: 1) taxonomic abundances; 2) gene 
expression; and 3) metabolite concentrations, between the first and third trimesters. Profile 
differences will then be utilized in the PERMANOVA and DESeq2 approaches as described 
above to identify associations between clinical variables and changes in microbiome structure 
and function. 
 
12 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
 
12.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
Research ethics committee/institutional review board approval will be obtained through Clinical 
Trials Ontario. All standard procedures related to consent will be followed. 
 
Informed consent will be obtained from all study participants. The trial will be verbally 
explained to participants by study personnel, who will then obtain informed consent. In the event 
that the participant does not speak English, a translator will be present either in person or through 
a phone call. Written informed consent will then be obtained and participants will be informed 
that they have the right to withdraw from the study at any time without any penalty in terms of 
care. Withdrawal from the study or refusal to participate will not in any way affect their ability to 

 
 
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receive any services offered to the community by the study. All signed informed consent forms 
will be retained in the study files. 
 
12.2 STUDY DISCONTINUATION AND CLOSURE 
 
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause.  Written notification, documenting the reason for study suspension or 
termination, will be provided by the suspending or terminating party to study participants, the 
Investigator, the CIHR, and regulatory authorities.  If the study is prematurely terminated or 
suspended, the PI will promptly inform study participants and the REB and will provide the 
reason(s) for the termination or suspension.  Study participants will be contacted, as applicable, 
and be informed of changes to the study visit schedule. 
  
Circumstances that may warrant termination or suspension include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to participants 
• Insufficient compliance to protocol requirements 
• Data that is not sufficiently complete and/or evaluable 
 
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the PI and REB 
 
12.3 CONFIDENTIALITY AND PRIVACY 
 
12.3.1 PARTICIPANT ID SYSTEM 
 
 
All participants will be assigned a unique study ID, the IDs will be assigned as follows: 
Maternal ID: 
Site ID- Participant number* 
Infant ID: 
Same as mom: Site ID- Participant number- 01  
 
Participants from SickKids will receive an ID: 01-XX 
Participants from St. Michael’s clinics will receive an ID: 02-XX 
Participants from St. Michael’s Affiliated Midwifery clinics will receive an ID: 03-XX 
*The participant number will be assigned consecutively. 
 
For example, the third participant enrolled at a St. Michael’s clinic, will be assigned the 
participant ID: 02-03. If this mother had twins the infant IDs would be: 02-03-01 and 02-03-02. 
 
Participant samples (stool, blood and rectal swabs), will be de-identified, and will only contain 
the participant ID, the visit number (1- baseline, 2- 30-34 weeks post conception, 3- 16wk-4 
months post partum and 4- 12 months post-partum), and the date and time of collection. The 
master linking log, which will link the participant name to the participant ID, will be stored as a 
password protected excel file. The master linking log for SickKids participants will be stored on 

 
 
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the SickKids Onedrive, and the master linking log for St. Michael’s participants will be stored as 
a password protected excel file on the St. Michael’s shared drive.  
 
Participant confidentiality and privacy is strictly held in trust by the participating Investigators 
and their staff. This confidentiality is extended to cover testing of biological samples and genetic 
tests in addition to the clinical information relating to participants. Therefore, the study protocol, 
documentation, data, and all other information generated will be held in strict confidence. Any 
research information obtained about the patient in this study will be kept confidential. A patient 
will not be identified by name, only by unique study ID number. The patient’s name or any 
identifying information will not appear in any reports published as a result of this study.  
 
All research activities will be conducted in as private a setting as possible. 
 
Any research information obtained about the patient in this study will be kept confidential. A 
patient will not be identified by name, only by unique study ID number. The patient’s name or 
any identifying information will not appear in any reports published as a result of this study. All 
identifying information will be kept behind 2 security measures or as per equivalent institutional 
policy, under the supervision of the study/site PI and will not be transferred outside of the 
hospital. 
 
The PI, study auditor, authorized representatives of CIHR, and representatives of the Research 
Ethics Board (REB), may inspect all documents and records required to be maintained by the 
Investigator, including but not limited to, medical records for the participants in this study. The 
clinical study site will permit access to such records. 
 
Study participant research data, for the purposes of statistical analysis and scientific reporting, 
will be transmitted to and stored at SickKids. This will not include the participant’s contact or 
identifying information. Rather, individual participants and their research data will be identified 
by a unique study identification number. The study data entry and study management systems 
used by clinical sites and by SickKids research staff will be secured and password protected. At 
the end of the study, all study databases will be de-identified and archived at SickKids. However, 
in order to facilitate sample processing at SickKids from St. Michael’s Hospital, identifiable 
labels on blood samples will be transferred to SickKids to assist with processing. 
 
 
12.4 FUTURE USE OF STORED SPECIMENS AND DATA 
 
Data collected for this study will be analyzed and stored at SickKids. After the study is 
completed, the de-identified, archived data will be transmitted to and stored at SickKids. 
Permission to transmit data to SickKids will be included in the informed consent.  
 
