Clinical trial NCT01857310
Conditions: Pregnancy Live Birth Spontaneous Abortion
Keywords: Folic Acid Zinc Semen In vitro fertilization Assisted reproductive technology Ovulation induction Intrauterine insemination Pregnancy Live Birth Abortion, spontaneous
Sponsor: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)
Start date: 2013-06-01; Completion date: 2019-06-01
Enrollment: 2370 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The overarching goal of this trial is to determine if an intervention comprising folic acid and zinc dietary supplementation improves semen quality and indirectly fertility outcomes (i.e., live birth rate) among couples trying to conceive and seeking assisted reproduction. The following study objectives underlie successful attainment of the overarching research goal:

1. To estimate the effect of folic acid and zinc dietary supplementation on semen quality parameters, including but not limited to concentration, motility, morphology, and sperm DNA integrity, relative to the placebo group.
2. To estimate the effect of folic acid and zinc dietary supplementation on fertility treatment outcomes \[fertilization, embryo quality, implantation/human Chorionic Gonadotropin (hCG) confirmed pregnancy, clinical pregnancy, live birth\], relative to the placebo group.
3. To estimate the association between semen quality parameters, sperm DNA integrity and fertility treatment outcomes (fertilization, embryo quality, clinical pregnancy, live birth) and to identify the best combination of semen quality parameters for prediction of clinical pregnancy and live birth.
4. To estimate the effect of folic acid and zinc dietary supplementation on fertilization rates among couples undergoing assisted reproductive technology procedures, relative to the placebo group.
5. To estimate the effect of folic acid and zinc dietary supplementation on embryonic quality among couples undergoing assisted reproductive technology procedures, relative to the placebo group.

Detailed description:
Two micronutrients fundamental to the process of spermatogenesis, folic acid (folate) and zinc, are of particular interest for fertility as they are of low cost and wide availability. Though the evidence has been inconsistent, small randomized trials and observational studies show that folate and zinc have biologically plausible effects on spermatogenesis and improved semen parameters. These results support the potential benefits of folate on spermatogenesis and suggest that dietary supplementation with folate and zinc may help maintain and improve semen quality, and perhaps, fertility rates.

The Epidemiology Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development intends to conduct a multi-site double-blind, randomized controlled clinical trial to evaluate the effect of folic acid and zinc dietary supplementation on semen quality and conception rates among male partners of couples seeking assisted reproduction. Randomization will be stratified (with random sequences of block sizes) by site and assisted reproduction technique (IVF, non-IVF receiving fertility treatment at a study site, and non-IVF receiving fertility treatment at a nonstudy site) to ensure that balance between the treatment groups is maintained within site and within fertility treatment type over the enrollment period.

The study is designed with a sample size of 2,400 randomized participants based on obtaining adequate power to detect meaningful differences in the live birth rate between cohorts. Since the comparison of sperm parameters are differences between continuous assay measurements, this sample size will be more than sufficient for the primary sperm parameter comparisons. Additionally, calculations were done to demonstrate adequate statistical power when stratified analysis is to be performed (i.e., sample size distributions among the strata and their corresponding live birth RRs detected at 80% statistical power, with an alpha level of 0.05 and a total sample size of 2400 couples divided among the folic acid/zinc and placebo arms of the trial).

Data collection will include screening male and female partners for eligibility, administering baseline questionnaires, and collecting biospecimens in both partners of the couple, body measurements for both partners, daily journal reporting for male partners, medical record abstraction related to required treatment and outcome data, and semen quality of four samples collected at baseline, two, four, and six months following study enrollment. A data coordinating center (DCC) will support the trial.

The primary analysis plan is based on an "intention-to-treat" (ITT) approach comparing the two cohorts based on the randomized assignment, both overall and by treatment strata (IVF, non-IVF receiving fertility treatment at a study site, and non-IVF receiving fertility treatment at a nonstudy site).This approach will be applied to the two primary endpoints (semen parameters and live birth rate) as well as designated secondary endpoints (number of follicles, number and proportion of oocytes fertilized).

The DCC will perform periodic safety analyses and present interim reports to the Data and Safety Monitoring Board (DSMB) as requested, during the recruitment phases of the trial. It is anticipated that safety analyses will be performed every 6-12 months. The final analysis will be performed upon completion of data collection and editing in the follow-up and close-out phase of the trial. Also one full formal interim analysis is planned and the power calculations with considerations for the choice of optimal time for the analysis have been conducted.

Arms: arm 1: 5 mg folic acid and 30 mg elemental zinc, taken orally, daily for 6 months. arm 2: Matching placebo, taken orally daily for 6 months.
Interventions: intervention 1: 5 mg folic acid and 30 mg elemental zinc intervention 2: Placebo Comparator: Placebo
Intervention descriptions: intervention 1: None intervention 2: None

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
November 6, 2018 
 
Folic Acid and Zinc Supplementation Trial (FAZST) 
 
ClinicalTrials.gov Identifier: NCT01857310 
 
Study Protocol and Statistical Analysis Plan (including summary of protocol amendments) 


 
 
 
Study Project Officers 
Dr. Enrique F. Schisterman, NICHD  
Dr. Sunni L. Mumford, NICHD 
 
Site Principal Investigators 
Dr. C. Matthew Peterson, University of Utah 
Dr. Erica Johnstone, University of Utah 
Dr. Douglas Carrell, University of Utah  
Dr. Bruce Campbell, Minneapolis, Minnesota 
Dr. Traci Clemons, The Emmes Corporation 
Dr. Brad Van Voorhis, University of Iowa  
Dr. Ginny Ryan, University of Iowa 
Dr. Jared Robins, Northwestern University 
Supported by 
Eunice Kennedy Shriver National Institute of Child 
Health and Human Development (NICHD) 
Study Dietary Supplements Provided by 
UPM Pharmaceuticals, Inc. 
 
Version 11.0 
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This study is being conducted in compliance with the protocol, FDA regulations (21 CFR Parts 
50, 54, 56 and 312 when applicable), Good Clinical Practices, applicable local regulations and 
the Declaration of Helsinki. 
 
 


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INVESTIGATOR STATEMENT OF APPROVAL 
FAZST Protocol Version 11.0 
 
I have read the Protocol and agree to follow the procedures as outlined in this Protocol. 
I will not start the study until I have obtained written approval by the governing Institutional Review 
Board/Ethics Committee. I will obtain written informed consent from all study participants prior to 
conducting any study procedures. 
I understand that my electronic or handwritten signature or that of a co-investigator on a case report form 
indicates that the data contained on that form have been reviewed and accepted as accurate by the signatory. 
I agree to conduct this study in compliance with the applicable local regulations, FDA regulations as 
applicable (21 CFR 50, 54, 56, 312), Good Clinical Practices, and the Declaration of Helsinki. 
I understand and am aware of my responsibilities as an Investigator as described in the applicable Good 
Clinical Practices guidelines. 
I agree to protect the confidentiality of my patients when allowing the Sponsor of this clinical trial, their 
authorized monitors and/or relevant regulatory authorities access to my medical records for FAZST 
participants.  
 
 
 
 Principal Investigator, Printed Name 
 
 
 
 
 
 
 
 Address: 
Telephone # 
 
 
 
 
 
 Principal Investigator, Signature 
Date 
 
Upon signing, send this page (original) to the DCC for their files and keep a copy for your Regulatory Binder. 
 
FAZST Data Coordinating Center 
c/o Kayla Chaney 401 N. Washington Street, Suite 700 
Rockville, MD 20850-1785


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Glossary of Abbreviations 
AE 
Adverse Event 
AMH 
Anti-Müllerian Hormone 
ART 
Assisted Reproductive Technology 
CDC 
Centers for Disease Control and Prevention 
DCC  
Data Coordinating Center 
DFI 
DNA Fragmentation Index 
DNA 
Deoxyribonucleic Acid  
DSMB 
Data and Safety Monitoring Board 
EC 
Ethics Committee  
eCRF 
Electronic Case Report Forms  
EPL 
Early Pregnancy Loss 
ER 
Egg Retrieval 
ET 
Embryo Transfer 
FDA 
Food and Drug Administration 
FET 
Frozen Embryo Transfer 
FF 
Follicular Fluid 
GCP 
Good Clinical Practice  
hCG 
Human Chorionic Gonadotropin 
HEPT 
Hamster Egg Penetration Test 
HSG 
Hysterosalpingogram 
ICH 
International Conference on Harmonization 
ICSI 
Intracytoplasmic Sperm Injection 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
IND 
Investigational New Drug 
ITT 
Intent to Treat 
IUI 
Intrauterine Insemination 
IVF 
In Vitro Fertilization 
MOP 
Manual of Procedures 
MSM 
Marginal Structural Models 
NICHD 
Eunice Kennedy Shriver National Institute of Child Health and 
Human Development 
NIH 
National Institutes of Health 


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OHRP 
Office of Human Research Protection 
OI 
Ovulation Induction 
OPIM 
Other Potentially Infectious Materials 
ORI 
Office of Research Integrity 
OSHA 
Occupational Safety and Health Administration 
P4 
Progesterone 
PHS 
Public Health Service 
PI 
Principal Investigator 
PLB 
Probability of Live Birth 
PPE 
Personal Protection Equipment 
RCT 
Randomized Controlled Trial 
ROC 
Receiving Operating Characteristic   
RR 
Rate Ratio 
SA 
Semen Analysis 
SAE 
Serious Adverse Event  
SART 
Society for Assisted Reproductive Technologies 
SCPS 
Specimen Collection, Processing and Storage 
SCSA 
Sperm Chromatin Structure Assay 
SDI 
Sperm Deformity Index 
SGA 
Small for Gestational Age 
SOP  
Standard Operating Procedures 
TCC 
Trial Coordinating Center 
TZI 
Teratozoospermic Index 
US 
Ultrasound 


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Table of Contents 
1.0 STUDY DESCRIPTION AND OBJECTIVES .................................................................................................. 6 
1.1 Primary Objective .......................................................................................................................... 6 
2.0 BACKGROUND ................................................................................................................................................... 6 
2.1. Couples seeking pregnancy through assisted reproduction ............................................................ 7 
3.0 STUDY DESIGN .................................................................................................................................................. 8 
3.1. Introduction .................................................................................................................................... 8 
3.2. Eligibility Criteria .......................................................................................................................... 8 
3.3. Study Dietary Supplement Formulation ....................................................................................... 10 
4.0 OUTCOME MEASURES .................................................................................................................................. 10 
5.0 PARTICIPANT MANAGEMENT ................................................................................................................... 13 
5.1. Recruitment .................................................................................................................................. 13 
5.2. Screening Process ......................................................................................................................... 14 
5.3. Baseline Study Visit ..................................................................................................................... 15 
5.4. Randomization ............................................................................................................................. 15 
5.5. Active Follow-up.......................................................................................................................... 15 
5.6. Female Partner Follow-up ............................................................................................................ 16 
5.7. Specimen Collection .................................................................................................................... 16 
5.8. Retention Plan and Compensation ............................................................................................... 19 
6.0 STUDY TIMELINE ........................................................................................................................................... 20 
7.0 STATISTICAL ANALYSIS .............................................................................................................................. 23 
7.1. Overview ...................................................................................................................................... 23 
7.2. Study Endpoints and ITT Analysis .............................................................................................. 23 
7.3. Sample Size and Power Determinations ...................................................................................... 25 
7.4. Interim Analysis ........................................................................................................................... 27 
7.5. Prediction Models and Secondary Analyses ................................................................................ 28 
7.6. Safety Analyses ............................................................................................................................ 29 
7.7. Evaluation of Drop Outs and Adherence ..................................................................................... 29 
7.8. Implementation of Statistical Analysis......................................................................................... 29 
8.0 DATA MANAGEMENT .................................................................................................................................... 30 
8.1. Web-based Clinical Trials ............................................................................................................ 30 
8.2. FAZST Web-based System .......................................................................................................... 30 
8.3. Questionnaires and Other Data Forms ......................................................................................... 31 
8.4. Adverse Experience Reporting ..................................................................................................... 32 
8.5. Confidentiality Procedures ........................................................................................................... 36 
8.6. Quality Control Procedures .......................................................................................................... 36 
8.7. Reports and Newsletter ................................................................................................................ 36 
8.8. Investigational Product ................................................................................................................. 36 
9.0 HUMAN SUBJECTS PROTECTION AND CONFIDENTIALITY PROCEDURES ................................. 37 
9.1. Regulatory Binders ....................................................................................................................... 39 
9.2. Retention of Records .................................................................................................................... 39 
9.3. Risks and Benefits ........................................................................................................................ 39 
9.4. Study Population .......................................................................................................................... 43 
9.5. Confidentiality Procedures ........................................................................................................... 43 
10.0 APPENDICES .................................................................................................................................................... 44 
11.0 REFERENCES ................................................................................................................................................... 47 
 
 
 


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PROTOCOL SYNOPSIS 
 
Study Title 
Folic Acid and Zinc Supplementation Trial (FAZST). 
Objectives 
To evaluate the effects of folic acid and zinc dietary supplementation in males on semen quality 
parameters, and indirectly on fertility and pregnancy outcomes among couples attempting to 
conceive and seeking assisted reproduction. 
Study Design 
This is a multi-center, double-blind, block randomized placebo-controlled trial which randomizes 
men of couples attempting to conceive and seeking assisted reproduction either to study dietary 
supplements or to placebo to assess the effects of folic acid and zinc dietary supplementation on 
semen quality and fertility outcomes.  The trial is sponsored by the Eunice Kennedy Shriver 
National Institute of Child Health and Human Development (NICHD). 
Cohorts and 
Sample Size 
A total of 2,400 male participants will be randomized equally (1:1) either to active study dietary 
supplements (5 mg folic acid and 30 mg elemental zinc daily) or to matching placebo. Only the 
males will be randomized to study dietary supplement or placebo. Randomization will be stratified 
by site and assisted reproduction technique (IVF, non-IVF/receiving fertility treatment at a study 
site, non-IVF/receiving fertility treatment at a non-study site). 
Supplement 
Duration 
6 months 
Participant 
Selection 
Male participants will be recruited from couples seeking assisted reproduction.  Additional 
inclusion/exclusion criteria are described in Section 3.2. 
Primary 
Hypothesis 
Folic acid and zinc dietary supplementation will maintain and improve normal semen quality 
parameters and increase live birth rate. 
Study 
Endpoints 
Primary 
a) Semen quality – Standardized quantification of volume, concentration, motility, 
morphology, sperm count, and deoxyribonucleic acid (DNA) fragmentation index. 
b) Live birth – based on hospital records. 
Secondary 
c) Human Chorionic Gonadotropin (hCG) detected pregnancy (implantation) – a 
quantitative hCG evaluation in serum > 5 mIU/mL.  
d) Clinical intrauterine pregnancy – visualized gestational sac in the uterus on ultrasound. 
e) Ectopic pregnancy – either visualization of no gestational sac in the uterus with a 
suspicious mass in the adnexa on ultrasound, an hCG level more than 1500 mIU/mL 
without visualization of an intrauterine gestational sac on ultrasound, or a slowly rising or 
plateauing serum hCG level without visualization of an intrauterine gestation on 
ultrasound. 
f) Early pregnancy loss (EPL) – hCG pregnancy loss will be defined as a serum hCG > 5 
mIU/mL followed by a decline. Clinically recognized pregnancy losses will be identified 
by visualization of an intrauterine gestational sac followed by a loss prior to 20 weeks 
gestation. 
g) Specific pregnancy outcomes – including, but not limited to Cesarean section, pre-
eclampsia,  gestational diabetes, growth restriction, gestational age, preterm birth, birth 
weight (small for gestational age), and major neonatal complications including death, and 
severe post-partum maternal morbidity. Outcomes will be determined based on hospital 
records and medical chart abstraction. 
h) Early embryonic development parameters  – Fertilization rates, method of fertilization, 
number of cells and embryo morphology on day 3 and day 5, number of good quality 
embryos on day 5, proportion of good quality embryos on day 5, number of embryos 
transferred, quality of embryos transferred, number of embryos cryopreserved, and  


