Clinical trial NCT03165747
Conditions: Menopausal Osteoporosis
Keywords: Probiotics Menopausal Osteoporosis
Sponsor: Emory University
Start date: 2017-10-01; Completion date: 2019-03-23
Enrollment: 35 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Osteoporosis has a devastating impact on quality of life of postmenopausal women, and is a significant cause of disability and morbidity. Many drugs are approved for the prevention and treatment of osteoporosis, but are associated with high costs and side effects. Some data from animal studies suggests that supplementation with probiotics can safely treat and prevent osteoporosis. The probiotic VSL#3 is commercially available, is safe for human consumption, and has been widely used in human clinical trials, and has known health-promoting effects in both children and adults.

The double-blind, randomized, placebo-controlled trial of VSL#3 will be conducted for 12 months in 40 postmenopausal women to determine if VSL#3 improves bone mineral density and related bone markers. Study visits will include all or some of the following procedures: a medical exam, urine collection, height and weight measurement, a blood draw to assess bone biomarkers, a DEXA (dual energy X-ray absorptiometry) scan to measure bone density, and health questionnaires.

This is one of the first clinical trials proposed to investigate the effects of probiotics in bone in humans. If successful, this proposal will provide the first evidence that nutritional supplementation with the probiotic VSL#3 is a safe and effective strategy for preventing postmenopausal bone loss.

Detailed description:
Osteoporosis has a devastating impact on quality of life of postmenopausal women, and is a significant cause of disability and morbidity. Many drugs are approved for the prevention and treatment of osteoporosis, but are associated with high costs and side effects. Some data from animal studies suggests that supplementation with probiotics can safely treat and prevent osteoporosis. The probiotic VSL#3 is commercially available, is safe for human consumption, and has been widely used in human clinical trials, and has known health-promoting effects in both children and adults.

The double-blind, randomized, placebo-controlled trial of VSL#3 will be conducted in a population of ambulatory, otherwise healthy, postmenopausal women for 12 months. Control and VSL#3-treated postmenopausal women will be matched by age (± 3 years). Study visits will include all or some of the following procedures: a medical exam, urine collection, height and weight measurement, a blood draw to assess bone biomarkers, a dual energy X-ray absorptiometry (DEXA) scan to measure bone density, and health questionnaires.

The primary endpoint is the change in bone mineral density (BMD) at the L1-4 lumbar spine over one year of study. Changes in BMD at the femoral neck and total hip area will be secondary endpoints. All BMD data will also be used as a tool for future studies power calculation and design. Additional endpoints will include changes in bone turnover markers and inflammatory/osteoclastogenic cytokines. Measurements of indices of bone turnover and cytokine levels will provide much needed mechanistic information.The data will allow to establish whether VSL#3 prevents bone loss and/or increases bone mass by regulating bone resorption, formation, or both.

Arms: arm 1: VSL#3 two active sachets \[containing 450x109 colony-forming units (CFU)/sachet\], taken daily in a single administration. arm 2: Placebo, no supplemental probiotics.
Interventions: intervention 1: VSL#3 intervention 2: Placebo
Intervention descriptions: intervention 1: 8 strains of live bacteria: Bifidobacterium breve, B. longum, B. infantis, L. acidophilus, L. plantarum, L. paracasei, L. bulgaricus and Streptococcus thermophilus. 900 billion CFU taken orally daily in a single administration. intervention 2: Two placebo sachets taken orally daily in a single administration.

Protocol documents:
Prot_SAP_000.pdf: 
Company Study 
 
Version   10/17/18 
 
 
 
Effect of VSL#3 on Bone Mineral Density in Postmenopausal Women: a Pilot Randomized, Placebo-
Controlled Trial 
 
 
 
Protocol date: October 17, 2018 
 
 
NCT number: NCT03165747 
 
 
Principal Investigator: 
 
 
 
 
Roberto Pacifici, M.D. 
 
 
 
 
Garland Herndon Professor of Medicine 
 
 
 
 
Director, Division of Endocrinology, Metabolism and Lipids 
 
 
 
 
Emory University School of Medicine 
 
 
 
 
101 Woodruff Circle, WMRB 1307 
 
 
 
 
Atlanta GA 30322 
 
 
 
 
 
 
 
Co-Investigators: 
 
 
 
 
Thomas R. Ziegler, M.D.  
 
 
 
 
Jessica A. Alvarez, Ph.D., R.D.  
 
 
 
 
Kirk Easley, M.S.  
 
 
 
 
Emory Hsu, MD  
 
 
 
 
David L. Roberts  
 
 
 
 
Sharon H. Bergquist, MD 
 
 
 
 
 
 
 


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1. INTRODUCTION AND BACKGROUND 
   Osteoporosis has a devastating impact on quality of life of postmenopausal women, and is a significant 
cause of disability and morbidity. Numerous drugs are currently FDA-approved for the prevention and 
treatment of postmenopausal osteoporosis, the most frequent type of osteoporotic syndromes. However, most 
cases in the US remain untreated or ineffectively treated because of cost and/or adverse events of currently 
available drugs, leading to inadequate prescriptions of and poor compliance with anti-osteoporosis medications 
1,2.  For example, despite clear efficacy of bisphosphonate drugs in the secondary prevention of hip fracture, a 
recent large study in patients with hip fracture found that use of bisphosphonates decreased from an already 
dismal 15% in 2004 to an abysmal 3% in 2013 3. These data underscore the critical need to identify 
inexpensive, safe and effective interventions for both the prevention and treatment of osteoporosis. Animal 
studies 4-13 and a report in humans 14 suggest that nutritional supplementation with probiotics may represent an 
effective, safe and inexpensive modality to prevent and treat osteoporosis. Studies from laboratories in the US 
and Europe have clearly shown that probiotics prevent the bone loss induced by ovariectomy (ovx) 4-6, Adding 
to this evidence, we recently demonstrated that the probiotics Lactobacillus rhamnosus GG (LGG) and VSL#3 
(containing 8 strains of live bacteria, namely Bifidobacterium breve, B. longum, B. infantis, L. acidophilus, L. 
plantarum, L. paracasei, L. bulgaricus and Streptococcus thermophilus) prevent ovariectomy (ovx)-induced 
bone loss by decreasing cytokine-driven bone resorption8. Furthermore, we showed that both LGG and VSL#3 
induce bone anabolism in estrogen-replete mice.  
 
2. OBJECTIVES AND STUDY PURPOSE 
   Based on the strong preclinical evidence from our laboratory and others, our primary hypothesis is that 
oral administration of VSL#3 is a feasible, well-tolerated and effective strategy to prevent bone loss in 
postmenopausal women.  We further hypothesize that this simple approach will favorably impact biomarkers 
relevant to bone turnover, including inflammatory and osteoclastogenic cytokine production. 
 
Specific Aim 1.  To conduct a placebo-controlled, double blind, prospective clinical trial in which the 
commercially available oral probiotic VSL#3 will be administered to otherwise healthy, 
postmenopausal women for 12 months. The primary objective will be to demonstrate that VSL#3 
administration safely maintains or improves bone density in the lumbar (L1-L4) spine, femoral neck, and total 
hip area in the study subjects at 1 year. 
 
