Fecal Microbial Transplantation and Fiber Supplementation in Subjects With Obesity and Metabolic Syndrome
Phase: PHASE2
Conditions: Obesity, Metabolic Syndrome
Sponsor: University of Alberta

FMT and fiber supplementation in metabolic syndrome 
  October 1, 2018 
 
 
STUDY PROTOCOL 
 
Version 5 
 
 
 
Fecal microbial transplantation and fiber supplementation in subjects with 
obesity and metabolic syndrome. 
 
 
 
 
 
PROTOCOL VERSION DATE  
 
October 1, 2018 
 
 
 
 
 
Principal Investigator 
 
Dr. Karen Madsen 
Department of Medicine  
Division of Gastroenterology 
7-142 Katz Group Center 
Edmonton, AB T6G 2X8 Canada 
Phone (780) 492-5257 
E-mail: Karen.madsen@ualberta.ca 
 
 
 
 
 
 
 
 

 
 
 
GENERAL INFORMATION 
 
 
Name and address of the funding sponsor of the study 
 
The W. Garfield Weston Foundation; The Weston Microbiome Initiative 
 
22 St. Clair Avenue East, Suite 2001 
 
Toronto, Ontario 
 
M4T 2S3 
 
Name and address of the person authorized to sign the protocol and amendments  
 
Dr. Karen Madsen 
University of Alberta 
780-492-5257 
7-142 Katz Group Center 
Edmonton, Alberta 
T6G 2E1 
 
 
Name and address of study monitor 
 
Clinical monitoring will be provided through the University of Alberta Quality Management in Clinical 
Research Department (QMCR) for the duration of the trial.  
 
Name, title, address and telephone number(s) of the medical expert for the trial 
 
Dr. Dina Kao 
University of Alberta 
780-492-8307 
Zeidler Ledcor Center 
Edmonton, Alberta 
T6G 2E1 
 
 
Name and title of the investigator(s) and sub-investigators responsible for the trial with address and 
phone number(s)   
 
Dr. Karen Madsen 
University of Alberta 
780-492-5257 
7-142 Katz Group Center 
Edmonton, Alberta 
T6G 2E1 
 

 
 
 
Dr. Shahzeer Karmali 
University of Alberta 
405 CSC, Royal Alexandra Hospital 
780-735-6650 
Edmonton, Alberta 
T5H 3V9 
 
Dr. Gane Wong 
University of Alberta 
780-492-8663 
4-126 Katz Group Centre 
Edmonton, Alberta 
T6G 2E1 
 
Dr. Jens Walter 
University of Alberta 
780-492-1182 
4-126 Li Ka Shing Research Centre 
Edmonton, Alberta 
T6G2E1 
 
Dr. Valentin Mocanu 
University of Alberta 
Alberta Heath Services 
780-735-6650 
405 CSC, Royal Alexandra Hospital 
Edmonton, Alberta 
T5H 3V9 
 
Dr. Jerry Dang 
University of Alberta 
Alberta Health Services 
780-735-6650 
405 CSC, Royal Alexandra Hospital 
Edmonton, Alberta 
T5H 3V9 
 
Dr. Mike Laffin 
University of Alberta 
Alberta Health Services 
780-492-7077 
7-142 Katz Group Center 
Edmonton, Alberta 
T6G 2E1 
 
Dr. Arya Sharma 
University of Alberta 
Li Ka Shing Building, Rm 1-116 
87th Avenue and 112th Street 
Edmonton, Alberta    
T6G 2E1 
 

 
 
 
Dr. Daniel Birch 
 
University of Alberta 
780-735-4786 
418 Royal Alexandra Hospital CSC 
T5H 3V9 
 
 
Name and addresses of the clinical laboratories and/or other institutions involved in the trial 
 
The Centre of Excellence for Gastrointestinal Inflammation  
and Immunity Research (CEGIIR) 
University of Alberta 
7-126 Katz Group Center 
Edmonton, Alberta 
T6G 2E2 
 
Zeidler Ledcor Centre  
130 University Campus  
112 Street and 85th Avenue 
Edmonton, Alberta, Canada T6G 2X8 
 
Royal Alexandra Hospital 
Community Services Center Bldg, 4th Floor 
10240 Kingsway Avenue NW 
Edmonton, Alberta 
T5H 3V9 
 
University of Alberta Hospital 
8440-112 Street 
Edmonton, Alberta 
T6G 2B7 
 
 
 

 
 
 
Study Summary 
Title 
Fecal microbial transplantation and fiber supplementation in subjects 
with obesity and metabolic syndrome 
 
Short Title 
FMT and fiber in metabolic syndrome  
Protocol Number 
Pro00076642 
Phase 
This is a phase II clinical trial.  
 
Methodology 
This is an exploratory, four-arm, parallel design, randomized placebo-
controlled intervention study.  
Study Duration 
12 weeks 
Study Center(s) 
This is a single center trial at the University of Alberta 
 
Objectives 
The objective of this study is to determine if fecal microbial 
transplantation (FMT) combined with supplementation with a 
combined fiber supplement of resistant starch type 4, acacia gum, 
and soluble corn fiber has a clinically significant effect on metabolic 
outcomes in obese subjects with metabolic syndrome  
Study Outcomes 
Primary Outcome 
 
Changes in insulin sensitivity between the time of screening and 6 
weeks following treatment. 
 
Secondary Outcomes 
 Changes in Body weight and anthropometric parameters 
between baseline and week 6.  
 Changes in HbA1C, fasting glucose, glucose tolerance test 
between baseline and week 6  
 Changes in fasting lipid profile between baseline and week 6 
 Changes in blood pressure between baseline and week 6 
 Quality of life and satiety between baseline and week 6 
 Changes in serum  levels of leptin, adiponectin, ghrelin, CRP, 
TNF-α, IL-6, LPS, LPS-binding protein and zonulin between 
baseline and week 6 
 Changes in stool microbiota composition between baseline and 
week 6 
 Changes in stool short chain fatty acid composition between 
baseline and week 6 
 
Number of 
Subjects 
 
68  
 

 
 
 
Diagnosis and 
Main Inclusion 
Criteria 
Primary Diagnosis:  
 BMI > 30 
 
Key Inclusion Criteria:  
 Age 18-64 years at screening  
 Total body weight fluctuation over the last 6 months <10% 
 Fasting plasma glucose > 5.6 mmol/L OR HgbA1c ≥ 5.5% OR 
patients receiving an antidiabetic medication 
 At least one of the following:  
o Fasting triglyceride ≥1.7 mmol/L OR receiving 
dyslipidemia medication  
o HDL cholesterol <1.03 mmol/L in males or <1.29 
mmol/L in females OR receiving dyslipidemia 
medication  
o Known diagnosis of hypertension OR systolic blood 
pressure ≥130 or diastolic blood pressure ≥85 mmHg 
OR receiving antihypertension medication  
Study Product, 
Dose, Route, 
Regimen 
FMT:  
 50 gm of screened and encapsulated single donor stool 
(approximately 20-30 capsules) taken by mouth on day 1 of the 
trial after having fasted overnight and completed a bowel prep 
with Pico-Salax®. 
 Placebo pills will contain microcrystalline cellulose 
 
Soluble corn fiber (PROMITOR®: Tate&Lyle) 
 Women: 4.5 gm of PROMITOR by mouth days 1-3 increased 
to 9 gm daily from day 4 until trial completion. 
 Men: 5.5 gm of PROMITOR by mouth days 1-3 increased to 
11 gm by mouth daily from day 4 until trial completion. 
 
Resistant Wheat Starch 4 (Fibersym®: MGP Ingredients):  
 Women: 4.5 gm of powdered RS4 by mouth days 1-3 
increased to 9 gm by mouth daily from day 4 until trial 
completion. 
 Men: 5.5gm of powdered RS4 by mouth days 1-3 increased to 
11 gm by mouth daily from day 4 until trial completion. 
 