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Study Title 
Folic Acid and Zinc Supplementation Trial (FAZST). 
 
sperm penetration assay results. When available, information regarding the chromosomal 
complement of embryo will be assessed. 
i) 
Reproductive hormones and other measured biomarkers – Measured urinary, serum, and 
salivary concentrations of reproductive hormones, particularly androgens, proteomic 
analysis of human sperm and cardiometabolic risk factors and markers of oxidative stress, 
as well as measures of trace elements in toenails. 
Plans for Data 
Analysis 
The primary analysis will be an intent to treat (ITT) analysis based on the total cohort of randomized 
participants. This approach will be applied to the primary endpoints (semen parameters, live births) 
as well as designated secondary endpoints (i.e., early pregnancy loss, specific pregnancy outcomes 
and early embryonic development parameters). The difference between the measures of semen 
quality parameters, including concentration, motility, and morphology, and sperm DNA 
fragmentation for the two study cohorts will be examined by two-sided t-tests. The common null 
hypothesis states that the effect of folic acid/zinc on the outcomes is null compared to the placebo. 
Multivariate techniques will also be employed to consider the effects of folic acid/zinc on overall 
semen quality. The semen parameters will also be assessed using analysis of covariance 
(ANCOVA) models with each semen parameter as the dependent variable, clinical center, IVF 
strata and treatment group as factors.  The outcome of live birth will also be assessed using a 
Mantel-Haenszel analysis to evaluate effects across fertility treatment strata. 
The secondary objectives with binary endpoints will be analyzed using the standard Z-score (using 
the normal approximation to the binomial distribution). For binary outcomes such as pregnancy 
loss, the time to event may be of interest. In this case, survival analysis methods will be applied 
with the log-rank test for comparison of the two study cohorts. A stratified log-rank test will be 
used to assess treatment effects across IVF strata. For continuous outcomes such as birth weight, 
either parametric or non-parametric methods will be used as appropriate. If transformations to 
approximate a normal distribution are feasible, they will be carried out prior to the parametric 
analysis. The normality of the data will be examined both graphically using Q-Q plots and by formal 
tests (Shapiro Wilks test).  For data (either on original or transformed scale) assumed to be normally 
distributed, the comparison of the two cohorts will be carried out by a t-test. Alternatively, if a non-
parametric test is required, the Wilcoxon-Mann-Whitney test will be used. Stratified analyses will 
also be performed for secondary outcomes. Specifically, continuous secondary outcomes stratified 
analyses will be conducted using ANCOVA models with IVF strata and clinical center as factors 
for the comparison of the two cohorts.  If a non-parametric test is required, van Elteren tests will 
be used to compare the two cohorts, accounting for strata, to detect differences in the distributions 
of the secondary endpoints. 
 
The trial is sponsored by the Eunice Kennedy Shriver National Institute of Child Health and Human 
Development (NICHD). The designated NICHD Study Project Officers for FAZST are Drs. 
Enrique Schisterman and Sunni Mumford. Drs. Schisterman and Mumford will be responsible for:  
• General design and conduct of the trial and preparation of the essential study documents, 
including the protocol, Manual of Procedures (MOP), and data collection forms  
• Obtaining necessary regulatory approvals including, if applicable, an Investigational New 
Drug (IND) application, Office of Management and Budget Clinical Exemption, and an 
Institutional Review Board (IRB) Reliance Agreement 
• Monitoring and overseeing the contractors’ performance 
• Reviewing data collection practices and procedures  


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• Reviewing and implementing any recommendations from the Data and Safety Monitoring 
Board (DSMB) 
• Changing study procedures as appropriate  
• Appointing to, and disbanding of, study implementation subcommittees  
• Reviewing study progress in achieving goals and taking necessary steps to enhance the 
likelihood of achieving those goals  
• Overseeing all study-related analyses and publications  
 
Drs. Schisterman and Mumford will have no contact with study participants nor will they have 
access to any personal identifying information. 
Drs. Matthew Peterson and Erica Johnstone are the University of Utah (Salt Lake City, UT) 
Principal Investigators. The co-Principal Investigators at the University of Iowa are Dr. Ginny 
Ryan and Dr. Brad Van Voorhis. The Principal Investigator at Northwestern University is Dr. 
Jared Robins. Dr. Bruce Campbell is the Principal Investigator at the Center for Reproductive 
Medicine (Minneapolis, MN). They each are responsible for the following: 
• Ensuring the study is conducted according to the protocol and MOP  
• Identifying, recruiting, screening, enrolling, and retaining participants  
• Protecting participants’ rights  
• Updating the IRB regarding changes in the study protocol and MOP 
• Obtaining signed informed consent from each participant  
• Collecting study data per protocol, and following participants through study completion, 
including on-site semen analysis and collection and storage of biospecimens  
• Distributing the study dietary supplements to participants and assuring safety and integrity 
of the dietary supplements when stored at the study sites 
• Retaining specific records (e.g., dietary supplement storage and distribution records, local 
participant information, etc.)  
• Preparing and sending required reports to the DCC (e.g., recruitment, adverse event reports, 
documentation of IRB review and approval)  
• Providing the DCC with de-identified data from the clinical questionnaire and SART 
databases for FAZST enrolled couples 
• Communicating questions, concerns, and/or observations to the NICHD Project Officer 
and/or DCC 
 
Dr. Traci Clemons is the Principal Investigator for the DCC at The EMMES Corporation. The 
roles and responsibilities of the DCC include:  
• Developing and maintaining  the study website and web-based data management system  
• Developing and amending the study protocol and MOP  
• Developing the randomization scheme and procedures as well as blinding/unblinding 
procedures 
• Developing and implementing  data management procedures, including data entry, edits, 
and error reporting  
• Coordinating and managing data collection, processing, and tracking (data and 
biospecimens), and data quality control for the duration of the study 
• Monitoring and reporting adverse events 


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• Communicating with clinical sites: scheduling of meetings and training sessions, 
responding to, and documenting ad hoc communications  
• Conducting site visits to ensure adherence to the protocol and procedures  
• Establishing and implementing quality control procedures  
• Developing periodic reports: enrollment, participant status, study progress, adverse events, 
participant status (e.g., drop-outs), etc.  
• Obtaining and distributing dietary supplements to the clinical sites  
• Creating statistical analysis and presentation of the study data  
• Providing resulting datasets and documentation to NICHD 
• Organizing DSMB meetings: preparing reports for DSMB review and preparing summary 
reports of DSMB recommendations  
 
 


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1.0  
STUDY DESCRIPTION AND OBJECTIVES  
This is a multi-center, double-blinded, block randomized placebo-controlled clinical trial to assess 
the effects of folic acid and zinc dietary supplementation in males on semen quality, and fertility 
rates among couples trying to conceive and seeking assisted reproduction.    
 
1.1 Primary Objective 
The primary objective of the study is to assess the impact of folic acid and zinc dietary 
supplementation in males on semen quality and indirectly fertility outcomes (i.e., live birth rate) 
among couples attempting to conceive and seeking assisted reproduction.  A double-blind, 
placebo-controlled block randomized trial of folic acid and zinc dietary supplementation will be 
conducted. Following a screening/randomization visit, participants will return at 2, 4, and 6 
months. The rate of adverse events will be noted throughout the double-blind trial.  
 
2.0  
BACKGROUND 
Two micronutrients fundamental to the process of spermatogenesis, folic acid (folate) and zinc, 
are of particular interest for fertility as they are of low cost and wide availability.  Though the 
evidence has been inconsistent, small randomized trials and observational studies show that folate 
and zinc have biologically plausible effects on spermatogenesis and improved semen parameters.  
These results support the potential benefits of folate on spermatogenesis and suggest that dietary 
supplementation with folate and zinc may help maintain and improve semen quality, and perhaps, 
fertility rates.  Healthy, “fertile” couples attempting to conceive face a normal success rate of only 
15-20% per month. Couples who attempt conception for more than one year without achieving 
pregnancy are thought to have low fertility and are likely to be ideal candidates for the study of 
the effect of dietary supplementation on semen parameters and fertility rate.  It is estimated that 
10 to 15% of couples fall under this category.  
Folate and zinc are of particular interest with respect to spermatogenesis through a likely effect on 
sperm DNA structure. Folate is a critical micronutrient for several reasons.  Regulation of DNA 
function takes place in part by methylation, and folate provides methyl groups that are transferred 
to the N5 position of cytosine in DNA via methionine.1  Folate is involved in the methylation of 
proteins as well.  In addition, folate plays a direct role in building DNA by providing carbon atoms 
for both purine and pyrimidine synthesis. These carbon atoms provide portions of the carbon rings 
of DNA and RNA.2 Since folate is essential for DNA, transfer RNA, and protein synthesis, it is 
clear that folate is critical for germ cell development, spermatogenesis, and reproduction.3  
Zinc is essential in spermatogenesis as a co-factor for more than 80 metalloenzymes involved in 
DNA transcription, expression of steroid receptors, and protein synthesis.4, 5 It has anti-apoptotic6 
and antioxidant properties7 by binding sulphydryl groups in proteins and by occupying binding 
sites for iron and copper in lipids, proteins, and DNA.8 These two nutrients, zinc and folate, are 
dependent on one another for proper utilization. Zinc is reported to be necessary for the conversion 
of polyglutamylfolates to the monoglutamate form of folate which can be absorbed in the 
intestine.9, 10 Moreover, the increase in methionine synthetase activity observed in zinc-deficient 
animals could induce alterations in folate metabolism further promoting their synergistic 
properties.11  
The effects of these nutrients on spermatogenesis have not only biological plausibility but have 
also been reported in recent literature. In the most recently conducted randomized controlled trial 


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(RCT), 94 infertile men and 99 fertile men were divided into four groups receiving either folic 
acid (5 mg/day)/zinc sulfate (66 mg/day), folic acid/placebo, zinc sulfate/placebo, or 
placebo/placebo for 6 months.12 Only concomitant administration of folic acid and zinc led to a 
significant 74% increase in sperm density and total count in subfertile patients (insignificant 
increase in fertile men). Furthermore, in a study of 251 men of couples undergoing IVF or ICSI, 
fertile men showed a significant inverse association between seminal plasma folate and sperm 
DNA fragmentation index (DFI).13 In a study of 89 healthy men, high levels of folate intake were 
associated with lower frequencies of sperm disomies X, 21, sex nullisomy, and a lower aggregate 
measure of sperm aneuploidy, further confirming the beneficial effects of folate on 
spermatogenesis.14   
2.1.  Couples seeking pregnancy through assisted reproduction 
An ideal population to study the effect of micronutrients in an important bodily function such as 
reproduction is patients seeking to conceive through assisted reproduction. In this population, 
patients are highly motivated to follow markers of reproductive function such as ovulation and 
early pregnancy, and to report pregnancy outcomes. Patients undergoing assisted reproduction may 
have multiple semen analyses, hormonal assays, and ultrasounds that would allow collection of 
information on important steps in human reproduction such as follicular growth, exact timing of 
ovulation, timing of implantation, and early pregnancy development.15  
 
Furthermore, the effect of micronutrients on sperm DNA integrity is expected to improve the 
quality of human embryos and maintain or improve normal conception. The measurement of sperm 
DNA integrity is an advanced technique that holds promise in predicting pregnancy outcomes in 
couples undergoing assisted reproduction. Sperm DNA integrity is necessary for normal 
fertilization and transmission of paternal genetic material and significant differences in DNA 
integrity have been observed between fertile and infertile men.16-18 Pilot studies showed that high 
levels of DFI (>27%) were associated with decreased fertility in patients using assisted 
reproductive technologies (ART),19 even in men with completely normal standard semen 
parameters.20 Among the numerous available tests of sperm DNA quality, the sperm chromatin 
structure assay (SCSA), which measures the stability of sperm chromatin in acid media with 
acridine orange, is proven to be a superior measurement of DNA integrity and useful for 
epidemiologic studies.21, 22 Additional knowledge of the causes of sperm DNA fragmentation and 
decreases in sperm quality are crucial for an improved understanding of the effect of dietary 
supplementation on fertility. 
Tests of sperm DNA quality generally correlate well with other semen parameters, including 
concentration and motility,17, 23 and time-to-pregnancy.18, 24 However, the associations between 
DNA damage and pregnancy outcomes in assisted reproduction are less consistent. A recent 
literature review of work completed on this association from 1997 to present revealed little 
association between DNA integrity and fertilization rates; however, there was a strong association 
evident between DNA damage, embryo/blastocyst quality, and pregnancy rates.22 While tests of 
DNA damage have been associated with pregnancy rates, their utility as a sensitive diagnostic tool 
for pregnancy outcomes for couples seeking assisted reproduction is questionable.25 Given the 
conflicting evidence to date, large prospective studies are needed to further examine the predictive 
ability of traditional semen parameters along with DNA fragmentation on human reproduction. 
Studies looking at the role of micronutrients on early embryonic development are practically 
nonexistent because of lack of access to the early developing human embryo in vivo.  Patients 
undergoing in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) represent a 


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unique opportunity as they represent a highly motivated population that would allow access to 
descriptive information regarding early embryonic development. Possible parameters accessible 
in this population are rate of oocyte fertilization, quality of early embryo development assessed 
using the SART embryo scoring system, and ability of embryos to implant resulting in pregnancies 
and live births. Current technology will also allow the study of the chromosomal complement in 
the developing human embryo without affecting its chance of a successful implantation.26  Such 
outcomes would be of utmost importance in the study of effects of dietary supplementation with 
folic acid and zinc in males on human reproduction.   
In summary, preliminary evidence from the aforementioned studies suggests that dietary 
supplementation with folic acid and zinc may help maintain or improve semen quality, and 
perhaps, downstream fertility rates. Large prospective studies are needed to further elucidate the 
effects of folic acid/zinc dietary supplementation on semen quality, fertility rates, and early 
embryonic development parameters.  
3.0  
STUDY DESIGN 
3.1. Introduction 
This is a multi-center, double-blind, block randomized placebo-controlled trial to assess the effects 
of folic acid and zinc dietary supplementation on semen quality and reproductive outcomes among 
2400 male partners of couples attempting to conceive and seeking assisted reproduction.   
 