 
3. CRITERIA FOR PATIENT SELECTION 
   3.1 Inclusion criteria: 1) Willing and able to give written informed consent for participation in the study, 2) 
Age range 50-65 years, 3) Menopausal status (defined by >1 yr since last menstrual period or FSH level in the 
postmenopausal range), 4) Ambulatory, 5) Body mass Index (BMI) must be  ≥ 18.0 and ≤ 32 kg/m2 at 
screening, 6) Bone mineral density (BMD), expressed as T-scores, must be > - 2.5 in the lumbar spine (L1-L4), 
the femoral neck, and the total hip, as measured by dual energy X-ray absorptiometry (DXA), 7) Commitment 
not to use any products that may influence the study outcome (see below), and 8) Ability to understand and 
comply with the requirements of the study. 
 
3.2 Exclusion criteria: 1) Premenopausal status, 2) History of >1 previous atraumatic bone fractures after age 
50; 3) Presence of established osteoporosis (T-score ≤ - 2.5, in the lumbar spine, femoral neck or total hip as 
measured by screening DXA), 4) History of immunological or bone-related disorders including: HIV infection, 
Type I diabetes mellitus, bone marrow or organ transplantation; Inflammatory bowel disease (ulcerative colitis, 
Crohn’s disease); multiple myeloma; osteomalacia; osteosarcoma; Paget’s disease; rheumatoid arthritis; 
systemic lupus erythematous; parathyroid disorders, 5) uncontrolled type II diabetes mellitus (HgbA1c ≥ 7% 
within the last 12 months), 6) History of bariatric surgery or other forms of malabsorption (including 
documented celiac disease, or chronic diarrhea), 7) Alcohol abuse, 8) Clinically significant chronic kidney 
disease (stage ≥ 2, with total serum creatinine level > 2.5 mg/dL and calculated glomerular filtration rate (GFR) 
< 60 mL/min by the Modification of Diet in Renal Disease (MDRD equation), 9) Clinically significant 
cardiovascular disease (myocardial infarction, cerebral vascular accident or acute congestive heart failure 
within the previous 12 months, 10) Any malignancies, other than localized skin squamous cell carcinoma, 
diagnosed within the previous 5 years, or any history of metastatic cancer, 11) History of use of oral 
supplement products containing probiotic bacteria (more than once per week) within four weeks prior to 


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baseline, 12) Current use (within the past 8 weeks) of any medication with known influences on the immune or 
skeletal system (e.g. immune modulation therapy, systemic glucocorticoids, systemic steroid hormones, 13) 
Use of oral or injectable bisphosphonates for more than 1 year within the last 5 years, 14) Current or past use 
(within 1 year) of Denosumab, Teriparatide, Raloxifene, hormone replacement therapy (HRT), calcitonin, or 
any other anti-resorptive agent other than bisphosphonates used for the prevention and treatment of 
osteoporosis, 15) Use of antibiotics during the previous two months or frequent user of antibiotics (>2 courses 
during the previous 12 months) for any cause, 16) Smoking or use of nicotine-containing products during the 
last six months, 17) Known hypersensitivity to any of the ingredients in the VSL#3 or the placebo study drug, 
18) serum or plasma 25-hydroxyvitamin D [25(OH)D] concentration < 12 ng/mL, 19) uncontrolled thyroid 
disease (abnormal blood TSH level within the last 12 months and/or changing dose of thyroid replacement 
therapy within the last 12 months). 
 
3.3. Number of patients to be studied:  We propose to study a total of 40 patients. A total of 20 subjects will 
receive a placebo (control, 2 placebo sachets/day in a single dose), and 20 subjects will receive VSL#3 (2 
sachets a day in a single dose). 
 
3.4. Subject recruitment plan: We will employ several methods for identifying potentially eligible 
participants: 
 
a) We will post approved flyers throughout Emory-affiliated hospitals and clinics, Emory University and 
 
the surrounding Atlanta metropolitan area. 
 
b) We will utilize the study subject recruitment resources of the NIH-funded Atlanta Clinical and 
 
Translational Science Institute (ACTSI), including its weekly list-serve, clinical trial websites of Emory 
 
HealthCare, and ResearchMatch.org. 
 
c) Subjects will also be recruited among participants in an existing cohort of Emory University and 
 
Emory  Healthcare employees, the Emory/Georgia Tech Predictive Health Initiative (PHI) Cohort, who 
 
have consented to be contacted for potential study participation in future research and who meet 
 
initial screening criteria based on the most recent data available (within previous 12 months)15. 
 
d) Participants may also be recruited by advertising (via flyers or in person) at health fairs and other 
 
Atlanta community events.   
 
e) We will approach potentially eligible subjects among patients seen in Emory HealthCare-affiliated 
 
Endocrinology, Obstetrics/Gynecology, Rheumatology and General Medicine clinics in Atlanta, GA. 
 
This will involve a prescreen of the electronic medical record facilitated by a partial HIPAA waiver 
 
for screening purposes. We will also identify potentially eligible subjects among patients who have 
 
undergone a recent bone density scan at Emory clinics and/or whose data is entered into the Emory 
 
BoneStation electronic record. 
 
f) Through the Emory Data Warehouse, we will receive a list of patients in the Emory system who meet 
 
eligibility criteria and who have agreed to be contacted about research studies either by phone or 
 
through a letter.  
 
g) Individual participating Emory clinics will generate a list of clinic patients who meet eligibility criteria 
 
and will send a letter (directly or through the study team) to potentially eligible patients describing 
 
the study and providing study contact information.  
 
h) A combination of the above three (e-g) strategies may be used, but the research team will only 
 
approach potential study participants in person (during clinic visits) or by letter unless there is 
 
available documentation that  a potential participant is agreeable to being contacted by phone.   
 
3.5 Study withdrawal: Participants may withdraw from the study at any time.  Automatic withdrawal occurs 
when a participant refuses or is unable to take study drug, or refuses or is unable to complete study 
procedures, including the return visits to the Clinical Research Center at 6 and 12 months and the twice 
monthly study-related telephone calls required to ensure tolerance/compliance with the VSL#3 and placebo.  
 
4. STUDY DESIGN AND METHODS 
 


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   4.1 Overview of the study:  This will be a 12-month, double-blind, randomized, placebo-controlled trial of 
VSL#3 or placebo (Figure 1). Participants will be randomized to the placebo group or the VSL#3 group and 
treated and followed serially for 12 months. Baseline and follow-up assessments will be performed during an 
outpatient visit in the Emory University Hospital (EUH) Clinical Research Network (CRN) site of the Atlanta 
Clinical and Translational Science Institute (ACTSI) before initiation of the study drug (entry), and 6, and 12 
months following baseline assessments.  Subjects will report to the CRN after having fasted for at least 8 
hours.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The study will be conducted in a population of ambulatory, postmenopausal women aged 50-65 (BMI >18.5 
and ≤ 30 kg/m2), with a lumbar spine (L1-L4), femoral neck, and total hip BMD T-score > - 2.5, as measured by 
DXA (see section 3 above).  Women with osteoporosis (T score < -2.5) will not be included in the study 
because at this stage of knowledge we find it unethical not to offer effective treatment to these individuals. We 
will recruit subjects of all ethnic groups. Covariates known to influence bone mass, including body mass index 
(BMI), history of smoking > 6 months prior to entry, alcohol use, baseline BMD of the lumbar spine, femoral 
neck and total hip area, bone fracture history and co-morbidities will be collected and controlled for in the final 
analysis. The primary endpoint is the change in bone mineral density (BMD) at the L1-4 lumbar spine over one 
year of study. Changes in BMD at the femoral neck and total hip area will be secondary endpoints. All BMD 
data will also be used as a tool for future studies power calculation and design. Additional endpoints will 
include changes in bone turnover markers and inflammatory/osteoclastogenic cytokines.  These indices will 
provide much needed mechanistic information.  To determine additional potential mechanisms of action of 
probiotics on health, we will conduct serial metabolomics analyses in the plasma, as well as serial assessment 
of plasma thiol/disulfide redox balance. The data will allow us to establish whether VSL#3 prevents bone loss 
and/or increases bone mass by regulating bone resorption, formation, or both.  Based on our own data in mice 
following ovx, we hypothesize that treatment with VSL#3 will quickly decrease bone resorption and then 
stimulate bone formation, resulting in improved net BMD at least one site over the 12-month period of study. 
 