Acacia Gum (Pre-Hydrated Gum Arabic: TIC GUMS):  
 Women: 4.5 gm of powdered acacia gum by mouth days 1-3 
increased to 9 gm by mouth daily from day 4 until trial 
completion. 
 Men: 5.5gm of powdered acacia gum by mouth days 1-3 
increased to 11 gr by mouth daily from day 4 until trial 
completion. 
 
 

 
 
 
Duration of 
administration 
FMT: Single dose of 50gm donor stool or placebo (microcrystalline 
cellulose) on day 1. 
 
Fiber: Daily administration until completion at week 6  
 
Reference therapy 
Both FMT and Fiber will be placebo matched as reference therapy. 
  
Statistical 
Methodology 
Study groups with by analyzed by pair-wise comparison with 
evaluation of means between and across groups using paired or 
unpaired t-tests for continuous outcomes and chi-squared tests for 
dichotomous ones. Multivariable predictors of change in relevant 
outcomes will be identified using appropriately constructed and 
calibrated linear regression models for continuous outcomes or logistic 
regression models for dichotomous ones.  
 
 
 
 
 
 
 
 
 
 

 
 
 
Table of Contents 
1  
BACKGROUND ............................................................................................................................................... 1 
1.1 
INVESTIGATIONAL AGENT ........................................................................................................................... 1 
1.2 
PRECLINICAL DATA ..................................................................................................................................... 1 
1.3 
RISK/BENEFITS ............................................................................................................................................ 2 
1.4 
DOSE RATIONALE ........................................................................................................................................ 3 
1.5 
TRIAL CONDUCT ......................................................................................................................................... 3 
1.6 
POPULATION................................................................................................................................................ 3 
1.7 
LITERATURE ................................................................................................................................................ 3 
2 
TRIAL OBJECTIVES...................................................................................................................................... 5 
3 
TRIAL DESIGN ............................................................................................................................................... 6 
3.1 
PRIMARY STUDY ENDPOINTS/SECONDARY ENDPOINTS .............................................................................. 6 
3.2 
STUDY DESIGN/TYPE .................................................................................................................................. 6 
3.3 
RANDOMIZATION ........................................................................................................................................ 7 
3.4 
MAINTENANCE ............................................................................................................................................ 7 
3.5 
TRIAL TREATMENT ...................................................................................................................................... 7 
3.6 
DURATION ................................................................................................................................................... 8 
3.7 
DISCONTINUATION ...................................................................................................................................... 8 
3.8 
PRODUCT ACCOUNTABILITY ....................................................................................................................... 8 
4 
SELECTION AND WITHDRAWAL OF SUBJECTS .................................................................................. 9 
4.1 
INCLUSION CRITERIA ................................................................................................................................... 9 
4.2 
EXCLUSION CRITERIA ................................................................................................................................. 9 
4.3 
SUBJECT WITHDRAWAL .............................................................................................................................. 9 
4.4 
TREATMENT OF SUBJECTS ......................................................................................................................... 10 
4.5 
MEDICATION ............................................................................................................................................. 13 
4.6 
MONITORING FOR SUBJECT COMPLIANCE .................................................................................................. 13 
5 
ASSESSMENT OF EFFICACY .................................................................................................................... 13 
5.1 
EFFICACY PARAMETERS ............................................................................................................................ 13 
5.2 
METHOD AND TIMING ............................................................................................................................... 14 
6 
ASSESSMENT OF SAFETY ......................................................................................................................... 15 
6.1 
SAFETY PARAMETERS ............................................................................................................................... 15 
6.2 
ADVERSE EVENT MONITORING ................................................................................................................. 16 
6.3 
SERIOUS ADVERSE EVENTS (SAES) AND SAFETY MONITORING  .............................................................. 16 
6.4 
ADVERSE EVENT FOLLOW-UP ................................................................................................................... 16 
7 
STATISTICAL PLAN .................................................................................................................................... 17 
7.1 
STATISTICAL METHODS............................................................................................................................. 17 
7.2 
SUBJECT POPULATION(S) FOR ANALYSIS .................................................................................................. 18 
7.3 
SIGNIFICANCE ........................................................................................................................................... 18 
7.4 
TERMINATION CRITERIA ........................................................................................................................... 18 
8 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTATION ................................................................ 18 
9 
QUALITY CONTROL AND QUALITY ASSURANCE ............................................................................ 18 
10 
ETHICAL CONSIDERATIONS ................................................................................................................... 19 
11 
DATA HANDLING AND RECORD KEEPING ......................................................................................... 19 
12 
FINANCE AND INSURANCE ...................................................................................................................... 19 

 
 
 
List of Abbreviations 
 
 
AE 
 
Adverse event 
ASM  
Anthropometric Standardization Reference Manual 
BMI 
 
Body mass index 
CEGIIR 
Center of Excellence in Gastrointestinal Inflammation and Immunity Research 
CRP                 C reactive protein 
DNA  
Deoxyribonucleic acid 
EQ-5D             EuroQol five dimensions questionnaire 
EtOH  
Ethanol 
FPG 
 
Fasting plasma glucose 
FMT                Fecal microbiota transplantation 
GI 
 
Gastrointestinal 
HAV                 hepatitis A virus 
HBV                 hepatitis B virus 
HBT                  human biotherapy 
HCV                 hepatitis C virus 
Hgb                  hemoglobin 
HIV                  human immunodeficiency virus 
HTLV               human T lymphotrophic virus 
HbA1C 
Glycated haemoglobin 
HDL  
High density lipoprotein 
HEC  
hyperinsulinemic euglycemic clamp 
HOMA-IR 
Homeostasis model assessment-insulin resistance  
HREB  
Health research ethics board 
IL 
 
Interleukin 
INR 
 
International normalizing ratio for pro-thrombin time 
IR 
 
Insulin resistance 
IWQOL 
Impact of weight on quality of life 
LPS 
 
Lipopolysaccharide 
NIH 
 
National Institutes of Health 
OGTT  
Oral glucose tolerance test 
RCT              Randomized controlled trial 
REDCap          Research Electronic Data Capture 
RS4 
 
Resistant wheat starch 4 
SAE 
 
Serious adverse event 
SCFA  
Short chain fatty acid 
TG 
 
Triglyceride 
TNF 
 
Tumor necrosis factor 
U of A              University of Alberta 
WHO  
World Health Organization 
 
 
 

 
P a g e  | 1 
1 Background 
 
1.1 Investigational Agent 
 Fecal microbial transplant(FMT) 
 Soluble corn fiber (PROMITOR®: Tate&Lyle) 
 Resistant Wheat Starch 4 (Fibersym®: MGP Ingredients):  
 Acacia Gum (Pre-Hydrated Gum Arabic: TIC GUMS):  
 Microcrystalline Cellulose (Microcel FG-200; Blanver Farmoquimica Ltd) (Placebo) 
 