FAZST will be conducted according to the guidelines described in the International Conference 
on Harmonisation (ICH) E6 Industry Guidance for Good Clinical Practice (CGP). These guidelines 
can be found at the following website:  
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E6_R1/Step4/
E6_R1__Guideline.pdf     
 
3.2. Eligibility Criteria 
Couples Inclusion Criteria: 
a. Heterosexual couples in a committed relationship with a female partner aged 18-45 
years and male partner aged 18 years and older attempting to conceive and seeking 
assisted reproduction at participating fertility clinics.  
b. Couples actively trying to conceive.  
c. Couples who are planning ovulation induction (OI), natural fertility optimization 
methods, or intrauterine insemination (IUI) should be willing to be on the study 
dietary supplement for at least 3 weeks before starting the next assisted 
reproduction cycle. Women with regular periods may initiate their fertility therapy 
at the start of the woman’s menstrual cycle following randomization if 
randomization occurred within the first 10 days of the cycle, but must wait one 
menstrual cycle if the visit occurred after day 10 of the cycle). For women with 
irregular periods or amenorrhea, the male must be on the study supplement for 3 
weeks prior to initiation of any ovulation induction medication (e.g., clomid, 
letrozole, gonadotropins). 


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Couples Exclusion Criteria:  
a. Female partner unwilling to participate (e.g., no abstraction of her assisted fertility 
treatment record or unwilling to complete baseline visit). 
b. Couples using donor, cryopreserved sperm, or sperm obtained via microsurgical or 
percutaneous epididymal sperm aspiration. 
c. Couples attempting to conceive with a gestational carrier (surrogate). 
d. Positive urine pregnancy test at screening. 
Male Inclusion Criteria:  
a. Willing to provide semen samples according to the proposed schedule at baseline, 
2, 4, and 6 months of follow-up. 
b. Able to complete regular study questionnaires and daily journals aimed at capturing 
ejaculation, sexual intercourse and lifestyle factors considered to affect male 
fecundity (e.g., cigarette smoking, fever, high temperature environment and other 
environmental exposures) and other data collection instruments (e.g., physical 
activity, food frequency questionnaire, stress). 
Male Exclusion Criteria: 
a. Age <18 years. 
b. Unwilling to abstain from use of non-study approved dietary supplements or 
medications containing folic acid or oral preparations containing zinc throughout 
the study. 
c. Unwilling to abstain from use of testosterone supplementation throughout the 
study. 
d. Diagnosis of Vitamin B12 deficiency or pernicious anemia. 
e. Consuming a vegan diet. 
f. A known genetic cause of male factor subfertility, including chromosomal 
disorders related to subfertility (e.g., Y chromosome deletions).  
g. Current use of drugs known to interact with folic acid or interfere with the 
biosynthesis of folic acid including all dihydrofolate reductase inhibitors, including 
but not limited to Trimethoprim, Triamterene, Bactrim, Iclaprim,  and 
Sulfonamides including diuretics, such as Hydrochlorothiazide (HCTZ), 
Metolazone, 
Indapamide, 
Lasix, 
Bumex, 
Torsemide, 
Chlorthalidone, 
Acetazolamide, Mefruside, Xipamide; Sulfonylureas, such as Glipizide and 
Glyburide; Cox-2 inhibitors, such as Celecoxib; Probenecid;  Sulfasalazine; 
Sumatriptan; Mafenide; Acetazolamide; Ethoxzolamide; Sulfiram; Zonisamide; 
optic agents including Dorzolamide and Dichlorphenamide; Valproic acid; 
Fluorouracil; Capecitabine; Methotrexate,  or other drugs known to interact with 
folic acid. 
h. History of organ transplantation 
i. Physician diagnosed: 
i. Current poorly controlled chronic diseases such as heart disease, diabetes 
mellitus, hypertension, cancer, inflammatory diseases, autoimmune, thyroid 
disease, endocrine dysfunction, liver disease, kidney disease, or HIV/AIDS 
or other immune-insufficient related illnesses. 
ii. Crohn’s disease, celiac disease, ulcerative colitis, gastric bypass surgery, 
lap band surgery or history of intestinal surgery to remove a portion of small 


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bowel. History of diseases/symptoms that require folic acid dietary 
supplementation, such as megaloblastic anemia, homocystinemia, and 
homocystinuria.  
iii. History of alcohol dependency disorder and/or other drug/substance 
dependency in the past 180 days. 
iv. History of psychoses or other mental conditions that would result in 
cognitive impairment and inability to participate in any part of this study 
including the informed consent process, as diagnosed by a physician within 
the past year. 
j. History of vasectomy without reversal, obstructive azoospermia such as CBAVD, 
or ejaculatory duct obstruction. 
k. Known allergy to folic acid or zinc dietary supplements. 
 
Female Exclusion Criteria: 
a. Age <18 or >45 years. 
 
3.3. Study Dietary Supplement Formulation   
Male participants will be randomized (1:1) into two groups receiving one of the following: 
• 
Folic acid 5 mg plus elemental zinc 30 mg (minimum, range 30-40 mg) taken orally 
 
daily  
• 
Placebo taken orally daily  
 
Randomization will be stratified by site and assisted reproduction technique (IVF, non-IVF 
receiving fertility treatment at a study site, and non-IVF receiving fertility treatment at a non-study 
site). Folic acid plus zinc and placebo tablets will be identical in appearance and weight to maintain 
blinding. The duration of dietary supplement regimens will be six months.  For the remainder of 
the protocol, the term “Study Supplement” will be used to refer to folic acid and zinc dietary 
supplements. 
4.0  
OUTCOME MEASURES 
Semen parameters 
Four fresh semen samples, collected at baseline and after 2, 4, and 6 months of study supplement 
or placebo, will be assessed for the following parameters (outcome measures) utilizing the World 
Health Organization (WHO) semen analysis procedure 5th edition27: volume, concentration, total 
sperm count, total motile count, percent motility, percent forward motility, percent normal 
morphology, and sperm DNA integrity. For sperm DNA integrity testing, sperm chromatin structure 
assay (SCSA) and other testing will be used.16  This assay measures the ability of sperm DNA to 
resist denaturation under acidic conditions. It is widely regarded as superior sperm DNA integrity 
test for epidemiologic studies given its high repeatability, efficiency, and prognostic value for 
men’s fertility potential.28 The test yields several parameters indicative of DNA denaturation and 
DNA fragmentation found to be predictive of fertility potential.    
Early embryonic development parameters 
Fertilization and embryo transfer will be based upon standard protocols within the trial clinical 
sites.  Oocytes will be assessed 16–18 hours after insemination or microinjection to determine 


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whether fertilization occurred. Fertilization will be considered normal if two pronuclei and two 
polar bodies are identified. Oocytes without visible pronuclei will be considered unfertilized. 
Oocytes with more than two pronuclei will be considered abnormally fertilized, and will thus be 
discarded. Embryos will be scored three days after fertilization according to the size and shape of 
blastomeres and to their degree of fragmentation.29   For couples who meet criteria for blastocyst 
culture, embryos will be graded 5 days after fertilization based on Society for Assisted 
Reproductive Technologies (SART) morphology criteria. When available, information from 
preimplantation genetic screening will be collected.  
hCG recognized pregnancy (implantation) 
For couples undergoing IVF/ICSI, serum quantitative hCG level will be measured at scheduled 
clinic visits at approximately 12 days post embryo transfer for day 5 embryo transfers, and 14 days 
post embryo transfer for day 3 embryo transfers in accordance with the usual care that the woman 
is receiving.  For couples undergoing other assisted reproduction techniques, self-report of a 
positive home pregnancy test or a missed menstruation at least 14 days after ovulation will be 
followed by measurement of serum hCG at the laboratory of the patient’s choice. 
Clinically recognized pregnancy 
Clinical recognition will be determined by the presence of a gestational sac on ultrasound scans. 
For the majority of patients, this will occur at the study site.  For patients who opt out of this 
element of standard care, documentation of intrauterine gestational sac or fetal heart tones as 
assessed by US or Doppler at the initial prenatal care visit will serve to confirm clinical pregnancy.  
 
Early (biochemical) pregnancy loss (EPL) 
Pregnancy loss is a measure of impaired human fecundity and encompasses two types of losses:  
1) EPL or hCG detected pregnancy loss, and 2) clinically recognized pregnancy loss.  
Approximately one-third of all pregnancies are spontaneously aborted, of which two-thirds are 
hCG pregnancy losses.30  Serum hCG will be used to detect early pregnancy and subsequent EPL.  
EPL will be defined as a decrease in serum hCG between any 2 sequential serum hCG levels, 
followed by a decrease in hCG to <5 mIU/mL without intervention.  
Clinically recognized spontaneous pregnancy loss 
Clinically recognized pregnancy loss is defined as a pregnancy loss before 20 weeks after a 
diagnosis of clinically recognized pregnancy as determined by presence of a gestational sac on 
ultrasound scan. Clinically recognized pregnancy losses affect approximately 10-15% of women 
with recognized pregnancies.  
 
Ectopic pregnancy 
 
Ectopic pregnancy will be defined as either visualization of no gestational sac in the uterus with a 
suspicious mass in the adnexa on ultrasound, an hCG level more than 1500 mIU/mL without 
visualization of an intrauterine gestational sac on ultrasound, or a slowly rising or plateauing serum 
hCG level without visualization of an intrauterine gestation on ultrasound.  
 
Multiple gestation 
 


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Multiple gestations will be identified by ultrasound scan at the study site or at the participant’s 
prenatal visits.  
Pregnancy outcomes 
The outcome of live birth will be defined as birth of a fetus with any sign of life (e.g., pulsation of 
umbilical cord, purposeful movements). 
Stillbirth will be defined as intrauterine fetal demise occurring at or after 20 completed weeks 
gestation or as the complete expulsion or extraction from its mother, after at least 20 completed 
weeks pregnancy, of a product of conception in which, after such expulsion or extraction, there is 
no breathing, beating of the heart, pulsation of the umbilical cord, or purposeful movement of 
voluntary muscle. 
For IVF patients, the gestational age will be based on the date of embryo transfer. For all other 
patients, gestational age will be based on the LMP using the guidelines defined in the table below. 
Method of estimating gestational age 
Variability (2 
standard 
deviations) 
Days from oocyte retrieval or co-incubation in in vitro fertilization + 14 days 
±1 day 
Days from estimated ovulation in Ovulation induction + 14 days 
±3 days 
Days from artificial insemination + 14 days 
±3 days 
Days from known single sexual intercourse + 14 days 
±3 days 
Days from estimated ovulation by basal body temperature record + 14 days 
±4 days 
First-trimester physical examination 
±2 weeks 
Second-trimester physical examination 
±4 weeks 
Third-trimester physical examination 
±6 weeks 
First-trimester obstetric ultrasonography (crown-rump length) 
±8% of the 
estimate 
Second-trimester obstetric ultrasonography (head circumference, femur length) 
±8% of the 
estimate 
Third-trimester obstetric ultrasonography (head circumference, femur length) 
±8% of the 
estimate 
 
As a general rule, the official gestational age should be based on the actual beginning of the last 
menstrual period, unless any of the above methods gives an estimated date that differs more than 
the variability for the method, in which case the difference cannot probably be explained by that 
variability alone. For example, if there is a gestational age based on the beginning of the last 
menstrual period of 9.0 weeks, and a first-trimester obstetric ultrasonography gives an estimated 
gestational age of 10.0 weeks (with a 2 SD variability of ±8% of the estimate thereby giving a 
variability of ±0.8 weeks), the difference of 1.0 weeks between the tests is larger than the 2 SD 
variability of the ultrasonography estimate, indicating that the gestational age estimated by 
ultrasonography should be used as the official gestational age. (Reference: A Simple Solution to 
Dating Discrepancies: The Rule of Eights Hunter, L. A. (2009). "Issues in Pregnancy Dating: 
Revisiting the Evidence". Journal of Midwifery & Women's Health 54 (3): 184–190. 
doi:10.1016/j.jmwh.2008.11.003) 


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Small for gestational age will be defined when infant birth weight is ≤ 10% for gestational age 
(weight 
< 
10th 
percentile), 
as 
defined 
by 
the 
standards 
of 
Kramer, 
et 
al. 
(http://www.pediatrics.org/cgi/content/full/108/2/e35). 
Preterm birth will be defined as delivery prior to 37 weeks of completed gestation.  Early preterm 
(up to <34 weeks) will be distinguished from late preterm 34 up to 37 weeks.  Spontaneous preterm 
labor and delivery and preterm premature rupture of membranes will be distinguished from 
indicated preterm birth due to maternal or fetal conditions (for example, preeclampsia or small for 
gestational age (SGA) fetus.  
Fetal intolerance of labor will be defined as Cesarean delivery due to abnormal fetal heart rate 
tracing. 
Abruption or vaginal bleeding will be defined as abruption or vaginal bleeding that result in 
hospitalization, Cesarean delivery, or pre-term birth. 
 
Biospecimen analysis 
Reproductive hormones and other biomarkers will be measured from stored urine, serum, and 
saliva samples for assessment of reproductive hormone concentrations, particularly androgens, 
cardiometabolic risk factors, markers of oxidative stress, and measures of trace metals in toenails. 
A proteomic analysis of human sperm will also provide valuable information regarding potential 
mechanisms. 
5.0  
PARTICIPANT MANAGEMENT 
5.1. Recruitment  
Potential study participants will be recruited from heterosexual couples seeking fertility evaluation 
and/or assisted reproduction at fertility clinics or other participating practices providing 
reproductive care. Recruitment methods will include attempts to enroll couples of various racial 
and socioeconomic backgrounds. This recruitment will happen in the clinical setting and may also 
occur within the community such as health fairs, radio ads, brochures and flyers, standardized 
phone messages while patients are “on hold” at the study site clinics, social media, community 
health clinics, student health centers, and holistic care centers; such as fertility yoga, fertility 
acupuncture and fertility massage etc. Couples seeking assisted conception will be approached 
regarding the study by their clinician, study coordinator or member of the research staff before, 
during, or after their initial consult or clinical visit. They will be given information regarding the 
study, and the opportunity to enroll on that day or given the opportunity to schedule an enrollment 
visit at a later time.  
 
Patients attending clinic visits who request information regarding the trial will obtain information 
via phone calls, email, posted mail or meet with research staff, who will then further explain the 
study and invite them to participate if appropriate. Couples seeking assisted conception at 
participating study centers may be given a brief information form regarding the study and the 
opportunity to schedule an enrollment visit.  Whenever possible, clinic patients will be provided 
basic study information and a consent form to review in the days prior to their clinic visit. This 
basic study information will be given, explained, and questions answered via phone, mail, or email. 
When discussing the study information with interested couples, the males will be asked to abstain 
from ejaculation for 2-5 days prior to presentation for their clinic visit so that, if eligible, the male 
partner can provide a semen sample for analysis and complete enrollment as well as randomization 


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on that first visit.  Because of couple time constraints of work or home, the couple may need to 
return to clinic on another day to complete the enrollment/baseline visit, semen analysis, and 
randomization. 
 
Men presenting to the primary clinical sites for semen analysis without their partner will be 
provided with study and contact information for research staff. Research staff will contact 
potentially eligible and willing participants who have agreed to be contacted to determine 
eligibility and provide information regarding study participation. Eligible couples who agree to 
participate will be scheduled for baseline data collection. Study information materials and contact 
information will be provided to outside and community clinics allowing recruitment of patients 
seen in these clinics for New Consultations. Study site websites will provide information regarding 
study participation as well as contact information for research staff. Patients attending clinic visits 
who request information regarding the trial will be provided with contact and study information. 
The study coordinator, research staff and principal investigator (PI) may provide 
inclusion/exclusion and enrollment criteria to local physicians and clinic nurse managers and be 
available to discuss the study parameters for potential study integration into their clinics.  
Facebook, web and radio announcements can be developed as needed. 
 