Figure 1: Study schema for protocol “Use of a Commercially Available Combination Probiotic, 
VSL#3, for Bone Health in Postmenopausal Women” 


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4.2. Screening for eligibility: For study subjects identified among patients seen at the Emory-affiliated 
Endocrinology, Ob/Gyn, Rheumatology and General Medicine clinics, patients will be pre-screened for 
eligibility using the Emory Healthcare electronic medical record, and potentially eligible subjects will be 
approached during their clinic visit by a study coordinator or they will be contacted by phone or letter (as 
described above) to conduct a full screen. For potential study subjects identified outside of Emory-affiliated 
Clinics, potential subjects will contact the study team by phone and undergo a pre-screening discussion.   
 
A full screen will consist of any screening medical/surgical and medication use information, blood and urine 
tests, screening DXA scan, and a physical examination. These may occur in the Emory-affiliated clinics by 
study personnel and/or in the Emory University Hospital CRN. Informed consent will be obtained prior to any 
research procedures. A verbal informed consent will be obtained from potential participants being pre-
screened by phone prior to collecting information regarding eligibility criteria. In order to reach our goal or 40 
randomized participants, up to 100 women may undergo a screening visit.  
 
4.3 Informed consent process: A written and signed informed consent will be obtained in person in a private 
area. After identification of a person as potentially eligible for the study, approved study personnel will explain 
the study in detail in the consent form as well as verbally. Potential participants will be informed of the purpose 
of the study, the study protocol, and the routine and potential risks associated with the study procedures. All 
risks, costs, and benefits will be discussed.  Participants will be assured of their right to withdraw at any time 
without prejudice to their care. They will be assured of confidentiality in maintaining records and reporting of 
results. Participants will have the option to provide permission to store biological samples for future studies. 
Any additional information regarding questions or concerns from potential or confirmed study participants will 
be provided.  
 
We will request a partial HIPAA waiver to be able to access the medical record for screening. 
 
For subjects recruited in Emory Clinics, the consent process will take place during a patient’s clinic visit. 
Potential study participants will have the option of taking the blank, unsigned consent forms home to consider 
prior to signing the consent forms, in which case patients who decide to participate will complete a verbal 
consent by telephone and will complete the full, written consent process during their first study visit (prior to 
any procedures).  For subjects recruited outside of Emory clinics, the consent process will take place during 
their EUH CRN screening visit. Participants will have ample time to call the study team with 
questions/concerns and make an informed decision about their participation in the study.  
 
An Institutional Review Board (IRB) approved study designee will provide verbal and written consent. The 
study is discussed thoroughly with each potential participant, and she will be encouraged to ask questions 
regarding the study and her participation. If the potential participant decides to enroll, she will sign the written 
informed consent, as will the study team member conducting informed consent. A copy of the signed consent 
form will be provided to enrolled study participants. Subjects must be able to read, write and understand the 
consent documents. Study personnel will subjectively assess the capacity to give informed consent through the 
use of questions to ensure understanding and comprehension of the study. Only adults will participate. 
 
4.4. Study Initiation and baseline assessments: Following screening and informed consent, baseline 
assessment and laboratory evaluation will be performed as outlined in Table 1. Dietary calcium and vitamin D 
intake will be reviewed by a registered dietitian. For participants not meeting at least the Recommended 
Dietary Allowance (RDA) for calcium (1200 mg) and vitamin D (600 IU), a recommendation will be provided for 
sufficient over-the-counter calcium citrate and/or vitamin D3 to meet the RDA. Participants will be allowed to 
continue their current regimens; however, they will be asked not to begin any new supplement, medical, or 
exercise therapies that could influence bone health. For individuals with a screening 25(OH)D < 20, a study 
physician will communicate with the participant’s primary MD (and/or the participant directly) and recommend a 
standardized dose of vitamin D replacement (daily vitamin D3 dose of 5,000 IU daily + 50,000 IU weekly for 12 
weeks with a recheck of 25(OH)D level in 3-4 months).  
 
 
Table 1. Study Endpoints by Visit 


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Assessments 
Screen 
in 
clinic or 
CRN unit 
Entry visit 
in CRN    
unit (max 4 
weeks after 
screen visit) 
6 mo visit 
in CRN    
unit (+/- 1 
month) 
12 mo visit in 
CRN 
Unit (+/- 1 
month) 
Medical/Surgical history, 
medication use, dietary 
calcium intake, alcohol 
intake, physical activity 
x 
 
x 
x 
Serum FSH  
(if indicated) 
x 
 
 
 
Physical Exam 
x 
 
x 
x 
Concomitant medications 
x 
x 
x 
x 
Gastrointestinal Symptom 
Rating Scale (GSRS) 
 
Determine GCRS every 2 weeks 
Study drug adherence 
assessment 
 
Determine adherence every 2 weeks 
Fasting serum markers of 
bone turnover (CTX and 
PINP) 
 
x 
x 
x 
Serum RANKL (free), OPG, 
TNF, IL-17 
 
x 
x 
x 
DXA scan: (lumbar  spine, 
femoral neck, and total hip, 
total and regional body fat, 
total and regional lean body 
mass, visceral fat)* 
x 
x 
 
x 
Dietary Intake 
3-day food record 
 
x 
x 
x 
BioElectrical Impendence  
Analysis (BIA) 
Body composition and total 
body water 
 
x 
x 
x 
Anthropometrics (height, 
weight, BMI, waist & hip 
circumference) 
 
 
 
 
Handgrip strength 
Hand-held dynamometer 
 
x 
x 
x 
Serum 25(OH)D 
x 
 
 
x 
Stool microbiota 
composition 
 
x 
x 
x 
 
X 
X 
X 
Plasma metabolomics 
 
x 
x 
x 
Plasma Aminothiols 
(GSH, GSSG, Cys, CySS) 
 
x 
x 
x 
Serum Intact PTH 
x 
 
 
 
Complete blood count 
(CBC) 
x 
 
x 
x 
Chemistry profile 
x 
 
x 
x 
Urinalysis 
x 
 
x 
x 
*Note: First DXA scan will be done at the screening visit or the entry visit (not both).  
 
4.5 Intervention:  A total of 40 matched postmenopausal women meeting all specific inclusion/exclusion 
criteria will be blindly randomized to one of two study arms. Arm A (n=20): VSL#3 two active sachets 
[containing 450x109 colony-forming units (CFU)/sachet], taken daily in a single administration; Arm B (n=20; 
control): two placebo sachets taken daily in a single administration. Thus, Arm A will receive 900 billion CFU 
daily and the control group (Arm B) will receive no supplemental probiotics (Figure 1).    
 