1.2 Preclinical Data 
Obesity is characterized by gut dysbiosis and a systemic low-grade inflammation that is 
implicated in development of insulin resistance and cardiovascular dysfunction [1].  A role 
for microbial produced metabolites including short chain fatty acids (SCFA) and modified 
bile acids on these parameters has been described in numerous publications [2-4]. Increased 
SCFA in particular have been shown to beneficial in weight loss possibly through a SCFA-
induced enhancement of gut barrier function, increased anti-inflammatory immune responses, 
and modulation of energy metabolism and production of gut hormones [5, 6].   
Studies using therapies aimed at altering gut microbes with fecal transplantation or antibiotics 
have demonstrated links between alterations in gut microbes and host metabolic parameters 
[7, 8].  In a study using fecal microbial transplantation (FMT), Vrieze et al showed that obese 
subjects who received fecal transplants from lean donors had improved insulin sensitivity and 
higher levels of butyrate-producing bacteria compared to obese individuals who received 
autologous FMT [7].  Previous studies have shown that FMT induces changes in metabolic 
parameters within 6 weeks but alterations in both gut microbes and metabolic parameters are 
not maintained suggesting an alternative strategy is required [7, 9].  In a second study, the 
same group showed that vancomycin acted to decrease gut microbiota diversity, bile acid 
dihydroxylation and peripheral insulin sensitivity in subjects with metabolic syndrome [8].  
These studies, along with numerous other animal studies, clearly indicate a role for gut 
microbes in the modulation of host metabolic parameters.  
Recent work has highlighted a necessary role for dietary fiber in the maintenance of microbes 
required for human health. In addition, studies suggest an inverse relationship between 
systemic inflammation, metabolic disease and dietary fiber intake [10]. This may be due to the 
fact that dietary fiber is not digested by the host but instead is fermented by the gut microbiota 
into SCFA and other metabolites. Individual differences in microbial composition can 
determine which fiber types are fermented and to what degree.  Individual microbes prefer 
different glycans and selective fermentation of different fibers can influence which microbial 
taxa proliferate [11].  In addition, the colonic microbiome exists as an ecosystem, where 
substantial cross-feeding occurs with metabolites produced by one organism being used by a 
second organism [12]. Therefore, to ensure growth of an entire ecosystem and enhance 
engraftment of an FMT, a variety of substrates needs to be available [13]. Resistant starch type 

 
P a g e  | 2 
4 (RS4), a prebiotic supplement derived from phosphorylated wheat-based resistant starch, 
resists host digestion and is fermented by the gut microbiota into anti-inflammatory SCFA 
[14]. RS4 has been used as a supplement in both human and animal studies to improve a 
number of metabolic parameters and is tolerated at high doses [15-18].  Human studies have 
shown RS4 to improve various aspects of host metabolic function including dyslipidemia, 
body composition, pro-inflammatory cytokines, glycosylated hemoglobin, and to attenuate 
post-prandial glucose and insulin responses [15, 17, 18].  RS4 has also been shown to 
modulate gut microbial composition and increase levels of SCFA [17]. Increases in SCFA 
have been shown to improve gut barrier function, induce anti-inflammatory regulatory T-cells, 
impact the production of gut hormones involved in energy metabolism, glucose control, and 
satiety as well as altering gut-brain neural circuits to enhance metabolic benefits [5, 19-22].  
Acacia gum is a water soluble polysaccharide dietary fiber that is resistant to host digestion 
and fermented in the distal colon resulting in the production of SCFA [23].  Soluble corn fiber 
is a maize-derived fiber that has a prebiotic effect in increasing numbers of bifidobacteria and 
increased acetate production [24].  Microcrystalline cellulose is largely resistant to microbial 
fermentation and is suitable for use as a placebo control [25].  
Overall these findings support the use of fiber supplements in patients with metabolic 
syndrome in helping to manage their condition.  However, in human trials involving fiber 
supplementation, a high degree of inter-individual variation in clinical response is seen [26, 
27]. This may occur due to the difference in microbial profiles between individuals and 
whether an individual possesses a particular microbial taxa capable of metabolizing any 
specific fiber.  Thus, a subject with a gut dysbiosis associated with an obese state may not 
possess the necessary microbes to fully utilize a fiber supplement.  If so, providing the 
recipient with specific microbes through FMT capable of metabolizing fiber supplements may 
improve clinical response. This concept is supported by a previous study that showed fiber 
supplementation to enhance responses to FMT in patients with ulcerative colitis [28].  This 
theory underlines the hypothesis of our trial investigation that supplementing individuals with 
a combination of fibers with enhance and maintain the beneficial effects of FMT on metabolic 
outcomes in obese subjects with metabolic syndrome.  
1.3  Risk/Benefits 
Members of the team have developed and refined the research protocols for this study, including 
fecal donor screening and collection, preparation of frozen fecal capsules, oral FMT delivery, 
prebiotic supplementation and patient safety. As such the intervention poses minimal risk to the 
patient. However, side effects from FMT and fibers include the potential for gastrointestinal 
intolerance. To mitigate this possibility, specific fibers were chosen that have documented 
limited GI side effects.   
 
The individual patients may derive benefit from improved metabolic parameters and decreased 
body weight. At the scientific level, benefits of this study will contribute to our understanding of 
barriers and facilitators to altering and sustaining changes in microbial communities within a 
human host and further elucidating an underlying interaction between metabolic syndrome and 
the gut. This information will be of utility in planning further large scale randomized controlled 
trials.  

 
 
 
1.4 Dose Rationale  
Fecal microbiome transplant (FMT) is provided in the form of oral encapsulated formulation as 
we have previously used in the treatment of C. difficile. Individuals will fast overnight and 
complete a bowel preparation using Pico-Salax®, an oral, routine colonoscopy preparation. This 
is necessary to reduce the individual’s microbiota to allow for the engraftment of the donor’s 
species.  The following day (Day 1) 50grams of FMT from a single, extensively screened 
universal donor will be administered in 20-30 capsules taken by mouth. This donor has been with 
the FMT program since 2012 and has high levels of microbes capable of producing SCFA. 
Orally administered encapsulated FMT will ease administration and limit risks to the patient and 
will only be administered once throughout the trial.  
 
RS4 (9 g women; 11 g men), soluble corn fiber (9 g women; 11 g men.), and acacia gum (9 g 
women; 11 g men) are provided as a powder supplement which facilitates addition to the 
patients’ daily diet. A combined total dose of 27 grams per day for women and 33 grams per day 
for men will be provided for the duration of the trial. Doses on days 1-3 of the trial will be half of 
that to minimize any possible gastrointestinal upset. These doses are based on guidelines from 
the U.S Institute of Medicine for adequate intake for dietary fiber.  All of these fibers are well 
tolerated at these doses and have minimal gastrointestinal side effects [16, 29]. RS4, corn fiber, 
and acacia gum will be provided to participants in powder form on a weekly basis in pre-
weighed foil packets.   
 
1.5 Trial Conduct 
This trial will be conducted in compliance with the study protocol as approved by University of 
Alberta Health Research Ethics Board (HREB) and in keeping with the Good Clinical Practice 
standards. The study protocol will be strictly adhered to with no changes taking place prior to 
review and approval by the University of Alberta Health Research Ethics Board (HREB) unless 
deviations are immediately necessary to avoid imminent hazard to patients. In the latter case, 
changes from the protocol will be conveyed to University of Alberta Health Research Ethics 
Board (HREB) as early as possible.  
1.6 Population 
The study population will include obese adults with a BMI ≥30 with evidence of insulin 
resistance and metabolic syndrome.  
 
1.7 Literature 
1. 
Asghar, A. and N. Sheikh, Role of immune cells in obesity induced low grade inflammation and 
insulin resistance. Cell Immunol, 2017. 315: p. 18-26. 
2. 
Tan, J., et al., The role of short-chain fatty acids in health and disease. Adv Immunol, 2014. 121: 
p. 91-119. 
3. 
Long, S.L., C.G.M. Gahan, and S.A. Joyce, Interactions between gut bacteria and bile in health 
and disease. Mol Aspects Med, 2017. 
4. 
van de Wouw, M., et al., Microbiota-Gut-Brain Axis: Modulator of Host Metabolism and 
Appetite. J Nutr, 2017. 147(5): p. 727-745. 