Couples seeking assisted reproduction will be approached at their first clinic visit.  Potential 
participants planning to undergo a care protocol other than IVF will be counseled that they may 
participate in the study if the male partner is willing to be on study dietary supplements for at least 
3 weeks prior to initiating their assisted reproduction method.  Women with regular periods may 
initiate their therapy at the start of the woman’s menstrual cycle following the visit if 
randomization occurred within the first 10 days of the cycle, or to skip one menstrual cycle if the 
visit occurred after day 10 of the cycle). For women with irregular periods or amenorrhea, the male 
must be on the study supplement for 3 weeks prior to initiation of any ovulation induction 
medication (e.g., clomid, letrozole, gonadotropins). 
5.2. Screening Process 
Couples will be asked screening questions by study personnel, and those that meet the initial 
inclusion/exclusion criteria will be invited to participate in the study.  These screening questions 
for inclusion and exclusion criteria can take place via phone, email, or in person. Testing for 
anemia (Hemoglobin <13 gm/dL) in males will be done using a point-of-care, hemoglobin meter, 
as a preliminary screen for men who require additional evaluation to rule out B12 deficiency.  Men 
who screen positive for anemia (hemoglobin <13) will have blood drawn for a serum B12 with 
reflex to methylmalonic acid (MMA), but may still be enrolled and randomized.  They will be 
advised of their abnormal lab result, and to discuss this with their primary care provider.  Written 
informed consent will be obtained prior to the initiation of any study-specific procedures. Female 
partners will provide informed consent for the baseline collection of study-related biospecimens 
(blood and urine) and a urine pregnancy test, medical chart abstraction, study questionnaires, 
anthropometric body measurements, vital sign measurement (blood pressure and pulse rate) and 
email or online contact for study related surveys.  If a female is pregnant at the screening visit, the 
couple will be excluded from participation in the study.  During the screening procedures, both the 
female and male participants must confirm that the female partner is not currently pregnant and a 
urine pregnancy test will be completed.  If participants remain interested, they will either be 
immediately enrolled (if they meet study inclusion and abstinence criteria), or will be scheduled 
for a later enrollment visit and reminded to abstain from ejaculation for 2-5 days. Participants will 


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be contacted by phone, text, mail, or email to remind them of their study visits, and study staff will 
provide instruction on study eligibility criteria.  
For men who screen positive for anemia, results of further testing will be available within 3 
business days. If serum B12 is ≥ 300 pg/mL, no further testing is needed and the male will continue 
study participation.  If serum B12 is <300 pg/ml, MMA will be determined from the same serum 
sample.  If MMA is > 0.4 µmol/L, the study supplement will be discontinued, and the participant 
will be advised to discuss likely B12 deficiency with his primary care provider.  Other aspects of 
study participation (questionnaires, semen analysis, and biospecimens) will continue. 
5.3. Baseline Study Visit 
Baseline data collection for males and females will consist of questionnaires (i.e., diet, physical 
activity, medication, and reproductive history), and anthropometric and vital sign (blood pressure 
and pulse rate) measurements. The baseline study visit will include an additional review of 
inclusion/exclusion criteria in the event of any changes during the intervening time period. The 
baseline interview will be conducted at study centers. Urine, blood, saliva, and semen samples will 
be collected from male participants, and urine and blood samples will be collected from female 
participants. Male participants will also be given access and instructed in the completion of an 
online daily journal. This will be the only study visit for the female participants. After all baseline 
data are collected, study personnel will randomize male participants to one of the two study 
cohorts.   
5.4. Randomization 
A computer-generated randomization schedule that assigns participants to the study supplement 
and placebo cohorts will be created by the Data Coordinating Center (DCC). A total of 2,400 
participants will be randomized (1:1) to active study supplements or matching placebo.   
 
Randomization will be stratified (with random sequences of block sizes) by site and assisted 
reproduction technique (IVF; non-IVF/receiving fertility treatment at a study site; and non-IVF/ 
receiving fertility treatment at a non-study site) to ensure that balance between groups is 
maintained within these factors over the enrollment period. The two Non-IVF strata will include 
natural fertility optimization methods, OI, and IUI. Participants who meet all criteria for 
randomization will be formally randomized during the baseline visit by entering enrollment 
information into the Advantage Electronic Data Capture (AdvantageEDCSM) system. Cohort 
assignments will be generated automatically by the AdvantageEDCSM system. 
 
Subsequent to randomization, male participants will be given bottles that will have a sufficient 
number of tablets for daily intake until the next scheduled visit (two and a half months).  
Participants will be counseled on how to take study supplements and will be instructed to begin 
taking study supplements starting on the day of randomization.  
5.5. Active Follow-up 
Male participants will have scheduled follow-up visits at 2, 4, and 6 months (± 1 week). Study 
personnel will collect anthropometric measurements, urine specimens, weigh remaining 
supplements, perform venipuncture for blood collection, and collect additional biospecimens 
including semen, saliva and, when appropriate, toenail clippings. During these scheduled follow-
up visits participants will be asked about their journal use to ensure appropriate recording of 
information. Additional supplement bottles will be provided as needed at the visits (i.e., 


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participants turn in previous bottle and receive a new one at the end of the visit). Participants will 
respond to questionnaires including, but not limited to, dietary intake, physical activity, 
reproductive health, and medication use, and basic anthropometrics will be measured. Prior to the 
follow-up visits, study staff will issue reminders via text message, email or phone to reinforce 
study participation, and to remind participants to abstain from ejaculation for 2-5 days before each 
study visit. A summary of assessments at each visit is described in Table 1. 
5.6. Female Partner Follow-up 
Female partners will complete a baseline questionnaire, anthropometric and vital sign 
measurements, and provide blood and urine specimens, and then will be passively followed by 
medical record abstraction at each visit to the clinic during assisted reproduction cycles for a period 
of up to 9 months after study enrollment. In addition, females will be asked to complete a short 
monthly questionnaire via online, phone or email to update their pregnancy status, and 
reproduction cycles progress. Once (if during this initial 9-month period) a female partner becomes 
pregnant, pregnancy outcomes will also be followed by chart abstraction and monthly updates. If 
a female partner has one or more pregnancies, followed by a loss, all pregnancies will be followed 
through the initial 9-month period. Couples initiating an IVF cycle in the sixth or seventh month 
after randomization will continue to be followed if pregnancy occurs for pregnancy outcomes 
during the initial 9-month period. A summary of assessments at each visit is described in Table 2.  
5.7. Specimen Collection 
All biospecimens collected during the FAZST study will be initially processed and stored at the 
local trial coordinating center sites in –80°C freezers. Eventually ALL specimens collected from 
the clinical sites will be sent to the NIH Tissue Repository (Fisher Bioservices: 627 Lofstrand 
Lane, Rockville, MD 20850; 301-762-1772). All samples will be tracked by the DCC from the 
clinic to the repository.  
In general, biospecimens will be collected from male participants at each clinic visit in the 
following order: blood, urine, semen, saliva, and toenail (at one follow-up visit).  For women 
partners, blood and urine are collected, and they will be obtained only at the baseline visit.  
Blood collection 
Each male participant will have blood collected by research personnel at their study visits. In 
general, participants will be seated for several minutes prior to blood draw to allow fluid shifts to 
equilibrate. Male participants will have blood collected at each study visit. Female partners will 
have blood collected at the baseline study visit only. A total of up to 50 mL of blood will be drawn 
at each blood draw using the following tubes:    
2 X 4.5 mL Light Blue Top, citrate anticoagulant  
2 X 10 mL Red Top, no anticoagulant 
1 X 10 mL Green Top, Sodium Heparin (freeze dried), 72 USP Units 
1 X 10 mL Lavender Top, Liquid K3 EDTA, 15% additive solution 
 
Blood samples will be processed into serum, aliquoted into cryovials, and frozen for future use.   
Urine collection 


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Male participants will be asked to provide a urine sample in a collection cup at each visit.  Female 
partners will be asked to provide a urine sample only at the baseline study visit.   
Semen collection 
Men will be instructed to abstain from ejaculation for at least 2 full days and not more than 5 days 
before attending the baseline clinic visit.  Samples will be collected by masturbation into 4.5-oz. 
Specimen Containers with screw cap. Sample volume will be measured after which semen aliquots 
will be removed for determination of motility, morphology, and frozen for later DNA 
fragmentation analysis (see Outcomes Section 4.0 for specifics). If a participant is unable to 
provide an adequate sample (e.g., due to spillage, forgetting abstinence, etc.), he will be asked to 
schedule another clinic visit to provide an additional sample.   
Saliva collection 
Male participants will be asked to donate a bolus sample (1 mL) of whole saliva at each clinic 
visit. Samples will be collected using the passive drool technique.31 
Toenail collection 
Toenails will be self-collected by the male participant at the four month follow-up visits to measure 
trace metals and minerals that have been implicated in the physiology of reproduction such as 
selenium and chromium.32, 33 Selenium is an essential trace element of importance to human 
biology and health. Increasing evidence suggests that this mineral plays an important role in 
normal growth and reproduction in animals and humans. Toenail levels of these measures reflect 
long-term (on average, up to 1 year) exposures which usually cannot be captured using blood and 
urine samples.   
 
 


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Table 1: Schedule of Events: Male Participant  
 
 
Screening 
Baseline 
Month 2 
Month 4 
Month 6 
Assessments 
 
 
 
 
 
Informed Consent process 
X 
 
 
 
 
Inclusion/Exclusion criteria review  
X 
X 
 
 
 
Demographic Collection 
X 
 
 
 
 
Concomitant Medication Review 
X 
X 
X 
X 
X 
Anthropometrics (body measurements) 
X 
 
 
 
X 
Vital Signs (blood pressure and pulse) 
X 
 
 
 
 
Screening for Anemia with reflex B12 and 
MMA testing 
X 
 
 
 
 
Medical History 
 
X 
 
 
 
Lifestyle Questionnaire 
 
X 
 
 
 
Food Frequency Questionnaire 
 
X 
 
 
X 
Soy/Caffeine Questionnaire 
 
X 
 
 
X 
24-Hour Dietary Recall 
 
 
X 
X 
 
Questionnaire Baseline 
 
X 
 
 
 
Questionnaire month 2 
 
 
X 
 
 
Questionnaire month 4 
 
 
 
X 
 
Questionnaire month 6 
 
 
 
 
X 
Daily Journal 
 
X 
X 
X 
X 
Dietary Supplement Randomization 
 
X 
 
 
 
Dietary Supplement Adherence Assessment  
 
 
X 
X 
X 
Dietary Supplement Safety Questionnaire 
 
X 
X 
X 
X* 
Biospecimen Collection 
 
 
 
 
 
Semen 
 
X 
X 
X 
X 
Blood 
 
X 
X 
X 
X 
Urine 
 
X 
X 
X 
X 
Saliva 
 
X 
X 
X 
X 
Toenail 
 
 
 
X 
 
*The Dietary Supplement Safety Questionnaire will also be administered via email 7 days after dietary 
supplement discontinuation. 


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Table 2: Schedule of Events: Female Partners  
 
Assessments 
 
Screening 
Baseline Visit 
Collection and 
Instruction 
During 
Assisted 
Conception  
Pregnancy 
Follow Up 
 
Ideally screening and baseline 
visit will be on the same day 
 
 
Informed Consent Process 
X 
 
 
 
Inclusion/Exclusion Criteria Review 
X 
X 
 
 
Demographic Collection 
X 
 
 
 
Food Frequency Questionnaire 
 
X 
 
 
Soy/Caffeine Questionnaire 
 
X 
 
 
Lifestyle Questionnaire 
 
X 
 
 
Anthropometrics (body measurements) 
 
X 
 
 
Vital Signs (blood pressure and pulse) 
 
X 
 
 
Baseline Questionnaire 
 
X 
 
 
New Couples Questionnaire – Clinic Baseline 
X 
 
 
 
Monthly Questionnaire 
 
 
X 
X 
Medical Chart Abstraction 
 
X 
X 
X 
Biospecimen Collection  
 
 
 
 
Urine Pregnancy Test 
X 
 
 
 
Blood 
 
X 
 
 
Urine 
 
X 
 
 
5.8. Retention Plan and Compensation 
Retention of the research participants will be one of the most difficult tasks of this study. There 
are multiple clinical visits each requiring blood, semen, saliva, and urine collection. Timing of 
these visits is important. As such, participant burden should be minimized as much as possible.  
Retention begins at the time of recruitment, and it is essential to establish a positive rapport with 
the research participants. This includes providing information in a friendly manner, being 
responsive to their concerns and time commitments of work and home, providing potential 
participants with enough detail to understand the requirements of participation, and stressing the 
importance of the potential research findings. The importance of timing and frequency of study 
visits and sample collection need to be stressed clearly. A gender-specific participant pamphlet 
will be provided to each participant with FAQs, instructions for filling out questionnaires and 
contact information of research staff at the baseline visit.  The male participant will be provided 
with an ASA 24 Dietary Recall instruction sheet at his month 2 visit. Awareness of the participants’ 
time commitment will be integral. Participants will be compensated for their participation in the 
trial.  
 
A total compensation amount of $350 will be provided to participants who complete all of the 
required procedures at the following visits: 
1. Baseline visit $50    
2. 2nd visit $50  Participants will receive an additional $15 if 80% of the daily journals have 
been submitted since the previous visit 
3. 3rd visit $75  Participants will receive an additional $15 if 80% of the daily journals have 
been submitted since the previous visit 
4. 4th visit $125  Participants will receive an additional $20 if 80% of the daily journals have 
been submitted since the previous visit 
 


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Required for Baseline visit participant reimbursement are Baseline and Study Questionnaires in 
AdvantageEDCSM.  For subsequent visits this will also include the ASA24. Compensation checks 
will be mailed to participants after they have completed the visit and signed the compensation 
form. Participants will also be required to complete a W9 form for personal tax purposes. The 
original form will remain in their research chart. If a participant is unable or unwilling to provide 
a social security number on the W9 form, he can still be in the study if interested but cannot be 
compensated monetarily. Females will not receive reimbursement for their participation.  
  
Exit Questionnaire (Withdrawal) 
 
As in any study, participants may choose not to follow the study protocol fully until its completion. 
Even with the utmost effort to retain study participants, some drop out is expected.  To remove the 
possible bias, information on reasons for study withdrawal, non-compliance, and drop out will be 
collected whenever possible.   
 
Study Supplement Retrieval 
All attempts will be made to retain participants at any level they feel comfortable participating in 
this study. In general, study staff will continue to work with participants as long as they are 
responsive, to find the best way to retrieve the supplement bottles dispensed to each participant.  
If the participant is not responsive after three attempts, the supplement bottles will be considered 
lost.  On a case-by-case basis, the number of attempts may vary to ensure that we do not 
unnecessarily burden or disturb participants.   All forms of attempted communication to recover 
IP bottles will be documented.   If the participant cannot be contacted by email, phone, or text a 
certified letter can be sent to the participant requesting the return of the dispensed supplement by 
whichever method is most convenient for the participant:  
• Staff may offer to pick up the bottle from physician’s office or participant’s home 
• Staff may send a stamped, self-addressed envelope to the participant if the current 
address is known. 
When the dispensed bottle is determined to be lost or thrown away, this will be documented in 
AdvantageEDC, on the last completed visit Supplement Accountability (ACT) Form.  
6.0  
STUDY TIMELINE 
The following figures display an overview of the study design and timelines and data collection 
scheduled throughout FAZST. Male participants will complete study visits at baseline and 2, 4, 
and 6 months of follow-up. Both questionnaires and biospecimens will be collected at each study 
visit. The duration of study supplementation or placebo will be 6 months. Female partners will 
complete a study visit at baseline and then be passively followed throughout the 6 months that the 
male partners are on the study supplement and an additional 3 months after supplement 
discontinuation. If she becomes pregnant during the 9 months of her follow up, her pregnancy and 
delivery will be followed and data collected via medical chart abstraction tools. Information 
regarding assisted reproduction methods and medications from female partners will be abstracted 
from medical records.   