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4.6. Randomization and Stratification: Personnel in the Department of Biostatistics and Bioinformatics at 
Emory University have developed a centralized electronic randomization system that has been successfully 
used in several clinical studies 16-19. Subjects will be blindly assigned in equal proportions, stratified by age 
(above and below 58 years) using a pseudo-random-number generator with randomly permuted blocks.  These 
assignments will be stored in a backend database table within the iDataFax database management system.  
This method serves to balance the treatment group assignments over the course of the study to ensure that 
the desired number of subjects will be allocated to each of the treatment groups at any time point during the 
randomization period.  Permuted block sizes will not be disclosed to the blinded study personnel to minimize 
the likelihood of their being able to predict the next randomization assignment in the series. Briefly, after 
obtaining informed consent, all eligibility confirmation data will be entered into the web-based data 
management system by a research coordinator.  If the patient meets all eligibility criteria, computer 
randomization will be performed and the coordinator will be provided with an audit trial of the process including 
a verifiable link between the unique study ID and treatment assignment.  The application will be password 
protected with access restricted to designated personnel, including the EUH Investigational Drug Pharmacy 
staff pharmacist who will dispense blinded study drug for subjects randomized to Arms A or B. 
 
4.7. Screening, Entry and Follow-up Evaluations: These will occur as outlined in Table 1. Adverse effects 
and safety data will be reviewed at least every 6 months by a Data Safety Officer, a gastroenterologist senior 
Emory faculty member with expertise in clinical trials who is unaffiliated with the study, as described below 
(Section 7.5).  
 
4.8. Procedures to be performed (all for research purposes only): 
 
 
Medical examination: Medical history and medications will be reviewed by a member of the research 
team, and a physical exam will be completed by a licensed physician, as required by the clinical research 
unit. Habitual alcohol intake and dietary calcium intake (based on reported supplements, dairy, and other 
high-calcium foods) will be assessed.   
 
Urine collection: Urine will be collected for standard urinalysis indexes.  
 
Anthropometric measurements: Height and weight will be measured without shoes. Height will be 
measured with a manual stadiometer. Weight will be measured with a digital scale. Body mass index 
(BMI) will be calculated from height and weight. Waist and hip circumference will be measured with a 
flexible measuring tape.  
 
Blood sampling: Up to approximately 70 mL of fasted blood will be collected at every visit from a 
peripheral vein. Blood will be drawn for determination of plasma and serum biomarkers, plasma 
metabolomics, plasma redox markers, and standard clinical blood tests listed in Table 1 above. Blood will 
also be stored for use for future studies, with consent from the study participant. 
 
Stool collection: Study subjects will be provided with a kit containing detailed instructions for stool 
collection and shipping. The kit contains a stool sample shipping tube, an adhesive paper for stool 
collection, a spatula for placing a small amount of stool into the tube, a biosafety bag and a pre-paid FedEx 
envelope for shipping the stool sample directly to the study lab within 48 hours. 
 
Bone density analysis: DXA will be used to measure bone density of the spine (L1-L4 segment) and the 
hip (non-dominant femoral neck and total hip area). Participants will be asked to lie still on a scanning 
table with their arms at their sides for approximately 20 minutes. All DXA scans will be performed on the 
same device within the Emory CRN by an experienced technician using a GE Lunar iDXA machine. Areas 
of interest will include the lumbar spine (L1-4), the femoral neck and the total hip). DEXA will also provide 
information about total and regional body fat, lean body mass.   
 
Three-day food records: Participants will be asked to complete a food diary for three days (2 weekdays 
and 1 weekend day) where they will record everything they eat and drink, including supplements. They will 
be given food records prior to their study visit and asked to return completed food records on the day of 
their visit. Alternatively, participants may choose to email or mail study investigators the completed food 
record. 
 
Body composition analysis: In addition to DEXA, body composition may also be assessed using 
bioelectrical impendence analysis (BIA); for this procedure participants will lie still on a table for <5 
minutes with electrodes placed on hands and feet.  


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 
Handgrip strength: A small, handheld dynamometer will be used to measure grip strength. Participants 
will be asked to squeeze the instrument as hard as they can for 3 seconds, which will measure the pounds 
of force applied. 
 
Questionnaires: Participants will be asked to answer a standardized, validated questionnaire (15 
questions) about GI symptoms segregated into 5 symptom domains (see below). They will also complete 
a questionnaire assessing their habitual physical activity (Modified Baecke Questionnaire)20. 
 
4.9. Primary Endpoint: The primary endpoint is change in BMD of the lumbar spine (L1-L4 segment), as 
measured by DXA. 
 
4.10 Secondary Endpoints (please see Table 1 above): Serial measures of: 1) bone density of the non-
dominant hip (femoral neck and total hip area) as measured by DXA, 2) serum collagen type 1 cross-linked C-
telopeptide (CTX) concentrations (a marker of bone resorption), 3) serum procollagen type I N propeptide 
(PINP) concentrations (a marker of bone formation), 4) serum free receptor activator of nuclear factor kappa-B 
ligand (RANKL) concentrations, 5) serum osteoprotegrin (OPG) concentrations, 6) serum tumor necrosis 
factor- (TNF), 7) serum interleukin-17 (IL-17) concentrations, 7) total and regional body fat (including 
visceral fat) and lean body mass as measured by DXA and BIA, 8) plasma thio/disulfide redox biomarkers, 9) 
compliance with study drug administration10) overall study recruitment and retention, and 11) ) stool 
microbiota composition. Measurements of indices of bone turnover and cytokine levels will provide much 
needed mechanistic information to establish whether VSL#3 may prevent bone loss and/or increases bone 
mass by regulating bone resorption, formation, or both.  Based on animal data, we hypothesize that treatment 
with VSL#3 will quickly decrease bone resorption and then stimulate bone formation. 
 
Study drug tolerance will be assessed by obtaining serial measures of the Gastrointestinal Symptom Rating 
Scale (GSRS) every two weeks during the 12-month placebo or probiotic regimen 21. These will be obtained 
with in-person interviews at the baseline and month 6 and 12 visits and by telephone contact with all subjects 
by the study coordinator every 2 weeks. Data will be analyzed within the 5 symptom domains depicting 
symptoms related to gastric reflux, abdominal pain, indigestion, diarrhea, and constipation. A GSRS total 
score, the sum of all 5 domains, will also be assessed. To our knowledge there is no published absolute or 
change in the GSRS score that is demonstrative of intolerance to a study drug or other treatment 21-23. 
Therefore we will a priori define intolerance to the study drug as a 50% increase in the total GSRS score from 
baseline at any two consecutive time points, or the new onset of a GSRS classifier score of 2 or 3 in at least 
three of the 15 gastrointestinal symptoms queried. If either of these signs of intolerance occurs, we will offer 
the subject the options of: 1) discontinuing from the study; 2) discontinuing the study drug but participating in 
the serial assessments (e.g. blood draws, DXA) in the research center, or 3) decreasing the dose of blinded 
study drug to one sachet/daily, with the option of resuming the two sachet/day study regimen upon resolution 
of symptoms. If intolerance symptoms recur, the same strategy will be followed. All episodes of intolerance to 
study drug and the twice-monthly GSRS data will be recorded by the study coordinator in the individual subject 
case report forms (CRFs) (see below). 
 
Compliance with the study placebo or VSL#3 doses will be assessed serially. This will be determined by 
contacts (twice monthly telephone or at research center visits) of the study coordinator with each subject and 
responses to three standardized question areas: 1) “Are you having any difficulty, problems or new symptoms 
with the study medication?” 2)  If yes, “What has the problem been?” and 3) “Have you missed any of your 
study drug doses, and if so how many in the previous 2 week period?” Appropriate notations based on subject 
responses will be documented in the CRF.   Subjects will be instructed at study entry and reminded via serial 
contacts to return all of their used and unused drug sachets at each CRN visit (6 months +/- 2 weeks and 12 
months +/- 2 weeks after entry).  Unused and used study drug satchets will be tallied and recorded in the CRF 
by the study coordinator serially for the entire study. All satchets (used and unused) will be given to the Emory 
University Investigational Drug Service (IDS) for disposal.  
 