 
 
 
5. 
Smith, P.M., et al., The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell 
homeostasis. Science, 2013. 341(6145): p. 569-73. 
6. 
Ha, C.W., Y.Y. Lam, and A.J. Holmes, Mechanistic links between gut microbial community 
dynamics, microbial functions and metabolic health. World J Gastroenterol, 2014. 20(44): p. 
16498-517. 
7. 
Vrieze, A., et al., Transfer of intestinal microbiota from lean donors increases insulin sensitivity in 
individuals with metabolic syndrome. Gastroenterology, 2012. 143(4): p. 913-6 e7. 
8. 
Vrieze, A., et al., Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin 
sensitivity. J Hepatol, 2014. 60(4): p. 824-31. 
9. 
Kootte, R.S., Levin E, Salojarvi J, Smits LP, Hartstra A, Udayapan SD, Hermes G, Bouer KE, Koopen 
Am, Holst JJ, Knop FK, Blaak EE, Zhao J, Smidt H, Harms AC, Hankemeijer T, Bergman J, Romijn 
HA, Schaap FG, Olde Damink S, Ackermans MT, Dallinga-Thie G, Zoetendal E, de Vos W, Serlie 
MJ, Stroes E, Groen AK, Nieuwdorp M. , Improvement of insulin sensitivity after lean donor feces 
in metabolic syndrome is driven by baseline intestinal microbiota composition. Cell Metabolism, 
2017. 26: p. 611-619. 
10. 
Grooms, K.N., et al., Dietary fiber intake and cardiometabolic risks among US adults, NHANES 
1999-2010. Am J Med, 2013. 126(12): p. 1059-67 e1-4. 
11. 
Koropatkin, N.M., E.A. Cameron, and E.C. Martens, How glycan metabolism shapes the human 
gut microbiota. Nat Rev Microbiol, 2012. 10(5): p. 323-35. 
12. 
Cockburn, D.W. and N.M. Koropatkin, Polysaccharide Degradation by the Intestinal Microbiota 
and Its Influence on Human Health and Disease. J Mol Biol, 2016. 428(16): p. 3230-3252. 
13. 
Walter, J., M.X. Maldonado-Gomez, and I. Martinez, To engraft or not to engraft: an ecological 
framework for gut microbiome modulation with live microbes. Curr Opin Biotechnol, 2017. 49: p. 
129-139. 
14. 
Bindels, L.B., J. Walter, and A.E. Ramer-Tait, Resistant starches for the management of metabolic 
diseases. Curr Opin Clin Nutr Metab Care, 2015. 18(6): p. 559-65. 
15. 
Al-Tamimi, E.K., et al., Consumption of Cross-Linked Resistant Starch (RS4(XL)) on Glucose and 
Insulin Responses in Humans. J Nutr Metab, 2010. 2010. 
16. 
Haub, M.D., et al., Different types of resistant starch elicit different glucose reponses in humans. 
J Nutr Metab, 2010. 2010. 
17. 
Upadhyaya, B., et al., Impact of dietary resistant starch type 4 on human gut microbiota and 
immunometabolic functions. Sci Rep, 2016. 6: p. 28797. 
18. 
Nichenametla, S.N., et al., Resistant starch type 4-enriched diet lowered blood cholesterols and 
improved body composition in a double blind controlled cross-over intervention. Mol Nutr Food 
Res, 2014. 58(6): p. 1365-9. 
19. 
De Vadder, F., et al., Microbiota-generated metabolites promote metabolic benefits via gut-brain 
neural circuits. Cell, 2014. 156(1-2): p. 84-96. 
20. 
Cani, P.D., et al., Gut microbiota fermentation of prebiotics increases satietogenic and incretin 
gut peptide production with consequences for appetite sensation and glucose response after a 
meal. Am J Clin Nutr, 2009. 90(5): p. 1236-43. 
21. 
Cani, P.D., et al., Changes in gut microbiota control inflammation in obese mice through a 
mechanism involving GLP-2-driven improvement of gut permeability. Gut, 2009. 58(8): p. 1091-
103. 
22. 
Everard, A. and P.D. Cani, Gut microbiota and GLP-1. Rev Endocr Metab Disord, 2014. 15(3): p. 
189-96. 
23. 
Koecher, K.J., et al., Estimation and interpretation of fermentation in the gut: coupling results 
from a 24 h batch in vitro system with fecal measurements from a human intervention feeding 

 
 
 
study using fructo-oligosaccharides, inulin, gum acacia, and pea fiber. J Agric Food Chem, 2014. 
62(6): p. 1332-7. 
24. 
Costabile, A., et al., Prebiotic Potential of a Maize-Based Soluble Fibre and Impact of Dose on the 
Human Gut Microbiota. PLoS One, 2016. 11(1): p. e0144457. 
25. 
Ferguson, M.a.J.G., Production of short-chain fatty acids following in vitro fermentation of 
saccharides, saccharide esters, fructo-oligosaccharides, starches, modified starches and non-
starch polysaccharides. J Science Food and Agriculture, 2000. 80: p. 166-170. 
26. 
Salonen, A., et al., Impact of diet and individual variation on intestinal microbiota composition 
and fermentation products in obese men. ISME J, 2014. 8(11): p. 2218-30. 
27. 
Korpela, K., et al., Gut microbiota signatures predict host and microbiota responses to dietary 
interventions in obese individuals. PLoS One, 2014. 9(6): p. e90702. 
28. 
Wei, Y., et al., Pectin enhances the effect of fecal microbiota transplantation in ulcerative colitis 
by delaying the loss of diversity of gut flora. BMC Microbiol, 2016. 16(1): p. 255. 
29. 
Martinez, I., et al., Resistant starches types 2 and 4 have differential effects on the composition 
of the fecal microbiota in human subjects. PLoS One, 2010. 5(11): p. e15046. 
30. 
Flint, A., et al., Reproducibility, power and validity of visual analogue scales in assessment of 
appetite sensations in single test meal studies. Int J Obes Relat Metab Disord, 2000. 24(1): p. 38-
48. 
31. 
Warkentin, L.M., et al., The effect of weight loss on health-related quality of life: systematic 
review and meta-analysis of randomized trials. Obes Rev, 2014. 15(3): p. 169-82. 
32. 
Wang, M.D., Sample size reestimation by Bayesian prediction. Biom J, 2007. 49(3): p. 365-77. 
33. 
Muniyappa, R., et al., Current approaches for assessing insulin sensitivity and resistance in vivo: 
advantages, limitations, and appropriate usage. Am J Physiol Endocrinol Metab, 2008. 294(1): p. 
E15-26. 
34. 
Edgar, R.C., UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat 
Methods, 2013. 10(10): p. 996-8. 
35. 
Wang, Q., et al., Naive Bayesian classifier for rapid assignment of rRNA sequences into the new 
bacterial taxonomy. Appl Environ Microbiol, 2007. 73(16): p. 5261-7. 
36. 
Caporaso, J.G., et al., QIIME allows analysis of high-throughput community sequencing data. Nat 
Methods, 2010. 7(5): p. 335-6. 
 
 
2 Trial Objectives 
The primary objective of this pilot feasibility study is to determine if fecal microbial 
transplantation (FMT) combined with supplementation with prebiotic fibers has a clinically 
significant effect on metabolic outcomes in obese subjects with metabolic syndrome.  The 
hypothesis is that FMT will modify the gut microbiota of the recipient to increase SCFA-
producing microbes. Supplementation with prebiotic fibers will then increase SCFA production 
and improve insulin sensitivity due to enhanced gut barrier function and reduced systemic 
inflammation.   
Specific aims include analyzing FMT and fiber-induced changes in host metabolic parameters 
including insulin sensitivity, fasting blood glucose levels, lipid profiles, and glycosylated 
hemoglobin levels; along with changes in anthropometric measurements; gut hormones; 
inflammatory markers; and hunger and satiety.  Compositional and functional changes in gut 
microbiota will be assessed and a correlation analysis between specific microbes, microbial 

 
 
 
functions and metabolic parameters will be carried out. The goals are to identify associations 
between the gut microbiome and host metabolism and to determine if prebiotic fiber therapy is 
effective in maintaining beneficial effects following FMT.  While these types of associations 
cannot define causality, results gained from this clinical trial have the potential to provide 
mechanistic insight in addition to an understanding of how supplemental prebiotic fiber may be 
beneficial in control of metabolic syndrome in an obese population.   
Results from this feasibility study will inform the design of a larger randomized double-blind 
placebo-controlled trial to provide rigorous scientific evidence necessary to invoke policy and 
practice changes in the management of metabolic syndrome. Further, this feasibility study 
will provide evidence regarding safety and any adverse outcomes due to the interventional 
therapy. This is critical information that will inform patient acceptance, tolerance, and 
compliance. The overall goal is to create a safe, well tolerated therapy based on manipulation 
of the gut microbiota that is an effective treatment for patients with metabolic syndrome. 
 