With the participant’s approval and as approved by the Research Ethics Boards (REBs), de-
identified biological samples will be stored at SickKids.  Extracted DNA and RNA will be batch 
shipped to SickKids from participating sites. These samples will be used to research the causal 
relationships between microbiome dynamics, pathogen exposure, nutritional status during 
pregnancy, undernutrition, its complications and other conditions for which study participants 

 
 
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52 
are at increased risk, and to improve treatment. De-identified samples and de-identified study 
data will be sent to research collaborators outside SickKids and Canada for analysis. The 
samples will not be sold. Blood and stool samples will be biobanked at SickKids. 
 
Established animal models will be used to study the casual relationships between microbiome 
dynamics, pathogen exposure and nutritional status during pregnancy and to examine whether 
manipulation of the microbiome can improve nutritional status. The gnotobiotic facilities are 
located at the University of Toronto and the University of Alberta. Human fecal microbiome 
transplants (FMTs) have been effectively applied to animals to study the role of human 
microbiota on nutritional status. Here we will perform FMT into germ-free mice and sterile 
piglets to first, recapitulate the disease-associated phenotypes in animals and second, to set the 
stage for therapeutic intervention strategies. We will investigate 10 patient microbiomes: 
correlated with positive and negative nutritional status, as indicated by primary and secondary 
outcomes. This is consistent with previous animal studies that identified key taxonomic 
associations with undernutrition[5]. Extracted microbes will also be sent to our collaborator Dr. 
Michael Grigg, the Chief of Molecular Parasitology at the NIH, Bethesda, and an expert at host-
parasite interactions. Dr. Grigg will oversee mouse experiments with the extracted microbes to 
investigate the parasite-microbiome interactions. Extracted microbes will also be sent to 
Dalhousie to investigate parasite-microbiome interactions.  
 
In addition to publishing findings in open access journals, we will ensure all sequences and 
metabolomics datasets are deposited in appropriate public repositories. SOPs, pathogen samples 
and statistical methods developed through this project will be shared with the IMPACTT 
research core. 
 
 
12.4.1.1 FUTURE GENETIC TESTING 
 
Participants will have the option to sign an additional section of the consent form for future 
genetic testing of their blood samples and their infant’s blood samples. Participants will have the 
option to opt out of this future testing, or to have genetic testing on their own samples but not on 
their infant’s. If they sign the consent form, the samples will be stored for future genetic testing 
to further dissect patterns of microbiome genotype associated traits.  
 
The focus of the current application is on the investigation of interactions between the bacterial 
and eukaryotic components of the microbiome and their downstream impact on host nutritional 
status. However, with the availability of host DNA, opportunities will exist to examine the 
interactions between host genetic factors and gut microbial communities. For example, defects in 
these interactions can result in imbalances in the microbial community, such as the loss of 
beneficial commensal microbes and also lead to impaired pathogen clearance[128]. In our own 
studies applying metatranscriptomics to fecal samples from mice, we showed that knockout of 
the gene encoding Perilipin-2, a protein involved in lipid absorption, in mice results in significant 
shifts in the expression of microbial genes involved in energy production [90]. Another study 
focused on gene-microbiome interactions in healthy individuals used genotyping to identify 58 
single nucleotide polymorphisms associated with the relative abundance of 33 taxa in their gut 
microbiomes[129]. Although not funded as part of this study, we envision that the availability of 
DNA samples from the mothers and children enrolled in this study, provides an unparalleled 

 
 
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53 
opportunity to apply whole genome sequencing or targeted resequencing methodology, to 
examine how the interactions between host genetic factors and the gut microbiome contribute to 
host nutritional status and birth outcomes.  Such studies would form the basis of future grant 
applications. 
 
During the conduct of the study, an individual participant can choose to withdraw consent to 
have biological specimens stored for future research. However, withdrawal of consent with 
regard to biosample storage may not be possible after the study is completed.  
 
12.4.1.2 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
 
Data collection is the responsibility of the study research staff at the site under the supervision of 
the site Investigator. The Investigator is responsible for ensuring the accuracy, completeness, 
legibility, and timeliness of the data reported. All source documents should be completed in a 
neat, legible manner to ensure accurate interpretation of data.   
 
In the data analysis, growth measurements, medication usage, health assessments and birth 
characteristics will be obtained from the patient’s clinical chart. Participants previous and recent 
data will be used to determine eligibility to participate in the study and/ or will be used as 
predictors, mediators, and/ or covariates in the analysis of their gut microbiota. 
 
For SickKids study participants, participant logs will be stored on SickKids Onedrive, using 
password protected excel files. For St. Michael’s participants, participant logs will be stored on 
the St. Michael’s shared drive using password protected excel files. 
 