Recruitment and retention will be documented via conventional CONSORT criteria and documentation of 
missed study visits, missed telephone communications and compliance with study drug administration. All data 
will be maintained in the CRFs. 
 


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4.13. Sample size and power considerations:  Longitudinal data from 380 postmenopausal women form the 
basis for estimating the sample size and power for lumbar spine BMD as the primary outcome endpoint.  BMD 
at the lumbar spine was available at baseline and 6 and 12 months after baseline (883 measurements across 
the 3 time points). Mean baseline BMD was 1.108 g/cm2 (within-subject standard deviation = 0.03 g/cm2 and 
the standard deviation on change = 0.02 g/cm2). Assuming no decline on average in BMD in the VSL#3 treated 
group and a decline on average of 0.03 g/cm2 in the control group and an estimated within-subject standard 
deviation in each group of 0.03 g/cm2, the proposed sample sizes (n=20 postmenopausal women per group) 
will provide more than 85% power to detect a difference on change of 0.03 g/cm2 at the two-sided 5% 
significance level if the true difference between treatment groups is 0.03 g/cm2 (two-sided two-sample equal 
variance t-test). 
 
4.14. Statistical analyses: Demographic data will be summarized with standard descriptive statistics. 
Repeated-measures analyses for each outcome (i.e., GSRS scale scores, BMD) will be performed with a 
means model via the SAS MIXED Procedure (version 9.4; SAS Institute, Cary, NC), providing separate 
estimates of the means by time on study (baseline, 1-year and 2-years) and treatment group.  The model will 
include three predictors (treatment arm, time on study and the statistical interaction between treatment arm 
and time on study).  A compound-symmetric variance-covariance form in repeated measurements will be 
assumed for each outcome and robust estimates of the standard errors of parameters will be used to perform 
statistical tests and construct 95% confidence intervals24. The model-based means are unbiased with 
unbalanced and missing data, so long as the missing data are non-informative (missing at random, MAR).  A 
P-value ≤0.05 will be considered statistically significant for the main effects (treatment and time on study) and 
for the treatment by time on study interaction effect from the repeated measures analysis. The statistical test 
for interaction between time on study and treatment will be the primary overall hypothesis test to determine 
whether an outcome in the two study groups changed in significantly different ways during follow-up (i.e., 
different temporal patterns over time).  If a mean outcome in the two groups is consistently different or similar 
over time (i.e., no statistical interaction) then the main effect test for treatment will be used as the primary test 
of VSL#3 efficacy.  If a significant interaction is detected, then t-tests will be used to compare the differences 
between the model-based intervention means at each time point and to compare differences over time within 
each treatment arm.  Specific statistical tests will be done within the framework of the mixed effects linear 
model. Analyses will control for risk factors for osteoporosis, such as age, gender, race, BMI, habitual alcohol 
consumption, smoking, physical activity, and fracture history, as necessary. All tests will be 2-sided and a P 
value ≤ 0.05 will be considered significant.  
 
5. DRUG DOSING 
5.1. Method for determining doses: Doses were based on the concentration of colony forming units (CFU) 
within a single, commercially available sachet, and doses used in previous trials in adults with inflammatory 
bowel diseases (IBD), irritable bowel syndrome, and other gastrointestinal (GI) disorders 25-31.  
 
5.2. Maximum Dosage: The maximum dosage will be 900x109 CFU orally daily.  This or higher doses have 
been used in most trials designed to assess the tolerability and efficacy of VSL#3 in IBD and irritable bowel 
syndrome in adults. 
 
5.3. Drug duration: Patients will take the drug for 1 year.  
 
5.4. Distribution and Storage of Investigational Products: The study drug/placebo will be delivered to the 
EUH Investigational Drug Service (IDS) directly from the manufacturer. The EUH IDS will store the study 
drug/placebo under the appropriate storage conditions, as indicated by the manufacturer. The Pharmacy will 
dispense the study drug/placebo to study subjects (who will remain blinded). Subjects will be provided with 
sufficient study drug product every two months. IDS will dispense a 2-month supply of the study drug sachets 
to the study team for each subject serially, who will over-night mail the resupply with a commercial carrier in 
sealed Styrofoam containers containing cold packs. The IDS will provide subjects with instructions for study 
drug storage at home. For all products provided, the lot number and expiration date will be recorded in 
individual subjects case report forms. All study drug products will be labeled with the following, “Caution: New 
Drug—Limited by Federal (United States) law to investigational use.” 
 


Version 10/17/18 
 
6. OBSERVATIONS AND MEASUREMENTS FOR STUDY OBJECTIVES 
 
Please see Sections 4.8-4.10 and Table 1 above. 
 
7. PROCEDURES TO MONITOR DRUG EFFECTS AND MINIMIZE RISK 
 
7.1 Potential Risks/Discomforts 
 
The risk of participating in this study is minimal.  
 
a. VSL#3: There are few reported side effects of this dietary supplement. Some studies have reported GI 
effects, such as bloating abdominal pain.  Of note, VSL#3 doses of up to 3600x109 CFU orally daily for 
several weeks or months have been reported to be well-tolerated in adults with irritable bowel 
syndrome, Crohn’s disease, and other GI disorders 32-37. 
b. Venipuncture: There is some minor discomfort and risk of mild bruising during venipuncture. Standard 
aseptic techniques will be used during phlebotomy; thus, infection is unlikely. Disposable pre-sterilized 
needles and syringes will be used for all blood drawing in this study; needles and syringes will not be 
reused. 
c. DXA: DXA involves exposure to small amounts of radiation. The radiation dose is equal to or less than 
the amount of background radiation received in a round-trip flight from New York to Los Angeles or the 
natural environmental radiation the average person receives in the United States annually. The risk 
from radiation exposure of this magnitude is considered to be negligible when compared to everyday 
risks. For screening purposes, if menopausal status is not clear, a urine pregnancy may be performed 
prior to DXA testing. 
d. Stool collection: There may be minor inconvenience from the process of collecting a small stool 
sample, placing a stool sample into the shipping tube and shaking it for a few seconds to mix. 
e. BIA: The BIA machine passes a weak electrical current through electrodes attached to hands and feet. 
Participants will not feel anything. There are no known risks associated with the procedure, although 
participants who have pacemakers or other implantable electronic devices will not undergo the BIA 
procedure. 
 
f. Other: Additional risks will be associated with confidentiality issues surrounding the collection/recording 
of data, but steps will be taken to minimize these risks, as described below. 
 
7.2. 
Assessment of Risk/Benefit 
  
a. Direct benefit: Participants will be compensated a stipend for their time and inconvenience. 
Participants will also receive information on their measured bone density after the 12-month period.  
Participants will be encouraged to share the results with their healthcare providers. There may be no 
specific health benefit to patients’ participation in the study.   
b. Indirect benefit: The long-term goal of this study is to identify a safe, well tolerated and simple 
intervention strategy, using a commercially available combination probiotic to minimize bone loss 
associated with menopause.  If our hypothesis is correct, VSL#3 would be an inexpensive, easily 
obtainable commercial probiotic that could be supplemented in this population.  
c. The anticipated benefits of the proposed research study outweigh the potential risks to participating 
subjects. The risks incurred are minimal relative to the potential benefit of finding an effective therapy to 
reduce bone loss in postmenopausal women. 
 