3 Trial Design 
 
 
 
3.1 Primary Study Endpoints/Secondary Endpoints 
 
Primary Endpoint:  
 
The primary endpoint is the change in insulin sensitivity between the time of screening and 6 
weeks following treatment.  
 
Secondary Endpoint:  
 
 Changes in body weight and anthropometric parameters between baseline and 6 and 12 
weeks 
 Changes in HbA1C, fasting glucose, and oral glucose tolerance between baseline and 6 
and 12 weeks  
 Changes in Fasting lipid profile between baseline and 6 and 12 weeks 
 Changes in blood pressure between baseline and 6 and 12 weeks 
 Effects on quality of life and satiety between baseline and 6 and 12 weeks 
 Changes in serum  levels of leptin, adiponectin, ghrelin, CRP, TNF-α, IL-6, LPS, and 
LPS-binding protein between baseline and 6 and 12 weeks 
 Changes in fecal microbiota composition between baseline and 6 and 12 weeks 
 Changes in stool short chain fatty acid composition between baseline and 6 and 12 weeks 
3.2 Study Design/Type 
 
This is an exploratory four-arm, parallel design, randomized placebo-controlled intervention 
study in obese individuals with metabolic syndrome to evaluate whether FMT from lean 

 
 
 
donors combined with supplementation with prebiotic fiber will have a clinically significant 
effect on metabolic parameters. The study includes a 2-week screening/baseline period 
followed by a single FMT and a 6 week study period in which prebiotic fiber or placebo will 
be added in powdered form to the subject’s normal diet for the duration of the trial (Appendix 
1).  A follow-up visit at 12 weeks will be done to determine if beneficial effects are 
maintained in the absence of ongoing fiber intake. A parallel arm design was chosen to avoid 
cross-over effects.   
 
The four groups are:   
1) Control (Placebo FMT and cellulose)  
2) FMT only (FMT followed by cellulose)  
3) Prebiotic only (Placebo FMT and prebiotic fiber)  
4) FMT + prebiotic fiber 
Schedule and Procedures: Subjects will attend a total of 5 clinic appointments throughout the 
study for anthropometric and blood pressure measurements, dietary intake, hunger and satiety, 
and quality of life assessment using questionnaires, and for collection of blood and fecal samples 
(Appendix 2).  The intervention will be stopped at 6 weeks.  A final follow-up visit will occur at 
12 weeks for anthropometric and blood pressure measurements, and collection of blood and stool 
samples.  
3.3 Randomization 
Subjects will be randomized to one of 4 groups via computer-generated numbers and stratified 
by gender. Individuals will be blinded as to their group allocation to reduce bias. Randomization 
concealment will be protected by several levels of security, including our secure website, 
password protection by only those authorized to randomize and a variable blocked 
randomization.  
 
3.4 Maintenance   
 
Randomized codes will be maintained in REDCAP.  Codes will be broken at the end of the trial.  
 
3.5 Trial Treatment 
 
Fecal microbiome transplant (FMT): 50grams of FMT from a single, universal donor will be 
administered in 20-30 capsules taken by mouth.  These capsules will be frozen at -700C until 
date of administration on day 1 of the trial. The FMT will be given after individuals have fasted 
overnight and completes a bowel preparation using Pico-Salax®, a routine colonoscopy 
preparation. We will use only one donor to reduce variability in donor profiles unless this donor 
becomes unavailable at which point we have backup donors available.  Placebo FMT will consist 
of cellulose pills. 
 
Fiber Supplementation:  
 

 
 
 
Soluble corn fiber (PROMITOR®: Tate&Lyle) 
 Women: 4.5 gm of PROMITOR by mouth days 1-3 increased to 9 gm daily from day 4 
until trial completion. 
 Men: 5.5 gm of PROMITOR by mouth days 1-3 increased to 11 gm by mouth daily from 
day 4 until trial completion. 
 
Resistant Wheat Starch 4 (Fibersym®: MGP Ingredients):  
 Women: 4.5 gm of powdered RS4 by mouth days 1-3 increased to 9 gm by mouth daily 
from day 4 until trial completion. 
 Men: 5.5gm of powdered RS4 by mouth days 1-3 increased to 11 gm by mouth daily 
from day 4 until trial completion. 
 
Acacia Gum (Pre-Hydrated Gum Arabic: TIC GUMS):  
 Women: 4.5 gm of powdered acacia gum by mouth days 1-3 increased to 9 gm by mouth 
daily from day 4 until trial completion. 
 Men: 5.5gm of powdered acacia gum by mouth days 1-3 increased to 11 gm by mouth 
daily from day 4 until trial completion. 
 
The product will be supplied to patients on a weekly basis in pre-weighed foil packets.  
 
Placebo: Placebo will consist of cellulose powder (Microcrystalline cellulose:Blanver) in 
identical foil packets.  
 
3.6 Duration 
 
The study duration is 12 weeks.  Subjects will be seen in the clinic at recruitment, and then at 2 
and 6 weeks following FMT.  Intervention will be from 1-6 weeks.  A final follow-up visit will 
occur at 12 weeks (Appendix 1) 
 
 
3.7 Discontinuation   
 
Enrollment in the study will be discontinued if a) 3 or more subjects receiving FMT or fiber 
supplementation experience similar SAEs and it is determined by the study investigator that such 
SAE’s are related to the study procedure; or b) if any one subject who receives FMT is reported to 
experience an SAE that had fatal or life-threatening outcome and it is concluded that such SAE 
could be reasonably related to FMT or fiber supplementation. . 
 
3.8  Product Accountability 
 
Prebiotic fibers will be obtained from the manufacturers and tracked in an electronic 
accountability log for the duration of the study. This log will include the date received, amount 
received, batch numbers, and conditions at receipt. When prebiotic fibers are dispensed to a 

 
 
 
participant, the date and amount provided will be documented in the accountability log. This will 
include the date, batch number, expiration date, unique kit identifier, and amount dispensed.  A 
balance of remaining fibers will be maintained and documented in the study’s accountability log.  
 
Prebiotic fibers will be stored in a limited-access location on the 7th Floor of Katz Group Center 
according to instructions received from each supplier.   
 
4 Selection and Withdrawal of Subjects 
4.1 Inclusion Criteria 
 Age ≥ 18 and < 65 years at the time of screening  
 BMI > 30 
 Total body weight fluctuation over the last 6 months less than 10% 
 Fasting plasma glucose > 5.6 mmol/L OR HgbA1c ≥5.5% OR patients receiving an 
antidiabetic medication 
 At least one of the following:  
o Fasting triglyceride ≥1.7 mmol/L OR receiving dyslipidemia medication  
o HDL cholesterol <1.03 mmol/L in males or <1.29 mmol/L in females OR 
receiving dyslipidemia medication  
o Known diagnosis of hypertension OR systolic blood pressure ≥130 or diastolic 
blood pressure ≥85 mmHg OR receiving antihypertension medication  
 
4.2 Exclusion Criteria 
 Systolic blood pressure ≥180 or diastolic blood pressure ≥110 mmHg at screening. 
 Triglyceride ≥6 mmol/L. 
 Acute infectious or inflammatory condition over the presiding 4 weeks. 
 Current or recent use (Previous 6 months) of insulin for diabetes control. 
 History of oropharyngeal or significant esophageal dysphagia, inflammatory bowel 
disease, colon cancer, or colonic polyps with high grade dysplasia. 
 History of autoimmune conditions or chronic inflammatory condition, such as 
rheumatoid arthritis, chronic active hepatitis B or C, HIV, chronic pancreatitis, advanced 
NASH, or liver cirrhosis. 
 Active malignancy. 
 Active substance abuse or excessive EtOH (defined as >2 X 8oz drinks/d). 
 