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The typical timelines for men and women undergoing IVF assisted conception are shown in Figure 
1 (female timeline is shown on the top in red, male timeline is shown on the bottom in blue). After 
the initial consultation, women may have a saline sonohysterogram and/or an ultrasound (US) 
completed.  Within 1 to 4 months, the first IVF assisted conception cycle is initiated. During the 
IVF cycle she will undergo 2 to 5 US and blood samples to assess uterine lining, follicular 
development and maturity. Egg retrieval (ER) is done and 3 or 5 days later embryo transfer (ET) 
is completed.  For the men, a SA is completed, along with a hamster egg penetration test (HEPT).  
Male partners collect a fresh semen sample on the day of ER.  Depending on the pregnancy 
outcome, an additional frozen embryo transfer (FET) or fresh cycle may be initiated within the 
following months.  During the six months of study follow-up it is anticipated that couples may 
complete between 1 and 3 IVF assisted reproduction cycles. 
Figure 1. Recruitment and subsequent clinic (non-study related) visits for new IVF 
consults 
 
 
The typical timelines for men and women initiating therapy with IUI are shown in Figure 2 (female 
timeline is shown on the top in red, male timeline is shown on the bottom in blue).  After the initial 
consultation women usually undergo an US, hysterosalpingogram (HSG), or saline 
sonohysterogram. Once IUI cycles are initiated they can be done for several months in a row as 
required. If unsuccessful after approximately 3 to 4 IUI assisted conception cycles, the patients 
may then undergo an IVF consultation. The men undergo a SA after the initial consultation and 
then are usually present at each IUI assisted conception. It is anticipated that couples may undergo 
up to 4 IUI cycles over the 6-month course of the study protocol. 


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Figure 2. Recruitment and subsequent clinic (non-study related) for patients initiating 
therapy with IUI 
 
 
 
 
The typical timelines for men and women undergoing OI are shown in Figure 3 (female timeline 
is shown on the top in red, male timeline is shown on the bottom in blue). After the initial 
consultation women usually undergo an US, HSG, or saline sonohysterogram. OI cycles are then 
followed-up by US and/or estrogen and progesterone (P4) measurements. If unsuccessful after 
approximately three OI assisted conception, the women may then undergo an additional 
consultation for further assisted conception methods. The men undergo a SA after the initial 
consultation. It is anticipated that couples may undergo up to three OI cycles over the 6-month 
course of the study protocol. 
 
Figure 3. Recruitment and subsequent clinic (non-study related) for patients undergoing 
OI 
 
 
 


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7.0  
STATISTICAL ANALYSIS 
7.1. Overview  
The FAZST trial is designed as a multi-center double-blinded block randomized controlled clinical 
trial with two cohorts: folic acid and zinc compared to placebo. The primary objectives are to 
compare the cohorts on four quantitative assessments of sperm quality (concentration, motility, 
morphology, and DFI) and live births in their partners. The study is designed with a sample size 
of 2,400 randomized participants based on obtaining adequate power to detect meaningful 
differences in the live birth rate between cohorts.  Since the comparison of sperm parameters are 
differences between continuous assay measurements, this sample size will be more than sufficient 
for the primary sperm parameter comparisons.   
The primary analysis plan is based on an “intention-to-treat” (ITT) approach comparing the two 
cohorts based on the randomized assignment, ignoring later changes in dietary supplement 
consumption. This analysis is described in Section 7.2. The DCC will perform periodic safety 
analyses and present interim reports to the Data and Safety Monitoring Board (DSMB) as 
requested, during the recruitment phases of the trial. It is anticipated that safety analyses will be 
performed every 6-12 months. The final analysis will be performed upon completion of data 
collection and editing in the follow-up and close-out phase of the trial. Also, one full formal interim 
analysis is planned and the power calculations with considerations for the choice of optimal time 
for the analysis are given in Section 7.3.  
7.2. Study Endpoints and ITT Analysis  
 
Study endpoints: operational definition and verification  
Study endpoints will be determined based on laboratory evaluation including standardized SA, 
clinical pregnancy tests, and medical record evaluations (including routine pregnancy follow-up, 
ultrasound). Whenever possible, endpoints will be verified using different sources of information. 
For example, self-reports of pregnancy loss will be ascertained by medical records (in case of a 
clinically recognized pregnancy) or by blood.  
Operational definitions for study outcome events are detailed in Section 4.0. The sources of 
information for study endpoints ascertainment are described below:  
Primary 
a) Semen quality – Standardized quantification of volume, concentration, motility, 
morphology, sperm count, and DFI fragmentation. 
b) Live birth – based on hospital records 
Secondary 
c) hCG detected pregnancy (implantation) – a quantitative hCG evaluation in serum > 5 
mIU/mL.  
d) Early pregnancy loss (EPL) – hCG pregnancy loss will be defined as a serum hCG > 5 
mIU/mL followed by a decline. Clinically recognized pregnancy losses will be identified 
by visualization of an intrauterine gestational sac followed by a loss prior to 20 weeks 
gestation. 
e) Clinical intrauterine pregnancy – visualized gestational sac in the uterus on ultrasound. 


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f) Ectopic pregnancy – either visualization of no gestational sac in the uterus with a 
suspicious mass in the adnexa on ultrasound, an hCG level more than 1500 mIU/mL 
without visualization of an intrauterine gestational sac on ultrasound, or a slowly rising 
or plateauing serum hCG level without visualization of an intrauterine gestation on 
ultrasound. 
g) Specific pregnancy outcomes -  including but not limited to  Cesarean section, 
preeclampsia, gestational diabetes, growth restriction, gestational age, preterm birth, 
birth weight (small for gestational age), major neonatal complications including death, 
and severe post-partum maternal morbidity will be determined based on hospital records 
and medical chart abstraction.  
h) Early embryonic development parameters - Proportion of ICSI, proportion of MII 
oocytes fertilized, number of cells and embryo morphology on day 3 and day 5, number 
of good quality embryos on day 5, proportion of good quality embryos on day 5, number 
of embryos transferred, quality of embryos transferred, number of embryos 
cryopreserved, sperm penetration assay results. 
i) Reproductive hormones and other measured biomarkers – Measured urinary, serum, and 
salivary concentrations of reproductive hormones, particularly androgens, proteomic 
analysis of human sperm and cardiometabolic risk factors and markers of oxidative 
stress, as well as measures of trace elements in toenails. 
All events will be reviewed and verified by a central outcome review that will be performed by 
the NICHD Project Officers and the Principal Investigator at the study clinical sites and the DCC.  
ITT analysis  
The primary analysis of the clinical trial will be ITT analysis based on the total cohort of 
randomized participants. This approach will be applied to the two primary endpoints (semen 
parameters and live birth rate) as well as designated secondary endpoints (number of follicles, 
number and proportion of oocytes fertilized). The difference between the measures of semen 
quality parameters, including concentration, motility, and morphology, and sperm DNA 
fragmentation for the two cohorts will be examined by two-sided t-tests. The common null 
hypothesis states that the effect of folic acid/zinc on the outcomes is null compared to the placebo. 
Multivariate techniques will also be employed to consider the effects of folic acid/zinc on overall 
semen “quality”. Specifically, a permutation test based on the sum of t-statistics across the four 
quality measurements will be conducted. The frequency of live births across cohorts using the 
standard Z-score (using the normal approximation to the binomial distribution) will be compared. 
Both of the primary analyses will be done with two-sided tests conducted at the 0.05 level.  The 
semen parameters will also be assessed using analysis of covariance (ANCOVA) models with each 
semen parameter as the dependent variable, clinical center, IVF strata and treatment group as 
factors.  The outcome of live birth will be assessed using a Mantel-Haenszel analysis to evaluate 
treatment effect across IVF strata. 
The secondary objectives with binary endpoints will be analyzed using the standard Z-score. For 
binary outcomes such as live births, the time to event may be of interest. In this case, survival 
analysis methods will be applied with the log-rank test for comparison of the two study cohorts.  
A stratified log-rank test will be used to assess treatment effect across IVF strata. For continuous 
outcomes such as birthweight either parametric or non-parametric methods will be used as 
appropriate. If transformations to approximate a normal distribution are feasible, they will be 
carried out prior to the parametric analysis. The normality of the data will be examined both 


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graphically using Q-Q plots and by formal tests (Wilks-Shapiro test). For data (either on original 
or transformed scale) assumed to be normally distributed, the comparison of the two cohorts will 
be carried out by a t-test. Alternatively if a non-parametric test is required, the Wilcoxon-Mann-
Whitney test will be used. For continuous secondary outcomes stratified analyses will be 
conducted using ANCOVA models with IVF strata and clinical center as factors for the 
comparison of the two cohorts.  If a non-parametric test is required, van Elteren tests will be used 
to compare the two cohorts, accounting for strata, to detect differences in the distributions of the 
secondary endpoints. 
7.3. Sample Size and Power Determinations 
Table 3 presents results of power calculations for FAZST. An alpha level of 0.05 and a sample 
size of 2,400 couples divided equally to the folic acid/zinc and placebo arms of the trial are 
assumed throughout the trial. Previously published studies suggest that for the control group, a 
probability of about 0.60 - 0.70 for live birth (PLB) can be expected, assuming up to three cycles 
of assisted conception.  
In the following, a range of probabilities for the placebo arm of 0.60, 0.63 and 0.65 is considered 
along with effect sizes of 1.05, 1.10 and 1.15. The effect of the intervention will be measured as 
the rate ratio (RR) of live birth in the folic acid/zinc arm compared to placebo. The calculations 
were performed using R statistical software based on a two-sample test of populations. Table 3 
presents the resulting power with no interim reviews.   
Table 3. Power for comparing live birth rates in the two cohorts with no interim looks 
based on an alpha level of 0.05 and a sample size of 2,400.  
PLB in placebo arm 
Live Birth RR to be Detected 
1.05 
1.10 
1.15 
0.60 
0.33 
0.86 
0.99 
0.63 
0.36 
0.90 
0.99 
0.65 
0.39 
0.93 
1.00 
 
The FAZST study is comprised of three strata (couples receiving IVF, couples receiving non-IVF 
at a FAZST site, couples receiving non-IVF at a non-FAZST site), which are block randomized. 
The power and sample size calculations for the analysis of the overall trial are presented 
previously.  The following calculations are to demonstrate adequate statistical power when 
stratified analysis is to be performed. In Table 4, we provide the results in sample size distributions 
among the strata and their corresponding live birth RRs that can be detected at 80% statistical 
power, with an alpha level of 0.05 and a total sample size of 2400 couples divided among the folic 
acid/zinc and placebo arms of the trial. These calculations assume that the cumulative live birth 
rate in the placebo arm for the IVF stratum is 0.63 and 0.50 for the placebo arms of the other two 
strata. For example, when the IVF stratum is expected to enroll 1000 participants and the two non-
IVF strata is each expected to enroll 700 participants, the detectable effect sizes in live birth RR 
are 1.13, 1.21, and 1.21, respectively. The lower effect size in the IVF stratum than that in the non-
IVF strata is consistent with clinical knowledge in that enhanced live birth rates attributable to 
folic acid/zinc supplementation is likely to be greater in the non-IVF strata assuming it is generally 
more fecund than the IVF strata and the IVF treatments are highly effective leaving less room for 
improvement. When multiple comparisons are corrected, we see slight elevation of effect sizes 


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(1.15, 1.24, and 1.24, respectively) that can be detected at the same 80% statistical power and with 
the same sample sizes.  
Table 4. Stratified power analysis for comparing live birth rate in treatment and placebo 
arms with no interim looks based on a total sample size of 2400, 80% statistical power, and 
an alpha level of 0.05. The three strata are:  A-IVF stratum, B-non-IVF FAZST site stratum 
and C-non-IVF non-FAZST site stratum. The PLB in placebo in these strata are 0.63, 0.50 
and 0.50 respectively. 
n per arm 
RR 
RR 
adjusting for 
multiple comparisons 
A 
B 
C 
A 
B 
C 
A 
B 
C 
300 
450 
450 
1.17 
1.19 
1.19 
1.19 
1.21 
1.21 
400 
400 
400 
1.15 
1.20 
1.20 
1.17 
1.23 
1.23 
500 
350 
350 
1.13 
1.21 
1.21 
1.15 
1.24 
1.24 
600 
300 
300 
1.12 
1.23 
1.23 
1.14 
1.26 
1.26 
 
While three strata have been conservatively block randomized by design, the Non-IVF FAZST 
and Non-IVF non-FAZST site are anticipated to be similar populations and could potentially be 
collapsed during analysis. The positive power and sample size of this collapsing of strata are 
displayed in Table 5. The gain of statistical power in the non-IVF strata allows us to shift some 
enrollment to the IVF stratum which is believed to have a smaller effect size to detect. For example, 
with a 700 and 500 sample sizes per arm for the IVF and non-IVF strata respectively, we are able 
to detect a live birth RR of 1.11 and 1.18, respectively. When multiple comparisons are corrected, 
the detectable live effect sizes are 1.12 and 1.19 respectively. 
If the strata are deemed collapsible while recruitment is still active then a re-alignment of sample 
size goals will allow for the detection of smaller effect sizes in the live birth RR, in the IVF stratum. 
This can be achieved by periodically assessing the distribution of characteristics of enrolled 
participants between non-IVF FAZST stratum and non-IVF non-FAZST stratum. On the other 
hand, if the strata are deemed collapsible after recruitment is completed, the sample size of the 
Non-IVF strata will obviously double resulting in a substantial reduction in effect estimates 
detectable with 80% statistical power and an alpha level of 0.05.  
 