7.3. Procedures to monitor drug effects 
 
Patients will be called every other week to maintain compliance/adherence with the study drug regimen and to 
monitor for any adverse effects. Questions will also focus on gastrointestinal symptoms (via the GSRS score), 
as these are the primary expected side effects of VSL#3. In addition to twice monthly phone calls, subjects will 
be asked to complete Subject Diary Cards to capture adverse events during the course of the trial and up to 1 


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month following the cessation of the study drug. Subjects will receive a final safety follow-up phone call 1 
month following the cessation of the study drug.  
 
Systemic reactions and adverse events (AEs) (e.g. diarrhea, headache, fatigue, rash, and myalgia) will be 
monitored every other week during study phone calls. The intensity of these events will be assessed on a 
standard toxicity grading scale for healthy volunteers38, as shown in Table 3. Standard laboratory values will 
be assessed at baseline, 6 month and 12 month visits and graded as shown in Table 4. Decisions to withdraw 
the study drug from individual subjects, will be at the discretion of both the PI and a Safety Officer (described 
below), based on a severity grade of 3 or 4 for Graded Adverse Events or Laboratory Values, and based on 
the likelihood of it being related to study. 
 
 
Table 3. Graded Adverse Events 
 
Adverse Event 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Nausea/vomiting 
No interference 
with activity or 1 – 2 
episodes/24 hours 
Some interference 
with activity or > 2 
episodes/24 hours 
Prevents daily 
activity, requires 
outpatient IV 
hydration 
ER visit or 
hospitalization 
for 
hypotensive 
shock 
Diarrhea 
2 – 3 loose stools 
or 
< 400 gms/24 hours 
4 – 5 stools or 
400 – 800 gms/24 
hours 
6 or more watery 
stools or 
> 800gms/24 
hours 
or requires 
outpatient IV 
hydration 
ER visit or 
hospitalization 
Headache 
No interference with 
activity 
Repeated use of 
non- 
narcotic pain 
reliever > 24 hours 
or 
some interference 
with activity 
Significant; any 
use of narcotic 
pain reliever or 
prevents daily 
activity 
ER visit or 
hospitalization 
Fatigue 
No interference with 
activity 
Some interference 
with activity 
Significant; 
prevents daily 
activity 
ER visit or 
hospitalization 
Myalgia 
No interference with 
activity 
Some interference 
with activity 
Significant; 
prevents daily 
activity 
ER visit or 
hospitalization 
Rash/Erythrema 
2.5 – 5 cm 
5.1 – 10 cm 
> 10 cm 
Necrosis or 
exfoliative 
dermatitis 
Pruitis/Itching 
No interference with 
activity 
Some interference 
with activity 
Significant; 
prevents daily 
activity 
ER visit or 
hospitalization 
Fever 
38.0 – 38.4 °C 
100.4 – 101.1°F 
 
38.5 – 38.9°C 
101.2 – 102.0°F 
 
39.0 – 40°C 
102.1 – 104 °F 
 
> 40°C 
> 104 °F 
 
Tachycardia - 
beats per minute 
101 – 115 
116 – 130 
> 130 
ER visit or 
hospitalization 
for arrhythmia 
Bradycardia - 
50 – 54 
45 – 49 
< 45 
ER visit or 


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beats per minute*** 
hospitalization 
for arrhythmia 
Hypertension 
(systolic) - mm Hg 
141 – 150 
151 – 155 
> 155 
ER visit or 
hospitalization 
for malignant 
hypertension 
Hypertension 
(diastolic) - mm Hg 
91 – 95 
96 – 100 
> 100 
ER visit or 
hospitalization 
for malignant 
hypertension 
Hypotension 
(systolic) – mm Hg 
85 – 89 
80 – 84 
< 80 
ER visit or 
hospitalization 
for hypotensive 
shock 
Respiratory Rate – 
breaths per minute 
17 – 20 
21 – 25 
> 25 
Intubation 
Other illness or 
clinical adverse 
event (as defined 
according to 
applicable 
regulations) 
No interference with 
activity 
 
Some interference 
with activity not 
requiring medical 
intervention 
 
Prevents daily 
activity and 
requires medical 
intervention 
 
ER visit or 
hospitalization 
 
Table 4. Laboratory Adverse Events  
Blood Parameter 
Reference 
Range* 
Mild/Moderate 
Severe/Critical 
Sodium – Hyponatremia  
mmol/L 
136-144 
126-135 
≤125 
Sodium – Hypernatremia mmol/L 
136-144 
145 – 155 
≥156 
Potassium – Hyperkalemia mmol/L  
3.6-5.1 
5.2-6.0 
>6.0 
Potassium – Hypokalemia mmol/L 
3.6-5.1 
3.0-3.5 
<3.0 
Glucose – Hypoglycemia mg/dL  
65 – 110 
45-64 
<45 
Glucose – Hyperglycemia mg/dL  
65 – 110 
111-450 
>450, or insulin requirement, or 
hyperosmolar coma 
Blood Urea Nitrogen, BUN mg/dL  
8-25 
26-150 
> 150 or requires dialysis 
Creatinine – mg/dL  
0.4-1.0 
1.1-5.8 
>5.8 or requires dialysis 
Calcium – hypocalcemia mg/dL  
8.9-10.3 
6.6-8.8 
≤6.5 
Calcium – hypercalcemia mg/dL  
8.9-10.3 
10.4-12.9 
≥13.0 
Albumin – Hypoalbuminemia g/dL  
3.5-4.8 
<3.5 
------ 
Total Protein – Hypoproteinemia g/dL  
6.1-7.9 
<6.1 
------ 
Alkaline phosphate unit/L 
32-91 
<91 
------ 
ALT unit/L 
≤ 33 
>33 
------ 
AST unit/L  
15-41 
>41 
------ 
Bilirubin mg/dL  
0.3-1.2 mg/dL 
1.3-20 
>20 
Cholesterol mg/dL 
≤ 199 
>199 
------ 
Hemoglobin (Female) - g/dL  
11.4-14.4 
6.6-11.3 
≤ 6.5 
WBC low x 103/mcL  
4-10 
2-3.9 
< 2.0 
WBC high x 103/mcL 
4-10 
11-50 
>50 
Eosinophils x 103/mcL 
<0.36 
>0.37 
Hypereosinophilic 
Platelets-high x 103/mcL 
150-400 
401-999 
>999 
Platelets-low x 103/mcL 
150-400 
51-149 
<50 


Version 10/17/18 
Urine Parameter 
Reference 
Range 
Mild/Moderate 
Severe/Critical 
Protein  
none 
trace 
Hospitalization or dialysis 
Glucose  
none 
trace 
Hospitalization for hyperglycemia 
Blood (microscopic) – red blood cells 
per high-power field (RBC/hpf)  
none 
trace 
gross blood or hospitalization or packed 
red blood cells (PRBC) transfusion 
*Specific to EUH Laboratories 
 
 
7.4. Additional protection against risks 
 
7.4.1 Subject identification and screening for eligibility: Our strict inclusion and exclusion criteria for entry 
will help to minimize potential risks. All subjects will be monitored closely by frequent clinical assessments and 
review of safety laboratory tests by experienced clinicians.   
 