 
4.3 Subject Withdrawal 
 
Subjects will be free to withdraw from the trial without prejudice to further medical care at any 
time.  Subjects will be withdrawn under the following circumstances: consent is withdrawn or 
subjects are lost to follow-up; requirement for antibiotic therapy for infection; pregnancy; 
symptomatic intolerance of the prebiotics as manifested by non-bloody diarrhea, abdominal pain, 
severe abdominal bloating or any other symptom deemed likely to be caused by the prebiotic; if 

 
 
 
any severe adverse event thought to be related to the study compound occurs. Data collected about 
the subject up to the time of withdrawal will remain in the trial database and be included in the 
data analysis.  
 
4.4 Treatment of Subjects 
 
All the information obtained during subject visits shall be reported in the subject’s Source 
Document (SD).  Visits are summarized in Table 1.  
 
Screening Visit 
 Obtainment of Informed Consent 
 Medical History 
 Hematology and Chemistry 
 Pregnancy Test 
 
Baseline Visit Week 0 
 Focused physical Exam with anthropometric assessment and vital signs 
 Fecal samples for microbiome analysis and levels of SCFA  
 Oral glucose tolerance test 
 Blood samples for levels of CRP, adipocytoquines and intestinal barrier assessment (LPS, 
LPBP, zonulin)  
 Food Frequency Questionnaire and 24-hours recall 
 Hunger and satiety questionnaire 
 Gastrointestinal Tolerance questionnaire 
 Quality of life questionnaire 
 Fecal microbial transplant 
 Dispense fiber supplements  
 
Week 1 – telephone interview 
 Telephone interview for adverse events and compliance monitoring 
 Hunger and satiety questionnaire 
 Quality of life questionnaire 
 Gastrointestinal Tolerance questionnaire 
 24 hrs food recall 
 
Visit at Week 2  
 Office visit for Adverse Event and Compliance Monitoring 
 Hunger and satiety questionnaire 
 Quality of life questionnaire 
 Gastrointestinal Tolerance questionnaire 
 24 hrs food recall 
 Fecal samples for microbiome analysis and levels of SCFA 
 Blood samples for levels of CRP, adipocytoquines and intestinal barrier assessment (LPS, 
LPBP, zonulin) 

 
 
 
 Dispense fiber supplements  
 
Week 4 – telephone interview 
 Telephone interview for adverse events and compliance monitoring 
 Hunger and satiety questionnaire 
 Quality of life questionnaire 
 Gastrointestinal Tolerance questionnaire 
 24 hrs food recall 
 
Visit at Week 6  
 Office visit for Adverse Event and Compliance Monitoring 
 Anthropometric assessment and vital signs 
 Hunger and satiety questionnaire 
 Quality of life questionnaire 
 Gastrointestinal Tolerance questionnaire 
 Oral glucose tolerance test 
 Fecal samples for microbiome analysis and levels of SCFA 
 Blood samples for levels of CRP, adipocytoquines and intestinal barrier assessment (LPS, 
LPBP, zonulin)  
 
Visit at Week 12  
 Anthropometric assessment and vital signs 
 Hunger and satiety questionnaire 
 Quality of life questionnaire 
 Gastrointestinal Tolerance questionnaire 
 Fecal samples for microbiome analysis and levels of SCFA 
 Blood samples for levels of CRP, adipocytoquines and intestinal barrier assessment (LPS, 
LPBP, zonulin)  
 
 
 

 
 
 
 
Table 1. General schedule for assessment and intervention. 
 
Visit 
S1 
S2 
 
0 
1 
(TC) 
2 
4 
(TC) 
6 
 
12 
Interview and 
assessment 
 
 
 
 
 
 
 
 
 
Interview 
X 
X 
X 
X 
X 
X 
X 
X 
Anthropometric assessment 
X 
X 
X 
 
 
 
X 
X 
Vital signs (BPx2) 
X 
X 
X 
 
X 
 
X 
X 
Consent 
X 
X 
 
 
 
 
 
 
Specimen sampling and Laboratory work 
TSH 
 
X
* 
 
 
 
 
 
 
CBCD 
X* 
X 
 
 
 
 
 
 
Renal Function (Cr, BUN) 
X* 
X 
 
 
 
 
 
 
Electrolytes 
X* 
X 
 
 
 
 
 
 
LFTs: (AST, ALT, ALP, 
Albumin, Bilirubin, INR)  
X* 
X 
 
 
 
 
 
 
Hs-CRP 
 
X 
 
 
X 
 
X 
 
Glucose homeostasis (FPG, 
HbA1c, Serum insulin, glucagon, 
c-peptide). 
X* 
X 
 
 
X 
 
X 
X 
Fasting lipids profile. 
X* 
X 
 
 
X 
 
X 
X 
Infectious markers: HIV, HVB, 
HVA, HVC. 
X* 
X 
 
 
 
 
 
 
Adipocytoquines: Leptin, 
Adiponectin, PYY, GLP-1, 
TNFα, IL-6, IL-1β. 
 
X 
 
 
X 
 
X 
 
X 
Intestinal Barrier: Plasma level of 
LPS, LPBP, zonulin 
 
X 
 
 
X 
 
X 
 
X 
Stool Sampling: SCFA, 16S 
rRNA. 
 
X 
 
 
X 
 
X 
 
X 
 
 
 
 
 
 
 
 
 
Investigations 
75g OGTT 
 
X 
 
 
 
 
X 
 
Hunger and satiety questionnaire 
 
X 
 
X 
X 
X 
X 
X 
Gastrointestinal Tolerance 
 
X 
 
X 
X 
X 
X 
X 
EQ-ED5 
 
X 
 
X 
X 
X 
X 
X 
Dietary Intake (24-h recalls) 
 
X 
 
X 
X 
X 
X 
 
Diet History Questionnaire 
(DHQ) 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
FMT 
 
 
X 
 
 
 
 
 
X: Sample collection  
* Not to be drawn if previous result available within 3 months 
 
 

 
 
 
4.5 Medication 
Medications permitted during this study include those under the category of oral diabetic agents, 
statins, fibrates and antihypertensives. 
 
Medications not permitted during this study include insulin, anti-neoplastic agents, antibiotics, 
anti-retrovirals, anti-virals or medications taken with the purpose of treating underling auto-
immune or inflammatory disorders.  
 
4.6 Monitoring for subject compliance 
 
We have designed regular clinic visits, telephone calls by the study monitor, self-documentation, 
and returning of treatment packages to help promote compliance. Study patients will attend a 
total of 6 clinic appointments throughout the 8 weeks of their participation in the study for 
anthropometric and blood pressure measurements, dietary intake, hunger and satiety, and quality 
of life assessment using questionnaires, and for collection of blood and fecal samples. Patients 
will be asked to keep 24-hour diet record including 2 weekdays and 1 weekend per week for 2 
weeks during screening and then for the duration of the trial. Patients will be offered the option 
to keep records using available digital devices (i.e. Myfitness pal®, Loseit®, etc) or conventional 
paper-based dairies. Digital or paper base records will be collected on each visit and reviewed by 
the research personnel. Additionally, patients will be asked to return treatment packages to 
provide supplementary measures of compliance. 
 
Patients will be provided all treatment and investigations for free under a highly trained team of 
investigators and reimbursed 10$ per visit for parking costs to attend follow-up clinic. Finally, 
we anticipate that the possibility of improving glycemic, metabolic and weight control and 
contributing to novel treatment methods will encourage compliance amongst our patients.  
 