Study data will be entered into REDCap (Research Electronic Data Capture), a secure, web-
based application designed exclusively to support data capture for research studies. REDCap is 
developed and maintained by a team at Vanderbilt University and licensed free of charge by the 
Research Institute at The Hospital for Sick Children. The application and data are housed on 
servers provided by The Hospital for Sick Children. These servers are located within SickKids 
secure data center. Local support for REDcap is provided by SickKids Research IT. All of the 
information entered into REDcap will be de-identified.   
 
The sample analysis information, including the sequencing data and metabolomics data will be 
de-identified and hosted on a network storage array in the Parkinson Lab. This will be protected 
by the Sickkids firewall. The patient sequence data will be removed, and the microbiome data 
will be uploaded on the National Centre for Biotechnology Information (NCBI). The NCBI 
facilitates the automated storage and analysis of databases for the research and medical 
community. 
  
12.4.1.3 DATA SECURITY AND CONFIDENTIALITY OF PARTICIPANTS USING THE 
ASA24 SYSTEM 
 
Researchers using the ASA24 recall system do not provide the NCI, Westat, or the ASA24  
system with any identifying data for participants of their studies. Rather, researchers specify a 

 
 
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54 
unique numeric identifier for each Respondent and download system-generated usernames and 
encrypted passwords that they provide to respondents so that they may access the application.  
 
The ASA24 system also does not collect any identifying data directly from respondents. 
However, IP address information is accessed for the purpose of routing information between the 
server and the respondent's computer—often the IP address is that of the user's Internet Service 
Provider (ISP). IP addresses are not stored or tracked by the ASA24 system. However, logs of 
connections are kept in the hosting environment for audit trail purposes. This information is not 
mined in any way but would be available if there were a legal obligation to release it. 
 
Response data is secured at the hosting site using industry standard security controls, including 
firewalls and encryption. All data entered into the ASA24 system at the Respondent’s 
computer is encrypted by the internet browser (e.g., Internet Explorer, Firefox) before they are 
transmitted to our servers using Secure Socket Layer (SSL) Technology. SSL allows for the 
authentication of the sending and receiving computers.  Only a particular study’s investigator(s) 
and the ASA24 operations team can access response data. Access is gained through usernames 
and strong passwords. 
 
The company that hosts the online survey is called Westat. The servers are located in Rockville, 
MD. Encrypted backups are stored offline in MD and no data is transferred to any other server, 
domestic or foreign. Email address is obtained for the Researcher conducting data collection 
using ASA24 and it is stored in the database. IP addresses of Researchers and Respondents are 
captured and logged by the web server. ASA24 does not capture any PII related to 
Respondents. ASA24 is subject to FISMA moderate level controls that protect the integrity, 
confidentiality, and availability of the system. Data is not shared by the ASA24NCI and 
Westat teams with other researchers. The system is a government system and subject to any 
lawful government purpose. 
 
12.4.1.4 STUDY RECORDS RETENTION  
  
To enable evaluations and/or audits, the Principal Investigator agrees to keep records, including 
the identity of all participating patients (sufficient information to link records, CRFs and hospital 
records), all original signed informed consent forms, copies of all CRFs, source documents, and 
detailed records in a secure location for a minimum of 7 years in accordance with SickKids 
policy.  
 
 
If the Principal Investigator relocates, retires, or for any reason withdraws from the study, then 
the study records must be transferred to an acceptable designee, such as another Investigator or 
another institution. 
 
12.5 PROTOCOL DEVIATIONS 
 
A protocol deviation is any noncompliance with the study protocol. The Principal Investigator 
will assure that no deviation from, or changes to the protocol will take place without documented 
approval from the Research Ethics Board (REB), except where necessary to eliminate an 

 
 
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55 
immediate hazard(s) to the trial participants. The noncompliance may be either on the part of the 
participant, the Investigator, or the study site staff. As a result of deviations, corrective actions 
are to be developed by the site and implemented promptly. All protocol deviations will be 
documented; the Principal Investigator will assess each protocol deviation to determine the 
impact to the patient’s rights, safety or welfare, study efficacy and data integrity. Protocol 
deviations must be sent to the reviewing REB in accordance with their policies. The Principal 
Investigator is responsible for knowing and adhering to the reviewing REB requirements. 
 
 
12.6 CONFLICT OF INTEREST 
 
The research team declares no conflict of interest. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12.7 ABBREVIATIONS 
 
AE 
Adverse Event 
AKU 
Aga Khan University 
ANCOVA 
Analysis of Covariance 

 
 
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56 
CIOMS 
Council for International Organizations of Medical Sciences 
CLIA 
Clinical Laboratory Improvement Amendments 
CONSORT Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
eCRF 
Electronic Case Report Forms 
GCP 
Good Clinical Practice 
ICMJE 
International Committee of Medical Journal Editors 
MOP 
Manual of Procedures 
MRP 
Most Responsible Physician 
MUAC 
Mid Upper Arm Circumference 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
REB 
Research Ethics Board 
SAE 
Serious Adverse Event 
SFT 
Triceps Skinfold Thickness  
SoA 
Schedule of Activities 
SOP 
Standard Operating Procedure 
 
 
 

 
 
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57 
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