7.4.2 Blood sampling: Experienced research nurses will use aseptic techniques for all study blood draws. 
Participants will be asked to stay hydrated with water the day before the study visit. Universal precautions will 
be employed in all instances involving human specimens and all blood processed under at least BSL2 
conditions. Discomforts associated with venipuncture are rapidly reversible. 
  
7.4.3 DXA: DXA will be performed by experienced technicians who are trained in radiation exposure 
minimization in patients. 
 
7.4.4 Privacy and confidentiality: The subjects’ extracted medical records and data from the study will be 
initially stored on paper prior to transfer to electronic format. The encrypted electronic record will remain on the 
principal investigator’s password protected computer in his locked office. Confidentiality will be assured by the 
use of subject codes rather than personal identifiers. The master list connecting the subject codes to identifying 
information will be secured in the PI’s computerized database. All paper subject records will be kept in locked 
file cabinets in the PI’s research office and will be accessible only to the PI and the investigative team. Only 
institutional review board (IRB)-approved study personnel will have access to individually identifiable 
information about human subjects. This may include the study PI, co-investigators, research coordinators, and 
other approved study personnel. All information and materials will be obtained for research purposes only and 
the data will be kept in strict confidence for use in this proposed research only. 
 
7.4.5 Other: Since the proposed tests are not inherently hazardous, hazard is likely to occur only as the result 
of impaired participant confidence or sudden unwillingness to complete a test. To avoid this possibility, study 
personnel will thoroughly explain all tests to potential participants prior to them signing the consent form. 
Potential participants will have the opportunity to see all test equipment and facilities before giving consent or 
undergoing testing. Trained personnel will be available at all times to monitor participants’ physical and 
emotional distress. Every effort will be taken to prevent injury or distress that may result from this study. A 
study physician will be on call for support. A qualified physician will have primary responsibility for outpatient 
care. 
 
7.5. Data and Safety Monitoring and Reporting: Trained study personnel will be with a participant at all 
times to monitor patient safety. Adverse events are expected to be uncommon and not pose more than 
minimal risk to the study participants. The Principal investigator (PI) will review all data collection forms at least 
semi-annually for completeness and accuracy of the data as well as protocol compliance. The PI will review 
this protocol on a continuing basis for subject safety and include the results of the review in annual progress 
reports submitted to the IRB.  
 
7.5.1 Data Safety Officer: A senior faculty Data Safety Officer, Dr. Jennifer Christie, MD, FASGE, Associate 
Professor of Medicine in the Emory University Division of Digestive Diseases, Clinical Director of 
Gastroenterology at the Emory Clinic, and Director of Gastrointestinal Motility has been designated for this 
study. Dr. Christie is an experienced physician-scientist with expertise in randomized controlled clinical trials 
and translational science. She will review semi-annual reports created by the Principal Investigator (PI) Dr. 


Version 10/17/18 
Roberto Pacifici and the Data Coordinating Committee, directed by Mr. Kirk Easley, MS, and make 
recommendations regarding the continuation of study. 
 
7.5.2 Participant Safety Data Examination, Monitoring Procedures/Oversight: Participant monitoring will 
be performed by the PI, Co-Investigators, and members of the study team. The IRB Reportable Events 
Guidelines will be followed. The standard Emory IRB reporting guidelines for AE and SAE reporting will also be 
followed. Any adverse events will be reported to the IRB within 10 days of the event if it is (a) an unanticipated 
problem involving risk to participants or others, (b) a death that is possibly, probably, or definitely related to the 
research, (c) an anticipated event occurring with a greater frequency, duration, or severity than what is 
described in the protocol related documents, or (d) any other information that suggests the research places 
participants or others at greater risk of harm than was previously known. A reportable events log will be 
maintained by the PI and reported annually to the IRB (or more frequently if required.) 
 
All adverse events will be graded as to their attribution (unrelated to protocol, or possibly, probably, or definitely 
related to protocol). Any AE that is reported to either the PI or the study team by a study subject or by medical 
staff caring for the subject and which meets the criteria will be documented as such. Adverse events will also 
be further labeled as “expected” AEs (which are predefined and listed as potential risks in the informed 
consent), “unanticipated” AEs, and serious adverse events. Serious adverse events (SAE) are predefined as: 
any experience that suggests a significant hazard, such as events which: a) are fatal, b) are life threatening, c) 
result in permanent disability, d) require inpatient hospitalization, or e) involve cancer, a congenital anomaly, or 
drug overdose.  
 
7.5.3 Performance Standards: The Data Coordinating Center (DCC) has worked closely with the clinical team 
during protocol development to specify performance standards that should be met to achieve high quality of 
trial conduct.  These standards will address screening and recruitment plans and rates, retention plans and 
rates, missing data, definition of dropout, the intention-to-treat principle (both as a design and analysis issue), 
completeness of data, procedural adherence, quality control plus plans to address problems that may arise 
among these interrelated issues. The proposed trial affords investigators an opportunity to revise and 
standardize the recruitment and retention plans that will be helpful for future trials and cohorts. 
  
7.5.4 Recruitment, Retention and an Intent-To-Treat Design: The clinical investigators and the DCC will 
work together to establish successful strategies for patient recruitment and patient retention.  Screening and 
recruitment reports will be generated monthly that include actual and expected recruitment statistics.  
Strategies to address potential problems with recruitment include every two-week telephone calls to each 
subject to address treatment/intervention adherence, medical record review of previous compliance with study 
visits, and patient education during the informed consent process about the scientific relevance of their data 
even if they discontinue the intervention, and the deleterious effect that missing data has on study integrity and 
credibility. Wording to this effect has been successfully added to informed consent forms to enable patients to 
make more informed decisions about their willingness to join a trial and to participate in continued follow-up 
without feeling inappropriately pressured to do so 39. 
 
Plans to minimize missed visits/assessments and minimize dropout will be an important focus during study 
design. Randomization alone is not sufficient to provide unbiased comparisons of interventions, but one 
sufficient condition to provide an unbiased comparison is to obtain complete data on all randomized 
participants. Therefore retention of the cohort is key to ensuring an intention-to-treat design and intention-to-
treat analysis (full compliance with treatment/intervention, no missing data and complete follow-up of all 
participants). Only intent-to-treat analyses fully preserve the validity of assumptions between intervention 
groups established by randomization. 
 
Valid reasons a patient can be off study include withdrawal of consent at any time during the study or the 
achievement of all required efficacy and safety end point information.  Withdrawal of consent will be used only 
when the patient no longer wishes to participate in the trial and no longer authorizes the investigators to make 
efforts to continue to obtain their outcome data.  Valid reasons for non-adherence to treatment include an 
inability to tolerate the intervention, toxicity, physician choice or need for other therapies.  All patients will 
continue to be followed with all scheduled outcome evaluations until the end of the study.  This strategy will 


Version 10/17/18 
allow a true all-inclusive, intent-to-treat design and analysis.  Patients will not be labeled ‘dropouts’ but as 
‘temporarily inactive’ and the patient will be welcomed back to the trial when possible.  The designation of ‘lost-
to-follow-up’ will be applied at the end of the trial to patients that remained inactive.  Monthly reports will 
summarize recruitment and retention rates, treatment adherence rates, observed and expected data on 
attendance to routine clinical visits and the degree of completeness of data capture.  The DCC will establish 
performance standards before the trial.  These standards will be reviewed and revised as needed during the 
trial to enhance the quality of trial conduct.  
 
7.5.5 Data Management Considerations: The DCC will establish processes and work-flow to create and 
maintain a high quality research database for the trial while preserving the confidentiality of all subjects’ data. It 
is difficult to implement a statistical analysis plan unless a data management plan has been followed to assure 
data quality and procedural adherence.  Quality control will be applied to each phase of data handling to 
ensure that all data are reliable and have been processed correctly.   
 