 
 
5 Assessment of Efficacy 
 
5.1 Efficacy Parameters 
The following efficacy parameters will be assessed at weeks 2, 4, and 6: 
 Quality of life  
 Dietary intake, hunger and satiety. 
 Body weight and anthropometric parameters.  
 Insulin Sensitivity Assessment: HbA1C, fasting glucose, OGTT, Fasting lipid profile  
 Blood pressure 
 Blood and Plasma investigations: Serum levels of leptin, adiponectin, ghrelin, CRP, TNF-
α, IL-6, LPS, LPS-binding protein, zonulin 
 Fecal microbiota composition  

 
 
 
 Stool short chain fatty acid composition 
5.2 Method and Timing 
 
Efficacy parameters will assessed during clinic visits at the times denoted in Appendix 1.   
Dietary Intake and Hunger and satiety assessment: Each subject will be asked to keep 24-hour 
diet record including 2 weekdays and 1 weekend per week for 2 weeks during screening and then 
for the duration of the trial. Patients will be offered the option to keep records using available 
digital devices (i.e. Myfitness pal®, Loseit®, etc) or conventional paper-based dairies. Digital or 
paper base records will be collected on each visit and reviewed by the research personnel. 
Hunger and satiety will be evaluated using a previously validated instrument [30].  
Health-related quality of life (HRQL): Assessment of the current health related quality of life 
will be accomplished by conducting standardized interviews and by using the validated 
questionnaires EQ-5D Index [31]. 
Anthropometric assessment: Anthropometric assessment will be performed by trained personnel 
and following the recommendations made by the CDC and published in the Anthropometry 
procedures Manual of the National Health and Nutrition Examination Survey (available at 
www.cdc.gov). Body weight will be measured using a validated, calibrated bariatric scale (Scale 
Tronix®) and recorded to the nearest 0.1 kg. Height will be measured to the nearest 0.1 cm using 
a wall-mounted stadiometer. Waist and hip circumferences will be measured following the 
recommendations of the Anthropometric Standardization Reference Manual (ASM), the World 
Health Organization (WHO) guidelines and the National Institutes of Health (NIH) guidelines. 
[32]. 
Blood and plasma samples collection: Subjects will be instructed to fast overnight prior to blood 
sampling. Aliquots of plasma and serum will be snap frozen in liquid nitrogen and stored in a -
80C freezer in the biobank.  
 Blood and plasma investigations: Routine laboratory investigations will be done using 
the AHS clinical trial lab and will include: Cell blood count and differential (CBCD), 
sodium (Na) potassium (K) chloride (Cl) and carbon dioxide (CO2), creatinine (Cr), 
blood urea nitrogen (BUN), aspartate transaminase (AST), alanine transaminase (ALT), 
alkaline phosphatase (ALP), albumin, C-Reactive protein (CRP), glycated Hemoglobin 
HbA1C (HbA1C), fasting blood glucose (FBG), fasting Insulin levels, plasma C-peptide, 
fasting lipid profile, and International Normalizing Ratio for pro-thrombin time (INR).  
Plasma glucose levels will be determined in fresh blood samples during the 60 minutes 
immediately after sample collections using standardized laboratory techniques and 
reported as mmol/L. Serum insulin levels will be determined in fresh or frozen samples 
using validated Human insulin ELISA kits following standardized laboratories 
techniques. Levels of leptin, ghrelin, adiponectin, TNFα, IL-6, LPS (lipopolysaccharide), 
LPS-binding protein and zonulin will be determined using currently accepted laboratory 
techniques.  

 
 
 
Insulin sensitivity assessment:  Insulin resistance (IR) is considered the cornerstone of the 
pathophysiology of metabolic syndrome and one of the leading drivers of the association 
between obesity and cardiovascular outcomes. The hyperinsulinemic euglycemic clamp (HEC) 
constitutes the gold standard for quantification of systemic insulin sensitivity, but it is an 
expensive and invasive approach.  As an alternative to the HEC, multiple indirect measurements 
of insulin resistance have been developed using less invasive techniques and have been validated 
in multiple clinical and research settings.  
 Oral Glucose tolerance test (OGTT) and surrogates of insulin resistance assessment: 
The oral glucose tolerance test (OGTT) is a dynamic test that reflects the efficiency of the 
body to dispose of glucose after an oral load. It is commonly used in clinical scenarios to 
diagnose glucose intolerance and diabetes. [33] After overnight fast (> 8 h), blood 
samples for determination of glucose and insulin concentration will be taken at -5, 0, 30, 
60, and 120 min following a standard oral glucose load.   
 Model Assessment-Insulin Resistance (HOMA-IR): The homeostasis Model 
Assessment-Insulin Resistance (HOMA-IR) is a mathematical model to predict the 
interaction between glucose and insulin dynamics across a range of glucose plasma levels 
assuming a feedback circuit between the liver and β-cells in the pancreas.  
 𝐻𝑂𝑀𝐴−𝐼𝑅=
fasting insulin (μIU/ml)] × [fasting glucose (mmol/l)
22.5
 
 
Anticipating a large variability in insulin sensitivities among participant of this study, 
logarithmic transformation may be applied  (logHOMA-IR) since it is known to increase 
the linear correlation of this measurement and clamp estimated insulin sensitivities.[33]  
Stool sample collection: Each subject will be instructed to collect and preserve at least 30 grams 
of stool and store at -200C in the freezer till the next clinic visit. Each subject will be supplied 
with stool and urine collection kits as well as written instructions. DNA will be extracted from 
stool samples using the QIAamp DNA Stool Mini Kit (Qiagen, CA).  Microbial composition will 
be characterized by 16S rRNA sequencing using MiSeq Illumina technology.  Read processing 
and quality control, chimera removal, primer trimming, and merging of pair-end reads will be 
done using fastx toolkit (http://hannonlab.cshl.edu/fastx_toolkit/), merge-Illumina-utils 
(https://github.com/merenlab/illumina-utils) and UPARSE pipeline [34].  Data will be analyzed 
using taxonomic based approaches  including the Ribosomal Database Project Multiclassifier 
tool [35] and non-taxonomic based clustering algorithms for Operational Taxonomic Unit 
determination available in pipelines such as UPARSE. Alpha-diversity indices and β-diversity 
indices will be calculated in QIIME [36]. SCFA will be quantified in fecal samples by gas 
chromatography. 
 
6 Assessment of Safety 
 
6.1 Safety Parameters 
Subjects will receive an information document about the study including the contact number of 
the principal investigator from the study coordinator. After subjects have received the full 
information about study participation, expected benefits and possible side effects and 

 
 
 
inconveniences related to study participation, written consent will be obtained.  The study will be 
explained by study investigators, and by the study coordinators. Informed consent can be obtained 
either by study coordinators or investigators.  The obtainment of informed consent will be 
documented in SD. Subject must commence study within 21 days of signing consent. Therefore, 
the screening visit and the Week 0 visit must be scheduled within 14 days of each other so that 
eligible patients can be randomized and have the FMT followed by starting the fiber supplement. 
 
6.2   Adverse Event Monitoring  
Subjects will be questioned by the research coordinator at weeks 1, 2, 4, and 6 about any Severe 
Adverse Events. These are defined in Section 6.4.  At each visit, the study coordinator will record 
the occurrence and severity of side effects: unacceptable flatulence, abdominal rumbling, bloating, 
abdominal pain or diarrhea thought to be related to the fiber supplementation.  
 
6.3  Serious Adverse Events (SAEs) and Safety Monitoring 
A serious adverse event is any untoward medical occurrence that at any dose: 
 results in death 
 is life-threatening 
 results in subject hospitalization or prolongation of existing hospitalization 
 results in persistent or significant disability/incapacity 
 is a congenital anomaly/ birth defect 
 
The principal investigator will notify the Health Research Ethics Board (HREB) about serious 
adverse events via a Local Serious Adverse Events Report.  This will occur within 7 days of the 
event.  The research coordinator will notify all subjects upon their next visit to clinic. 
 