 


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Table 5. Stratified power analysis for comparing live birth rate in treatment and placebo 
arms with no interim looks based on a total sample size of 2400, 80% statistical power, and 
an alpha level of 0.05, when the two non-IVF strata are collapsed. The two strata are:  A-
IVF stratum, B-non-stratum. The PLB in placebo in these strata are 0.63 and 0.50 
respectively. 
n per arm 
RR 
RR 
adjusting for 
multiple comparisons 
A 
B 
A 
B 
A 
B 
300 
900 
1.17 
1.13 
1.18 
1.14 
400 
800 
1.15 
1.14 
1.16 
1.15 
500 
700 
1.13 
1.15 
1.15 
1.16 
600 
600 
1.12 
1.16 
1.13 
1.18 
700 
500 
1.11 
1.18 
1.12 
1.19 
800 
400 
1.10 
1.20 
1.11 
1.22 
 
7.4. Interim Analysis  
For a multi-year trial such as this one it is useful to examine the data as it accumulates in order to 
see if the study should be ended early or to catch any surprising developments.  One of the most 
widely used procedures for interim analysis of a clinical trial is the alpha spending function 
approach of Lan and DeMets.49 This method uses a function of time to specify the rate at which 
the total Type I error probability will be spent during the trial interim reviews so that this quantity 
does not exceed the desired overall alpha level. For FAZST, a two-stage interim analysis plan will 
be implemented. The following group sequential two-stage test plan describes how alpha (α) will 
be spent across the interim and final analyses of the data.   
The interim analyses will be performed based first on the sequential interim monitoring of the 
semen quality parameters (sperm concentration; total sperm motility; and sperm morphology 
(abnormal form)) after 50% of the FAZST participants have completed their six months follow-
up.  The analyses will be unadjusted by any covariates, in line with the ITT approach.  The overall 
Type I error rate will be controlled at a pre-specified level, accounting for both multiple semen 
outcomes and the interim analysis.  As the effects of the nutritional supplementation are not clearly 
understood, the study should be stopped early only if there is strong evidence of harm.  Given this 
a one-sided alpha spending function, which approximates the O’Brien-Fleming50 boundary, will 
be developed.   First, a Bonferroni adjustment will be used to distribute the (sometimes called 
experiment-wise) Type I error alpha among the multiple semen parameters.  A α=0.025 (one-
sided) is to be assigned to the experiment-wide alpha level for the assessment of semen parameters, 
where α3=α/3 = 0.0083 will be used to compute the boundary for each individual test.  For each 
semen parameter, the amount of α3 available for the interim analysis performed at time t is α3(t) 
and will be obtained from the O’Brien-Fleming spending function.  Time t for the first stage of the 
FAZST interim analysis will be once 1200 (50% of the participants) have completed six months 
of follow-up.   Using these assumptions the test statistic for lower bound of harm will be 3.5547 
for each of the 3 comparisons with α3(t) = 0.00019 where t represents 50% of the participants 
completing 6 months of follow-up.    The DSMB will consider the interim analysis results as a 
resource to evaluate the risk of study treatment.  When a stopping boundary is crossed, there will 
be an indication that the treatment has an increased risk of harm for at least one of the semen 


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parameters.  The DSMB will consider the consistency of all semen analysis data to determine 
whether the second stage of the interim analysis should be considered.   
If the DSMB determines that the second stage of the interim analysis is applicable, it will be 
conducted based on the available data and the outcome of live birth.  For this stage, the estimated 
information fraction will be defined as the total number of participants completing 6 months of 
follow-up at the time of database closure for the second stage interim analysis.   Similarly, the 
alpha spending function approach will be applied to the sequential monitoring of live birth.  An 
overall alpha of 0.05 and a one-sided procedure in data monitoring will be used.  The hypothesis 
will be tested by a one-sided O’Brien-Fleming alpha spending design.   Assuming PLB = 0.63 in 
the placebo arm and a RR of 1.100, interim test boundaries using the O’Brien-Fleming alpha 
spending design based on various values of the information fraction are provided in the table 
below. When a stopping boundary is crossed, there will be an indication that the treatment has an 
increased risk of harm for live birth.  The DSMB will consider this result and all live birth data to 
determine whether a suspension of the trial intervention should be considered.   
Table 6. Examples of Critical Values of the Test Statistics for Live Birth 
Information 
Fraction 
Lower Bound 
0.20 
4.8769 
0.25 
4.3326 
0.30 
3.9286 
0.35 
3.6128 
0.40 
3.3569 
0.50 
2.9626 
 
7.5. Prediction Models and Secondary Analyses 
In addition to the ITT analysis the data collected in FAZST will be utilized to develop prediction 
models for various endpoints for couples undergoing IVF and/or ICSI procedures. These models 
will enhance understanding of these processes and the factors that may affect outcomes. Thus, the 
study will provide important information beyond the evaluation of the effects of folic acid/zinc on 
semen quality alone. 
The prediction models approach is described here only briefly. 
Prediction of live birth 
Various prediction models can be built for this binary event considering baseline and follow-up 
information (including, though not limited to, sperm concentration, motility, morphology, sperm 
DNA fragmentation, female age, female anti-müllerian hormone (AMH) and/or antral follicular 
count, female diagnoses, female assisted conception type, the number of embryos transferred, and 
embryo quality). Three methods can be used to build a prediction model: (a) logistic regression, 
(b) neural networks, and (c) regression trees. While logistic regression is a standard technique, the 


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other two methods will allow consideration for more complicated associations between risk factors 
and outcomes (e.g., interactions and optimal cut-off points) as well as discrete outcomes with 
several possible categories (e.g., early pregnancy loss, pregnancy complications, and birth).  
Validation methods will be used to compare these approaches whereby the data will be divided 
randomly into training (e.g., 2/3 of the data) and validation sets (e.g., 1/3 of the data). The models 
will be fit using the training set and their predictive ability will be compared on the validation set. 
Receiving operating characteristic (ROC) curves will be used to compare the different models and 
determine which is  preferred.34, 35  The optimal probability threshold for the logistic and neural 
network models will be chosen using the Youden Index.36-38 
7.6. Safety Analyses 
All reported adverse events will be listed by term, frequency, severity, and cohort.  Standard 
statistical tests using normal theory will be readily applicable for assessing these data. Adverse 
events will be collected in the males receiving study supplements though important female 
associated study endpoints, including adverse pregnancy outcomes and pregnancy loss, will be 
collected throughout the study and reported as adverse events via questionnaire and medical chart 
abstraction. 
7.7. Evaluation of Drop Outs and Adherence 
The DCC will co-develop with the Project Officer statistical methods for evaluation of drop out 
and compliance for future analyses of the study results. Both drop-outs and non-compliance 
introduce a problem when the processes do not occur at random. Drop-outs potentially lead to 
selectively missing data that may bias crude ITT comparisons. Non-compliance to assigned study 
supplements can also be influenced by intermediate study outcomes and therefore introduces 
additional bias. Marginal structural models (MSM) suggested in the causal inference framework39-
41 have been used in observational studies to account for time-dependent covariates that are on the 
pathway to disease outcome or self-selection into treatment.42, 43 MSMs have also been useful in 
clinical trial for handling non-compliance to provide unbiased estimates of the causal effect of 
cohort assignment building upon the theory of causal inference and the idea of ‘potential 
outcomes’.44, 45 A key component to these models is inverse probability weighting to model the 
exposure (e.g., folic acid and zinc supplementation) with the end result providing an estimate of 
the relative risk of the outcome of interest among the actual dietary supplements compared to 
placebo, with appropriate confidence intervals and appropriately adjusting for time-dependent 
confounders affected by prior exposure. 
Compliance 
All bottles, both study supplement and placebo bottles, will be weighed as a measure of participant 
adherence at the 2-, 4-, and 6-month study visits. Participants will be asked to bring all bottles 
(both unopened and opened) and unused tablets to each study visit. The original weight of the 
bottles when distributed should be recorded on the appropriate visit checklist. The bottles will be 
weighed using a calibrated scale. The bottles should be weighed outside the room where the 
participant is located, when possible.   
7.8. Implementation of Statistical Analysis 
Descriptive data analysis will be performed by the DCC. Questions related to data analysis and 
methodological issues will be discussed periodically at staff meetings at the DCC. Interim results 
will be summarized and presented at the DSMB meetings and discussed with the Project Officer. 


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Holding regular discussions of study analysis is crucially important to ensure that study aims are 
met and that the interpretation of data is valid. Publication quality summaries of the results of the 
statistical analyses will be prepared by the DCC team for interim and final reports; presentations 
and manuscripts in conjunction with statistical investigators at NICHD. 
8.0  
DATA MANAGEMENT 
8.1. Web-based Clinical Trials  
The DCC will develop an internet-based system for the FAZST in close collaboration with the 
investigators and staff members at the TCC and clinical sites.  
 
Over the last decade increasing numbers of RCTs as well as other clinical multi-center studies are 
performed using internet-based technologies. Some of the advantages of such systems include 
minimization of errors, real-time data reporting, saving in resources for recording of the data, less 
paper and less storage space required.46, 47 
8.2. FAZST Web-based System  
Data collection  
Source Documents: Source documents are defined as original documents, data and records.  They 
may include hospital records, clinical and/or office charts, laboratory data/information, 
participants’ journals or evaluation checklists, pharmacy dispensing records, recorded data from 
automated instruments, microfiches, photographic negatives, microfilm or magnetic media and x-
rays.  Study staff will clearly define the various source documents used to support the study as part 
of their local data management support. 
 
The investigator(s)/institution(s) will permit study-related monitoring, audits, IRB/ Independent 
Ethics Committee (IEC) review, regulatory inspection(s), and will provide direct access to all 
source data documents at the time of monitoring and/or upon request by the DCC. 
 
Web-based data collection and management system 
Data collection will occur via a web-based data entry system (AdvantageEDC for study specific 
needs such as enrollment and participant ID assignment) provided by the DCC for FAZST to allow 
easy access to enrollment 24 hours a day, seven days a week. Upon enrollment in AdvantageEDC, 
a form submission schedule is generated for each participant and displayed as a grid of forms by 
study visit that permits direct access to each electronic case report form for data entry. As data are 
entered, they are validated through range and within-form consistency checks. 
 
Data storage and quality control 
Data for individual participants will be recorded on electronic case report forms (eCRF) in the 
AdvantageEDC system. All participants screened for the study, including the screen failures, must 
be entered into AdvantageEDC. Participants will be randomized in the AdvantageEDC system, 
and the eCRFs must be current to reflect participant status at each phase during the course of the 
study. Participants will not be identified on the eCRFs by name or initials; instead, once 
participants are entered into AdvantageEDC, the system will assign them unique participant 


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identification numbers. Investigators are required to keep a separate log of participant names and 
addresses.   
 
Because of the potential for errors and inaccuracies in entering data into eCRFs, laboratory and 
other test results must be kept on file with the participant’s study dossier. Study records should be 
maintained for at least three years post completion of the final study report or publication of the 
primary endpoints or per local site IRB requirements  (whichever is longer). 
 
Data processing and data management 
Clinical data processing and management will be employed based on the procedures developed in 
conjunction with the DCC. All of the data entered into the AdvantageEDC system will be checked 
for valid values and ranges, between item logical consistency, and within-participant variation. 
Prior to any analyses the distributions of the measures will be examined to aid in selecting 
appropriate statistical techniques and data transformations. Data transformations (e.g., log 
transformations) will be used in an attempt to normalize non-normally distributed data so as to still 
be able to use statistical techniques appropriate for normal data. In addition, nonparametric and 
semi-parametric techniques may be used with non-normally distributed data. 
 
Data plan for participants with incomplete data 
Participants will be included in the data analysis provided that they complete the testing procedures 
listed and have completed at least one follow-up visit, and do not receive additional interacting 
medications. The DCC will work with site staff to ensure that the study records for all participants 
who terminate early are as up to date and as possible, with field and form exceptions reviewed and 
accepted to account for all required data. 
8.3. Questionnaires and Other Data Forms  
All necessary forms and documentation material will be available online through the study website 
and can also be printed on location at the clinical sites as needed. The availability of information 
online ensures that all are working with the same most updated version  
 
Forms will be designed to be clear, concise, and convenient to use and for the web-based forms 
also include imbedded coding and initial error checking mechanisms. Data collection for the 
FAZST trial will begin with screening, continue through the baseline visit, active follow-up (with 
daily journals and biospecimens collections) for six months, and end with passive follow-up during 
pregnancy. The principal mode of data collection for FAZST will be web-based.   
 
Questionnaires  
 
 
The male study questionnaires include the baseline questionnaire, 24-hour dietary recall, food 
frequency questionnaires, and 2-, 4-, and 6-month follow-up questionnaires and daily journals. 
Refer to Table 1 for specific questionnaire content. Briefly, baseline questionnaires will elicit 
socio-demographic information, stress, medical and reproductive history and health-related habits. 
Follow-up visits and questionnaires will obtain information on adherence to and safety of study 
supplement and brief behavior and lifestyle information. Questionnaires are self-administered and 
entered into the AdvantageEDC system. All questionnaires, except the 24-hour dietary recall, will 
be programmed by the DCC and incorporated into the AdvantageEDC system. For the 24-hour 


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dietary recall, the ASA24 Automated Self-administered 24-hour Recall (v. 2011 or 2014) 
developed by the National Cancer Institute will be used.  The ASA24 is a freely available, secure, 
Web-based 
tool 
that 
enables 
self-administered 
24-hour 
recalls 
(http://riskfactor.cancer.gov/tools/instruments/asa24/). All questionnaires will be reviewed by the 
study nurse/staff to verify understanding and completion.  
 
The female study questionnaires include the baseline questionnaire, food frequency 
questionnaires, and monthly questionnaires. Refer to Table 2 for specific questionnaire content. 
Briefly, baseline questionnaires will elicit socio-demographic information, stress, medical and 
reproductive history and health-related habits. Monthly on-line follow-up questionnaires will 
obtain information on any current fertility treatment she has received and pregnancy outcomes. 
Questionnaires are self-administered and entered into the AdvantageEDC system. Questionnaires 
will be reviewed by the study nurse/staff to verify understanding and completion.  
 
Daily journals 
  
These will be completed daily via the internet, smartphone, or a telephone system by each male 
participant during the six months on study dietary supplements. These forms, eliciting information 
on dietary supplement compliance, intercourse, exposures to alcohol and stress, and possible 
adverse effects are designed to be simple and convenient to encourage response. 
 
Abstraction forms  
 
Information on the reproductive history and demographics of the female partner and the course of 
fertility treatments she may have received, pregnancy and delivery will be obtained through 
abstraction of medical records from routine pre-natal clinic visits, routine tests (e.g., blood tests, 
ultra-sound, amniocentesis) and any hospitalization, OBGYN, other primary medical doctor, or 
emergency room visits.  
 
Biospecimen tracking  
For each visit where specimens are collected, pre-printed labels that include barcodes will be 
provided and placed on tubes and containers per the Manual of Procedures (MOP). Each specimen 
sample after processing will be labeled with a barcode and dot matrix code, and all biospecimens 
will be entered and tracked in the Global Trace System, a component of the AdvantageEDC 
System.    The barcodes contain the participant identification number, the visit number and the 
type of specimen. Mapping of the repository boxes in Global Trace will allow the location of any 
specific specimen as needed. Shipment from the local repository to the central study repository 
will be documented and performed according to the Standard Operating Procedures (SOP) for the 
lab specimen and in collaboration with the central repository. The specimen tracking information 
will be saved in Global Trace which is maintained by the DCC.  
 
8.4. Adverse Experience Reporting 
Definitions of adverse event (AE) and serious adverse event (SAE) 
Standard definitions for AEs and SAEs, their identification, characterization regarding severity 
and relationship to study interventions, and processing are included in Appendix A. 


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• AE collection period and follow-up 
All AEs will be recorded from the first day the study supplement was taken through seven days 
following the last dose of the supplement. All reportable events and all suspected adverse 
reactions will be followed until resolution or medically stable.   
All AEs, either observed by the Investigator or one of his/her medical collaborators, or reported 
by the participant spontaneously, or in response to direct questioning, will be reported.  Any 
SAE regardless of severity or potential association with the study supplements must be 
documented in study records by the site Investigator and promptly reported to the DCC (within 
24 hours of learning about the event).  Non-serious adverse events can be collected in a routine 
manner using case report forms.  Adverse events will be collected in the males receiving study 
supplements.  Important female associated study endpoints, including adverse pregnancy 
outcomes and pregnancy loss, will be collected throughout the study and reported as adverse 
events via questionnaires and medical record abstraction forms. 
• Obligations of Site Investigators 
 
FAZST requires the site investigators to report all AEs, regardless of their severity or potential 
association with the study supplements. When submitting adverse event information the DCC, 
a site investigator may not delegate someone other than a listed study physician the 
responsibility for reviewing the accuracy of the contents of the adverse event report. When 
reporting an AE, the site investigator must assign a severity grade to each event and also 
declare an opinion on the relatedness of the event to the study supplements. 
 