7.5.5.1 Case Report Forms (CRFs): The clinical investigators and the DCC will collaborate on the design of 
CRFs for each study.  All CRFs will be designed using Adobe InDesign.  Forms questions and lay-out will be 
optimized for ease of forms completion and ease of data entry. 
 
7.5.5.2 Design of the Data Management System: CRF data as defined and dictated by protocol will be 
collected and managed using iDataFax or web-based tools (DF Web). iDataFax is a commercially available 
electronic data capture (EDC) system used to enter, review and modify patient data, and to submit it over the 
internet to a DataFax server maintained at the Rollins School of Public Health at Emory University. iDataFax 
provides secure 128 bit SSL encryption of all data and document transmissions over the internet.  DataFax is 
fully 21 CFR Part 11 compliant and its use has been fully validated to FDA standards.  The DCC has used 
iDataFax since 1998 for several NIH funded multicenter clinical trials and multicenter cohort studies. The EDC 
system has extensive data management features including a data query system to help ensure study 
credibility.  Beyond EDC, DataFax allows data worksheets, source documents, and other participant records to 
be scanned and securely attached to any data record.  
 
Data queries are reduced through the use of DataFax’s powerful edit check features. Extensive data checks, 
both within and across forms, will be built into the computerized data capture system developed for the trial. 
Data are checked by comparing values of a field to predefined specifications (for example, minimum and 
maximum values or specific codes) and by programs written in an internal C-like language that allow for 
intricate comparisons among multiple fields on the same or on different CRFs. System generated queries 
requesting correction or clarification of values can be responded to on-line or by faxing/ emailing a corrected 
version of the CRF. The system provides a mechanism for defining the prescribed patient visit schedule so that 
queries indicating overdue CRFs are automatically generated.  DataFax has a module for exporting CRF data 
by creating Statistical Analysis System (SAS) datasets. Department of Biostatistics personnel have developed 
S-Plus functions that produce the code for making tables and graphs in LaTeX, a mark-up language commonly 
used in mathematical fields for document processing. This set of tools are used by the DCC for generating 
automated DSMB reports. 
 
7.5.5.3 Data Acquisition Plans and Reports:  Data entry will be distributed and the study database will be 
centralized. For the proposed studies, personnel at the clinical center will enter patient data from the screening, 
baseline, and follow-up visits into eCRFs on the server at the DCC via the internet. The iDataFax system will 
generate regular reports (overall and by clinical center) that summarize and track routine data collection.  
These reports will help the investigative team monitor and maintain data completeness during follow-up and 
achieve high data capture performance standards by minimizing missed clinical visits, preventing missing data 
and maintaining high cohort retention rates. Monthly reports will be generated for clinical sites on the status of 
patient screening and enrollment and on the status of data queries issued by the DCC. These reports will be 
available to appropriate study personnel on a website developed and maintained by the DCC. 
 
7.5.5.4 Study Material Access:  The DCC will maintain a web dashboard. The investigators will access study-
related documents through this dashboard including CRFs, the Protocol, meeting minutes, monthly data 
reports, enrollment and recruitment curves, presentations and publications. 


Version 10/17/18 
 
7.5.5.5 Quality Control 
 
Quality control (QC) is the responsibility of all study personnel. QC covers all phases of the studies from 
developing good data collection instruments, to training of clinical center staff, and monitoring adherence to 
study protocols, procedures and progress of the studies. The quality control process includes data entry plus 
all data is reviewed for completeness and accuracy. 
 
7.5.5.6 Training: The DCC will train clinical personnel in study procedures for data collection. Training will 
include providing instruction on completion of CRFs, use of the iDataFax system for data entry, data quality 
control via QC reports generated by iDataFax, and use of the web dashboard. 
 
7.5.6 Policies and Methods for Ensuring Blinding of Study Results: Only the IDS pharmacy will know 
which study drug was given to the subject. The study team, parents, subjects, individuals entering the data, 
statistician, individuals performing laboratory tests and DXA scans, research center nurses and investigators 
will remain blinded as to which treatment the subjects received.  Each subject will be assigned a unique 
identifier, which will be used in the database. In addition, all lab specimens will only include the patient’s unique 
identifier. Permuted block sizes will not be disclosed to the blinded study personnel to minimize the likelihood 
of their being able to predict the next randomization assignment in the series. Investigators and all study 
personnel will remain blinded until the final subject has completed her final visit and primary and secondary 
endpoints have been analyzed.   
 
8. INCIDENTAL FINDINGS 
 
Subjects will be receiving a DXA scan at screening/entry and again at 12 months for research purposes 
only. The scans will not be used for clinical or diagnostic purposes.  If a potential subject is screened and found 
to have a T-score ≤ - 2.5, in the lumbar spine, femoral neck or total hip as measured by DXA, the individual will 
be provided with a referral to an endocrinologist and/or primary care physician and will have an option to have 
the scan sent directly to the physician of choice for review. The DXA procedure generates a report that 
includes bone mineral density of the spine and hip. For enrolled study participants, they will be given their 12-
month DXA report, and they will be encouraged to share this information with their healthcare providers in 
making a diagnosis about the state of her bones. For enrolled subjects found to have a 12-month T-score ≤ - 
2.5, in the lumbar spine, femoral neck or total hip as measured by DXA, the individual will be provided with a 
referral to an endocrinologist and/or primary care physician and will have an option to have the scan sent 
directly to the physician of choice for review. Study participants may receive a copy of DEXA results on their 12 
month study visit. They will be reminded that the DEXA scan is for research only, and participants will be 
encouraged to share the results with their healthcare provider.  
 
9. COMPENSATION 
Participants will be paid $25 for a screening visit and $50 for each additional study visit, for a total of up to 
$175 for full completion of the study, in the form of cash. Participants will be paid upon the completion of each 
visit. Participants will only be paid for visits in which they participated. There will be no compensation or 
reimbursement for travel, parking or other expenses. 
 
10. PROTOCOL BIBLIOGRAPHY 
 
1. 
Khosla S, Shane E. A Crisis in the Treatment of Osteoporosis. J Bone Miner Res. 2016;31(8):1485-1487  
2. 
Jha S, Wang Z, Laucis N, Bhattacharyya T. Trends in Media Reports, Oral Bisphosphonate 
Prescriptions, and Hip Fractures 1996-2012: An Ecological Analysis. J Bone Miner Res. 
2015;30(12):2179-2187  
3. 
Kim SC, Kim DH, Mogun H, Eddings W, Polinski JM, Franklin JM, Solomon DH. Impact of the U.S. 
Food and Drug Administration's Safety-Related Announcements on the Use of Bisphosphonates After 
Hip Fracture. J Bone Miner Res. 2016;31(8):1536-1540 PMCID:PMCPMC5040596 


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4. 
Chiang SS, Pan TM. Antiosteoporotic effects of Lactobacillus -fermented soy skim milk on bone 
mineral density and the microstructure of femoral bone in ovariectomized mice. Journal of agricultural 
and food chemistry. 2011;59(14):7734-7742  
5. 
Ohlsson C, Engdahl C, Fak F, Andersson A, Windahl SH, Farman HH, Moverare-Skrtic S, Islander U, 
Sjogren K. Probiotics protect mice from ovariectomy-induced cortical bone loss. PloS one. 
2014;9(3):e92368 PMCID:PMC3956931 
6. 
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