Clinical monitoring will be provided through the University of Alberta Quality Management in Clinical 
Research Department (QMCR) for the duration of the trial.  All serious adverse events will be 
reviewed by the QMCR and the study team to determine if a pattern exists which could be 
related to the study. If the events are deemed to be attributable to the study treatment the 
treatment arms will be unmasked. If it is concluded that the SAEs could be related to the study 
treatment, a recommendation will be made to halt enrolment in the study.  
 
 
6.5  Adverse Event Follow-up 
 
All SAEs will be recorded in the SD.  For SAEs, the investigator will obtain sufficient information 
to determine the causality of the adverse event. Follow-up of the adverse event, after the date of 
therapy discontinuation, will be required if the adverse event persists. Follow-up is required until 
the event is resolved. 
 
All observed or reported adverse events considered to be related to FMT or fiber supplementation 
will be recorded on the adverse event page(s) of the Source Document. These adverse events will 
be recorded in detail; onset during the study, duration, nature, severity, action taken and outcome. 
 
Severity will be defined as: 
 Mild 

 
 
 
 Moderate 
 Severe 
 
Action taken will be defined as: 
 
None 
 
Supplementation temporarily interrupted 
 
Supplementation definitely stopped 
 
Other treatment 
 
Outcome will be defined as: 
 Resolved 
 Ongoing 
 Lost to follow up 
 
In addition, abnormal findings that result in a change in study supplement dosage or in 
discontinuation of the supplement, or require intervention or diagnostic evaluation to assess the 
risk to the subject, will be recorded as adverse events.  
 
7 Statistical Plan 
 
7.1 Statistical Methods 
Data analyses will be performed using Stata Statistical Software® Release 13.0 (College Station, 
TX, Stata Corporation) and [SAS/STAT] software, Version 9.2 of the SAS System for UNIX. 
Pair-wise comparisons between the study groups will be performed separately.  Variables will be 
examined descriptively and graphically, including assessments of temporal trends and tests of 
normality.  Mean change in each outcome will be calculated and compared within each study group 
and between study groups using paired or unpaired t-tests for continuous outcomes and chi-squared 
tests for dichotomous ones.  Multivariable predictors of change in relevant outcomes will be 
identified using appropriately constructed and calibrated linear regression models for continuous 
outcomes or logistic regression models for dichotomous ones (i.e., change in the prevalence of a 
given dichotomous variable will be the dependent variable in the model).  Statistical tests for 
treatment effects of FMT and prebiotcs on the abundance of bacterial populations and SCFA will 
be performed using Statistical Analysis Systems (SAS Institute Inc., Cary, NC). Data following 
normal distribution will be analyzed by ANOVA. Correlations between bacterial populations and 
SCFA and inflammation measurements will be assessed by multivariate statistical methods such 
as principle component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) 
and further confirmed by Spearman correlation tests.  
 
All analyses related to the clinical outcome will be by intention to treat. In the case of drop-outs 
or missing data, where possible “a last value carried forward” approach will be used. Analyses will 
be conducted by a biostatistician at EPICORE Centre who is blinded to treatment allocation. 
Prebiotic adherence will be reported as a simple proportion of prebiotics actually taken divided by 
the prebiotics dispensed to form a prebiotics possession ratio – a standard method of reporting 
medication adherence. 

 
 
 
 
7.2 Subject Population(s) for Analysis 
 
Sample size and power calculation: Basic anthropometric and metabolic characteristics of 
subjects enrolled in the Edmonton Adult Bariatric Clinic 2011 and 2012 are presented in Table 
1. Based on the reported baseline parameters and assuming a treatment allocation factor of 
1:1, with repeated measurements design including 1 baseline and at least 3 follow-up 
measurements, accepting a type 1 error of 0.05 and a correlation between repeated 
measurements of 50%, a sample size of 15 subjects per arm will give the study a power of 
80% to detect a change of 0.8 log [SD=1] in the logHOMA-IR value, or 80% power to detect 
a minimal difference of 1.1 [SD 1.1] in HbA1c levels between groups.  Assuming a potential 
10% drop-out rate, we will enroll a total of 68 subjects for this feasibility study. All 
randomized subjects who complete 6 weeks of follow-up will be selected for analysis.  
7.3 Significance 
The significance level used in this study will be 5% (0.05).  
7.4 Termination Criteria 
 
The study will be discontinued if a) 3 or more subjects receiving FMT or fiber supplementation 
experience similar SAEs and it is determined by the study investigator that such SAE’s are related 
to the study procedure; or b) if any one subject who receives FMT is reported to experience an 
SAE that had fatal or life-threatening outcome and it is concluded that such SAE could be 
reasonably related to FMT. In this pilot feasibility trial there will be no interim analysis.  
. 
8 Direct Access to Source Data/Documentation 
The investigator and the University of Alberta will permit trial-related monitoring, audits, 
IHREB review and regulatory inspection(s) by providing direct access to source 
data/documentation. 
 
9 Ethical Considerations 
 
Our study will be carried out in keeping with the Canadian Good Clinical Practice standards. 
Current University of Alberta research policies and government regulations pertaining to this 
study will be followed at all times.  
 
The current study protocol and any amendments made here forth will be submitted to the 
University of Alberta Health Research Ethics Board (HREB) for approval who will submit 
written approval or disapproval of conduct of the study.  
 
Prior to enrolment, study subject will be provided consent forms describing the purpose and 
procedures of the trial. Said consent forms will contain adequate information from which patients 
will be able to make an informed decision regarding their choice to participate or not. A copy of 

 
 
 
the consent form will be submitted in our application to the University of Alberta Health 
Research Ethics Board (HREB. Prior to enrolment in the trial, this consent form will need to be 
signed by the study subject and investigator obtaining consent from the individual.  
 
10 Data Handling and Record Keeping 
 
The principal investigator and her designees shall have responsibility to perform data management 
and statistical analysis of clinical data. As part of the responsibilities assumed by participating in 
the study, the investigators agree to maintain Source Documents which will serve as case histories 
for the subjects treated as part of the research under this protocol. The investigator agrees to 
maintain accurate source documentation as part of the case histories. Study records are comprised 
of the Source Documents and all other administrative documents, such as HREB correspondence, 
clinical trial material, supply shipment documents etc. Source documentation is defined as any 
hand-written or computer generated document that contains medical information or test results that 
have been collected. These may include: lab reports, clinic notes, drug disbursement log, patient 
sign-in sheets, patient completed questionnaires, telephone logs, etc. All draft, preliminary and 
pre-final iterations of a final report are also considered to be source documents, e.g., fax lab reports 
and hard copy lab reports initial results and hard copy full, final report. 
 
11 Finance and Insurance 
This project will be financed by combined sources with the majority of funding granted from 
The W. Garfield Weston Foundation – Weston Family Microbiome Initiative. The University of 
Alberta proper, University of Alberta CIP program, Alberta Innovates and Canadian Institutes 
for Health will cover salary costs of participating researchers. Alberta Health Services has 
donated in-kind the use of their FMT program including donor screening and supplies for FMT 
encapsulation.  
 
 
12 Supplements 
 
Appendix 1: Trial Design 
Appendix 2: Fiber Supplements 
Appendix 3;    Questionnaires 
 
 

 
 
 
Appendix 1    Trial Design 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Placebo (N = 17) 
       Subjects (N = 68) 
Stratified Random Assignment 
Screen/Baseline 
-1 
0 
Visit 1 
Visit 2 
Day 1 
Day 2 
 TC                Visit 3                                         Visit 4                                     Visit 5              Visit 6   
1                2               3               4              5                 6            12 
 
 
 
Female dose: 
Male dose: 
12.5 g 
17.5 g 
25.0 g 
35.0 g  
Week 1 Fiber Titration 
FMT (N = 17) 
Fiber (N = 17) 
FMT + Fiber (N = 17)