For any SAE, the DCC must be notified within 24 hours of when the site investigator first 
learns of the occurrence of the event. Adequate information must be collected with supporting 
documentation and entered in the AdvantageEDC data collection system.  This data entry 
serves as notification of the DCC. The 24-hour reporting deadline is necessary to provide 
adequate time to investigate the report and determine if the SAE requires further reporting. The 
DCC and/or NICHD would then prepare materials to submit, if necessary, to regulatory 
authorities and IRBs within the timeframes required by regulation for expedited safety reports. 
 
For AEs considered non-serious, timely reporting is considered acceptable within seven days 
of learning about the event. 
 
• SAE Reporting Responsibilities of the Site 
 
When an SAE is identified, the site investigator (or the study coordinator) shall promptly: 
 
1. Notify the site PI (if a different person) in person or by telephone about the SAE. 
2. Use the AdvantageEDC system to prepare the available SAE information on a study 
adverse event case report form. If supplementary information is to be supplied, this may 
be attached to the case report form in AdvantageEDC.  If the data system is not available, 
use a MedWatch Form FDA 3500A and fax to the DCC.  The event must be entered into 
AdvantageEDC as soon as the system is available. 
3. The site PI (or another designated study physician) is responsible for reviewing and 
approving the report contents (including the event description, grading of event severity, 
and relatedness to study supplements). 


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4. Submit the initial report to the DCC within 24 hours of recognizing the event.  Submission 
to the local IRB is based on the site’s responsibilities per the site’s IRB. 
5. The adverse event report and each page of any attached materials must describe the study 
participant only by their coded study identifier(s). Any personally identifying information 
(e.g., name, telephone number, address, etc.) must be removed or obscured before delivery 
to the DCC.  Transmission may be done electronically via the AdvantageEDC system or 
by facsimile transmission to the DCC.  Any information that cannot be submitted via the 
AdvantageEDC system to the DCC must be sent by facsimile transmission (1-800-576-
5790). 
6. If the DCC requests additional information or if further pertinent details become available 
(e.g., laboratory reports, follow-up evaluations, discharge summaries, autopsy reports, 
etc.), promptly submit them to the DCC. 
 
• Submitting an expedited Safety Report to the Local IRB 
 
When the DCC or NICHD informs a site that expedited safety reporting to regulatory 
authorities is required, the PI should review and update any previously reported materials as 
needed.   
 
Each expedited safety report should routinely include a brief cover memorandum, the 
completed MedWatch Form FDA 3500A, and any additional pertinent information 
recommended by the DCC or the Medical Monitor. Once a report is assembled by the DCC 
and provided to each of the sites, the site PI must submit the expedited safety report to the local 
IRB within the required reporting timeframe.  Follow-up reports will be submitted when 
required or when pertinent information becomes available.  The PI must retain a complete copy 
of each expedited safety report as it was submitted to their IRB, and forward a copy of each 
report to the DCC. The DCC will be responsible for further dissemination of expedited safety 
reports as described below. 
 
• Obligations of Study Sponsor and Data Coordinating Center 
 
The NICHD, or the DCC on behalf of the NICHD, must immediately investigate each reported 
serious adverse event. Recommendations for reporting AEs for dietary supplements are 
described in the FDA “Guidance for Industry: Questions and Answers Regarding Adverse 
Event Reporting and Recordkeeping for Dietary Supplements as Required by the Dietary 
Supplement and Nonprescription Drug Consumer Protection Act” (2007) and if applicable, 
may be covered in the FDA regulations regarding drug products. FDA (and other relevant 
regulatory authorities), the DSMB and all participating investigators must be notified within 
15 days of any adverse experience that is associated with the use of study supplements and that 
is both serious and unexpected.  If the reported AE is an unexpected fatal or life-threatening 
experience associated with the use of the study supplements, the NICHD must notify the FDA 
and the DSMB as soon as possible but no later than seven calendar days after the sponsor’s 
initial receipt of the information.  
 
All SAEs must be reported to the DCC through AdvantageEDC within 24 hours of the site 
becoming aware of the event.  SAEs not requiring expedited reporting will be summarized in 
annual reports to regulatory authorities and disseminated to the DSMB, study investigators, 
and IRBs active in the study. 


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When the DCC or NICHD has determined that expedited reporting of an AE is required, the 
DCC will be responsible for performing the following procedures and for reporting the AE to 
regulatory authorities within the required timeframes: 
 
1. Notify the site that expedited reporting will be required and request any additional 
information needed to complete an appropriate report. This may include completion or 
updating the information in AdvantageEDC and the submission of supporting 
documentation, laboratory reports, discharge summaries, etc. The DCC will prepare a 
cover memorandum for reporting the event to regulatory authorities. 
2. When the cover memorandum, MedWatch Form FDA 3500A, and any pertinent 
attachments are ready, the DCC will submit a copy of the completed report, by fax or 
courier delivery before the regulatory reporting deadline, to the following persons: 
a. FDA (addressed to FDA, Center for Food Safety and Applied Nutrition, Office of 
Food Defense, Communication and Emergency Response, CAERS Team, HFS-11, 
5100 Paint Branch Parkway, College Park, MD 20740, or if applicable, to the 
designated Medical Officer for any covering IND in effect for the study);  
b.Site PIs at each active participating site (who is responsible for forwarding the 
 
report to the local IRB); 
c. NICHD Project Officers; 
d. DSMB Chair.  
3. If relevant follow-up information becomes available, the DCC will be responsible for 
obtaining the details from the site. This information will be reviewed by the Medical 
Monitor. A follow-up MedWatch form will be completed and forwarded to all parties that 
received the earlier safety report. 
4. The DCC will also transmit copies of all expedited safety reports to the designated 
individual from any cross-referenced IND sponsors, if applicable. A copy of the safety 
sections of the annual FDA reports, if applicable, will be forwarded to the NICHD and will 
be provided to the designated individual from any cross-referenced IND sponsors. 
• Terminology to Use for AE Description  
 
When reporting an AE, the event description should use the best matching terminology 
describing the event as found in the “Common Terminology Criteria for Adverse Events” 
(CTCAE, v4.0). If an available CTCAE term fits the event well, no additional descriptors may 
be needed.  However, necessary descriptions should be added in order to clarify the event or 
to place it in an appropriate context.  The AE name should ideally be 1-3 words in length with 
additional description provided elsewhere on the adverse event report. A copy of the CTCAE 
is posted on the study website.  Standardized terms from CTCAE are used the DCC and the 
study sponsor to categorize events for reporting to regulatory authorities using the “Medical 
Dictionary for Regulatory Activities” (MedDRA).  In most cases, the CTCAE terms match the 
MedDRA coding terminology.  If an appropriate term matching the AE cannot be found in the 
CTCAE and the preferred MedDRA term is unknown, the AE description should include a 
diagnosis, sign or symptom with additional information to facilitate subsequent categorizations 
into MedDRA coding terms. 


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8.5. Confidentiality Procedures  
Strict confidentiality procedures ensure that information collected for the FAZST trial could not 
be linked to the participant’s identifier except for cases where medical attention may be needed. 
Participants’ personal information with exception of date of birth will be kept locally at each 
clinical center and kept separately from the data collected during the study where only the 
Participant ID for each participant will appear. The locator information will be kept in a secured 
local server with paper forms in a locked secure cabinet/area.  
8.6. Quality Control Procedures  
The DCC will perform ongoing checks of all data entered into the database. These will be part of 
the AdvantageEDC Suite (Integrity) and will include checks for discrepancy between information 
obtained through different forms and/or at different follow-up times. These procedures are 
essential for the success of the trial. Quality control processes will be established to identify any 
deviations and discrepancies in information obtained from various sources. Routine error and 
completeness reports will be sent to the clinical center and should be returned to the DCC in a 
timely manner to resolve identified problems and correction of errors. Concise summaries of the 
findings of the quality control procedures will be presented periodically on the study web-site to 
alert study personnel of common problems.  The DCC will suggest resolutions as well as any 
modifications to the study protocol.  
 
Quality control for clinical monitoring  
The DCC with the NICHD will conduct annual field audits of the clinical sites to assure 
adherence to the study protocol and requirements. The audits will include examination of the data 
collection process, specimen collection and processing, data entry procedures, local data 
management and record keeping.  
8.7. Reports and Newsletter  
The DCC will be responsible for preparation and distribution of interim and final reports, annual 
newsletter, presentations and other publications in coordination with the Project Officers at the 
NICHD and in collaboration with the study investigators. Reports will include summaries of 
recruitment and follow-up status, by clinical site, graphical and tabular presentations and results 
of interim statistical analysis. The reports will have clear explanations of the methods used, 
interpretations and point out questions or problems that need special attention.  
 
Special reports of AEs occurring during the course of the study will be prepared periodically and 
submitted to the Project Officer and the DSMB.  
 
The annual newsletter will be made available on the study website and contain information on the 
progress and performance of the clinical centers. 
8.8. Investigational Product 
Identity of Investigational Product 
The study supplement and matching placebo is supplied by UPM Pharmaceuticals Inc. Each tablet 
of the active study supplement contains 5 mg of folic acid and 30 mg elemental zinc (as zinc sulfate 
monohydrate). Inactive ingredients include silicified microcrystalline cellulose, isomalt, fumed 
silica, croscamellose sodium, fumed silica and magnesium stearate.    The same inactive 


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ingredients are used to make the placebo tablets. All tablets are coated with hypromellose, 
polyethylene glycol, polysorbate 80 titanium dioxide for a uniform white appearance. The study 
supplement and placebo tablets are manufactured and tested to meet USP specifications and quality 
standards for nutritional and dietary supplements. 
 A sufficient supply of study supplement will be provided for each participant and will be sent to 
the study site. Study product labels will be printed with the protocol number, applicable storage 
conditions, lot number and the instruction to take one tablet each day. The label will also include 
space for participant number and visit number. 
 
Blinding and placebo tablets 
 
The AdvantageEDC system will be used for randomization and assignment of blinded study 
supplies. Matching placebo tablets will be supplied by UPM Pharmaceuticals Inc. to maintain the 
blinding of folic acid and zinc tablets and the placebo tablets.   
 
If required, emergency unblinding of the participant can be completed by authorized site staff.  
Only a physician at the site will be authorized to unblind participants in emergent situations. In 
case of an emergency, the DCC should be notified before unblinding any participant. The DCC 
will keep a copy of the randomization code in a secure place. All unblinding events should be 
recorded on a protocol violation form in EDC within 24 hours of the unblinding. 
 
Storage and Disposition of Supplies 
A specified number of bottles containing 77 tablets of 5 mg folic acid and 30 mg elemental zinc 
or matching placebo will be shipped to the PI’s designated pharmacy or other acceptable controlled 
storage location for inventory control and dispensing to study participants. All tablets will be stored 
at room temperature, in a tightly-closed container, away from heat and light, and in a locked 
storage area. Room temperature of 77°F is ideal; excursions permitted from 59° to 86°F (15 -
30°C).  A temperature log will be kept on a weekly basis.   
 
The dietary supplements and matching placebo products used in this study are designated 
specifically for and are to be used only within the context of this study under the supervision of 
the PI.  Returned study supplies may not be re-dispensed. As specified in the Manual of 
Procedures, all used and expired study dietary supplements will be destroyed in accordance with 
the site SOP on study product destruction.  Following study closeout, all other unused clinical 
supplies will be discarded.  
Supplement Accountability  
The PI, or a qualified designee, will verify that study supplies are received intact and in the correct 
amounts. An accurate inventory of study supplements will be kept by the site. Each site will be 
provided with electronic supplement accountability forms to document receipt of supplies from 
UPM Pharmaceuticals, Inc. Supplement dispensation and accountability will be verified by the 
DCC and study monitors throughout the study and at the site close out visit.   
 
 
9.0  
HUMAN SUBJECTS PROTECTION AND CONFIDENTIALITY PROCEDURES 


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This section details the issues relevant to protection of human subjects involved in the FAZST 
trial.  
 
DSMB 
This study will be overseen by a DSMB. The DSMB will receive tabulated data relating to safety 
of study participants. This evaluation will also assess data quality and timeliness, participant 
recruitment, accrual, and retention. These reviews will allow the DSMB to determine whether 
there is any change to the anticipated benefit-to-risk ratio of study participation. These reviews 
will also allow the DSMB to determine whether the study should: 1) continue as originally 
designed, 2) implement a protocol change, or 3) be terminated.  If a recommendation is made to 
change the research study design, an adequate rationale for this decision must be provided. In 
addition, members of the DSMB will review data from individual study participants on a quarterly 
basis to evaluate the progress of the study and the safety and confidentiality of study participants. 
Any SAEs or breaches in confidentiality will be reviewed by the DCC and reported immediately 
to the DSMB for review throughout the study.  
 
 
 IRB review and informed consent 
This protocol and the informed consent document and any subsequent modifications will be 
reviewed and approved by the IRB responsible for oversight of the study at each participating site. 
Four IRBs will review this protocol: 1) University of Utah, and 2) The Emmes Corporation 3) 
University of Iowa 4) Northwestern University. NICHD will be covered under a reliance 
agreement with the University of Utah.   
 
The principles of informed consent in the current edition of the Declaration of Helsinki will be 
implemented before any protocol-specific procedures are carried out. Informed consent will be 
obtained in accordance with U.S. 21 CFR Part 50.2 and all other applicable regulatory 
requirements. A signed consent form will be obtained from the participant. The consent form will 
describe the purpose of the study, the procedures to be followed, and the risks and benefits of 
participation.  A copy of the consent form will be given to the participants. Study participants will 
be informed he/she is free to refuse any aspect of study participation without repercussion or to 
withdraw at any time.    
 
All investigators will have completed the federally required training in research ethics and will 
emphasize the voluntary nature of this study. Approval from the IRB will be in place prior to 
beginning recruitment.   
 
Participant confidentiality 
Raw data will be stored in locked cabinets in a locked office. Social security numbers may be 
collected for payment purposes only but will not be submitted to a central database.  De-identified 
data will be submitted to a central database designed by the DCC. The database will be password 
protected. All laboratory specimens, evaluation forms, reports, and other records that leave the site 
will be identified only by the participant ID to maintain participant confidentiality.  All records 
will be kept in a locked file cabinet.  Clinical information will not be released without written 
permission of the participant, except as necessary for monitoring by the IRB, NICHD, or the 
NICHD’s designee. Any information uncovered through the interviews that might indicate abuse 
or neglect requires the investigators to inform appropriate local agencies in accordance with State 
law.  Otherwise, all information will remain confidential. 
 


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Statement regarding scientific misconduct 
This study will be conducted using good clinical practice (GCP), as delineated in Guidance for 
Industry: E6 Good Clinical Practice Consolidated Guidance, and according to the criteria specified 
in this study protocol. Before study initiation, the protocol and the informed consent documents 
will be reviewed and approved by an appropriate IRB. Any amendments to the protocol or to the 
consent materials must also be approved before they are implemented. 
 
Compliance with 42 CFR Part 93, Public Health Service (PHS) Policies on Scientific Misconduct 
is implicit in the application for this proposal. The academic institutions participating in this 
proposal have approved assurances and required renewals on file with the Office of Research 
Integrity (ORI) and compliance with these policies and procedures and the requirements of Part 
93 are in place. The academic institutions participating in this proposal understand and abide by 
the definitions of research misconduct per PHS policies (fabrication, falsification, or plagiarism in 
proposing, performing, or reviewing research, or in reporting research results). 
9.1. Regulatory Binders 
9.2. Retention of Records 
Each site will maintain a confidential regulatory binder that contains site-specific and study-wide 
documentation. The regulatory binder does not contain clinical records, CRFs or other source 
documents regarding individual participants. The site PI or designee is required to maintain this 
documentation and make it available for review by authorized study monitors, auditors and 
regulatory authorities.  Both current and outdated study documents must be maintained. Older 
versions of documents may be stored elsewhere in a secure location, provided a reference to the 
actual storage location remains in the binder. If the binder contents become too voluminous, 
multiple volumes may be maintained.  The regulatory binder may also be kept in electronic 
format; file structure will be provided by the DCC.   If kept electronically, the binder must be 
stored on a secure network server.    
The site PI is responsible for maintaining intact study records for a period of at least two years 
after the investigation is discontinued and the sponsor has notified any applicable regulatory 
authorities. Local policies for records retention may require longer periods, or the NICHD may 
request a longer retention period. The NICHD should inform the Investigator/Institution in writing 
when trial-related records are no longer needed. 
9.3. Risks and Benefits  
 
Procedure risks  
Folic acid/zinc supplement and placebo: Any dietary supplement (including folic acid and zinc) 
can have harmful side effects, especially when mixed with other products or if the participant is 
allergic to folic acid or zinc. Participants who are allergic to the folic acid/zinc dietary supplements 
will be ineligible to participate. Research personnel will monitor participants’ pill intake (including 
supplements and herbal remedies) through text/email reminders and the information provided in 
the daily journal. Folic acid usually has very few side effects, but symptoms reported with the use 
of folic acid include fever, general weakness or discomfort, reddened skin, shortness of breath, 
skin rash or itching, tightness in chest, trouble breathing, and wheezing.  Although many people 
who take zinc do not experience side effects, nausea, vomiting and upset stomach may occur.  Rare 


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but more severe symptoms reported with the use of zinc include fever, general weakness or 
discomfort, sore throat, reddened skin, shortness of breath, skin rash or itching, tightness in chest, 
trouble breathing, and wheezing.  Although it is possible that folic acid supplementation may mask 
some symptoms of pernicious anemia, specifically the hematological disorder, but did not improve 
the associated neuropathy.48 The neuropathy is reversible if treatment begins promptly, and the 
symptoms of anemia are not necessary to diagnose the deficiency and the brief length of study 
dietary supplement administration, 6 months, should ensure prompt treatment.    
 
Blood Sample: This procedure is likely to involve discomfort or minimal pain as well as bruising. 
Rarely, blood draws may lead to clotting, infection, and fainting. Blood is drawn by certified 
healthcare personnel in research and/or medical facilities.  
 
Anthropometric Measurements and vital signs: There are no anticipated physical risks associated 
with anthropometric measurements and the measurement of vital signs (i.e., blood pressure, pulse 
rate). However, there could be some discomfort and/or embarrassment when having the 
measurements taken. This procedure will be conducted by trained research personnel.  
 
Urine Collection: There are no known risks associated with this procedure. This is a standard test.  
 
Semen Collection: There are no known risks associated with this procedure. However, there could 
be some discomfort and/or embarrassment when providing semen samples. 
 
Saliva Collection: There are no known risks associated with this procedure.  
 
Toenail Collection: There are no known risks associated with this procedure. 
 
Interview: Men will be interviewed at baseline, coinciding with initial evaluation at the fertility 
clinic, in order to obtain information on demographics, reproductive history, occupational history, 
stress, depression, medical history, and lifestyle factors that may be related to their fertility. This 
could be uncomfortable and or embarrassing. This interview will be conducted by trained 
personnel experienced in interviewing participants.  
 
Daily journal: Recording information every day can be difficult and frustrating at times. However, 
the diary has been designed to collect information in a concise fashion to minimize the time spent 
on this component of the study. The risks associated with this procedure are minimal and include 
the accidental or incidental disclosure of sensitive personal information.  This risk is minimized 
by adhering to strict confidentiality policies (see below). 
 
Medical Records Review: The risks associated with this procedure are minimal and include the 
accidental or incidental disclosure of medical information. This risk is minimized by adhering to 
strict confidentiality policies (see below). 
 
 
Benefits to participants and others 
Couples enrolled in this study may not realize a direct benefit as a result of their participation. 
However, during the medical evaluations and interviews, information about their medical care and 
fertility enhancement may be discovered that may not have been without participating in this study. 
In this event, that information will be relayed to the couple and their referring physician so that 
management of these issues can be pursued.  


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It is possible that couples with nutritional deficiencies may have more positive fertility outcomes 
as a result of participation in this study. However, this cannot be guaranteed.  
 
Information derived from this study may potentially identify whether folic acid and zinc dietary 
supplementation may significantly impact semen quality and fertility outcomes for many couples 
seeking assisted conception in the future.  
 
 
Benefits and importance of the knowledge to be gained  
Recent literature suggests that dietary supplementation with folic acid and zinc may be beneficial 
for maintaining semen quality, and perhaps, downstream fertility and pregnancy outcomes. Folic 
acid/zinc supplementation may be an ideal dietary supplement; it is safe, widely available, has few 
side effects, and is inexpensive. The possibility that these dietary supplements could have a major 
impact on semen quality and fertility is very important to study but there are still many unknowns. 
It is not clear which participants would benefit from folic acid/zinc supplementation, whether other 
factors that can influence semen parameters add to the effects of folic acid/zinc supplementation, 
and the potential side effects of this dietary supplementation. Earlier studies have only focused on 
specific effects of folic acid/zinc supplementation on semen parameters, and not necessarily with 
downstream fertility and pregnancy outcomes. As essential micronutrients in the spermatogenesis 
process, the effects of folic acid/zinc supplementation are expected to have an impact not only 
semen quality, but also may impact fertilization rates, embryo quality, implantation, and thus, 
increasing chance of conception. 
 
The potential risks to the participants in this study are relatively low and are reasonable in relation 
to the importance of the knowledge gained. Procedures outlined in this protocol such as semen, 
blood and urine collection, anthropometric measurements, and interviews have minimal risk and 
it is not anticipated that the risks are above those in the course of a person’s daily life.  
 
 
Alternative procedures  
Men and women may choose not to participate in this study and continue attempts at conceiving a 
pregnancy on their own or after consultation with a physician.  
 
Protection of Human Subjects from Research Risks  
 
 
DCC responsibilities  
The DCC responsibilities to protect human subjects from research risk will focus on the 
information aspects. The potential risks involved in this study include clinical ones (e.g., adverse 
allergic reaction to the folic acid/zinc supplement), emotional ones (e.g., increased anxiety, 
embarrassment due to questions related to sexual behavior) and risks to privacy. The clinical sites, 
along with the DCC, will help develop a clear informed consent document to make sure that 
participants understand the study and know the risks involved, however minimal. Participants’ 
personal information, test results and other data will be kept confidential throughout the trial and 
stored in secured data archives. Personal information will be kept in a locked file cabinet with 
limited access to specific study staff. Interviews (personal and by telephone) and specimen 
collection will be conducted in privacy. Personal information is never released without prior 
permission from participants. Locator information is stored separately from the data identified by 


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participants study ID number. Linking documents are kept secure by authorized staff members. 
Procedures to release information in case of an adverse event will be established in coordination 
with the Project Officers and the DSMB.  
 
All DCC personnel will complete human subject protection training. Any harm suffered by a 
participant, either physical or emotional, or alleged infringement on privacy will be documented 
and reported to the IRB and to the DSMB.  
 
 
NICHD responsibilities 
The NICHD responsibilities to protect human subjects from research risk will focus on maintaining 
confidentiality for study participants by having access to no personally identifiable information.  
The NICHD will have no contact with study participants. All data that the NICHD will obtain will 
be de-identified. All information collected in the study will be kept strictly confidential and be 
used solely for research purposes. A Certificate of Confidentiality will be obtained. 
 
All NICHD personnel will complete human subject protection training. Any harm suffered by a 
participant, either physical or emotional, or alleged infringement on privacy will be documented 
and reported to the IRB and to the DSMB.  
 
The Division of Intramural Population Health Research, NICHD, is committed to reproducible 
research and fully supports the NIH data sharing (including biospecimens) policy.  The Division 
has a protocol for requesting such information that was publicly posted in the Federal Register and 
approved by the NICHD.  In short, potential collaborators submit a concept protocol using a 
standardized methodology that undergoes peer review. If found acceptable and contingent upon 
having the resources for the research, the Division prepares an analytic file and supporting 
biospecimens for distribution to the collaborating institution once IRB approval is in place at the 
destination. All information is de-identified, and may undergo additional security measures for 
ensuring confidentiality of data and participants’ privacy, depending upon the research question. 
Every effort is afforded for the continued protection of study participants, their data and 
biospecimens during all aspects of research and beyond. There is no expiration of our commitment 
to full human subjects’ protection through all phases of research including follow on studies 
relying on the biospecimen repository. 
 
 
TCC responsibilities 
The University of Utah’s responsibilities to protect human subjects from research risk will focus 
on ensuring the informed consent, safety of participants, and confidentiality of data collection 
processes for its and subcontracted clinical sites. 
  
All University of Utah personnel and personnel from subcontracted sites will complete human 
subject protection training from CITI or an equivalent documented training program. Any harm 
suffered by a participant, either physical or emotional, or alleged infringement on privacy will be 
documented and reported to the IRB and to the DSMB.  


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9.4. Study Population  
 
Men and minorities 
Couples attempting to conceive and seeking assisted conception at participating fertility clinics 
will be recruited for participation this study. No efforts will be made to exclude any men based on 
minority, racial, or ethnic status.  
 
Participants will be requested to complete a brief questionnaire at the time of informed consent 
that identifies ethnicity and racial designation(s). The participant will be asked for ethnicity first 
and then will be able to identify one or more racial designation.  
The trial is potentially open to all male partners of heterosexual couples attempting to conceive 
and seeking assisted conception at participating fertility clinics who fit the inclusion/exclusion 
criteria. The DCC will work with the clinical sites to identify potential sources of data to 
characterize as best as possible the race/ethnicity composition of the population to be screened for 
participation in the trial. Typically, trial populations do not accurately represent the target 
population. However, effort will be made to direct the screening to allow a heterogeneous group 
to be included. The DCC will generate monthly summaries to each site with the age and ethnic 
composition of the population screened and the group recruited. If an imbalance develops, the 
issue will be discussed with the clinical sites and with the project officer to come up with necessary 
changes in recruitment strategies to minimize exclusion of minorities for any reason. No sub-group 
analysis is planned but statistical modeling of the data will assess the impact of age and 
race/ethnicity on study endpoints.  
 
Special populations and circumstances  
The study will not be recruiting and enrolling special classes or participants such as children, 
prisoners, institutionalized individuals, or others who are likely to be vulnerable populations. This 
study does not include plans to include special conditions such as the use of recombinant DNA 
molecules, human embryonic germ cells, or human embryonic stem cells.  
 
 
Pregnant women, fetuses, placenta  
Women are excluded from enrolling in the study as partners if they are already pregnant. However, 
since this study is focused on improving the outcome of reproductive health, it will be necessary 
to follow women if they become pregnant during the course of the study. All stipulations outlined 
in 45 CFR 46.204 (Research Involving Pregnant Women and 45 CFR 46.206 (Research Involving, 
After Delivery, the Placenta, the Dead Fetus, or Fetal Material) will be followed to maintain 
research integrity and protection to the participants.  
 
 
Exclusion if unable to consent  
Although this is identified in the inclusion/exclusion above, it is important to reiterate that anyone 
who does not have the ability to provide effective informed consent will not be enrolled.  
9.5. Confidentiality Procedures  
To ensure the strict confidentiality of participants’ information and data, the following procedures 
will be followed:  
 
a) 
Participant IDs – upon recruitment, each participant will be assigned a study 
identification number (Participant ID). This ID will be used by project staff on all 
data forms, laboratory specimens and in the study’s main databases. In this manner, 


Folic Acid and Zinc Supplementation Trial Protocol 
 
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FAZST Version 11.0  
 
44 
 
 
the identity of the participant will not be linked directly to the data collected during 
the study without a locator file. The assignment of ID numbers will be done by the 
DCC using random components that will make the sequence difficult to predict.  
b)  
A separate locator file will have the identifying information for each participant 
(including name and address) linked to the Participant ID number. This file will be 
kept in a secure place and will only be available to the site personnel, not the DCC 
or sponsor.  
c)  
Reports generated based on the collected data will not include any identifying 
information and thus preserve the confidentiality of the participants.  
d)  
The only occasion where participant identity may be linked to the collected data is 
in case of an adverse event that requires medical intervention; these cases will be 
discussed with the Project Officer on a case-by-case basis.  
e)  
The need to ensure strict confidentiality of the data collected from participants will 
be stressed as part of the training of the study personnel involved in data collection. 
The importance of preserving confidentiality as a moral duty of researchers will be 
explained. The data may include sensitive information about sexual history and 
contacts and other personal data such as medical history. The willingness of 
participants to give such information is based on the trust that this data will be used 
for the research purposes only. Therefore, it is essential that study staff understand 
this and protects the data collected not only through the study protocols but also 
information gathered through incidental observations of study participants.  
f)  
The data collected during the study will be presented in an aggregated form as totals 
(means, medians, etc.) or percentages and will not include specific individual 
results. 
g)  
Access to the database will be limited to authorized study staff and will be protected 
with a changing password.  
 
The DCC will not have any street-level locator information in the study database. All locator 
information will be kept at the local clinical sites. The participant’s ID number and identity will 
be kept in locked files in the research center of the study PIs. Only key staff and investigators will 
have access to the records. In order to monitor this research study, authorized representatives from 
the clinical sites’ IRBs, and other federal agencies such as NIH (National Institutes of Health), 
FDA (Food and Drug Administration) and OHRP (Office of Human Research Protection), and 
qualified monitors from the DCC may inspect the research records. A Certificate of Confidentiality 
(#CC-HD-13-19) has been issued by the federal government that provides further confidentiality 
protection by authorizing study staff and contractors to protect the privacy of participants in this 
study.  
 
10.0 
APPENDICES 
Appendix A: Safety Monitoring 
Definitions 
An adverse event (AE) is any untoward medical occurrence in humans, whether or not considered 
study supplement related which occurs during the conduct of a clinical trial. Any change in clinical 


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FAZST Version 11.0  
 
45 
 
 
status, ECGs, routine labs, x-rays, physical examinations, etc., that is considered clinically 
significant by the study investigator is considered an adverse event.    
Suspected adverse reaction is any adverse event for which there is a reasonable possibility that 
the study supplement caused the adverse event. A reasonable possibility implies that there is 
evidence that the study supplement caused the event. 
Adverse reaction is any adverse event caused by the study supplement.  
Serious Events (Serious Adverse Events, Serious Suspected Adverse Reactions or Serious 
Adverse Reactions)  
A serious adverse event or serious suspected adverse reaction or serious adverse reaction as 
determined by the DSMB is any event that results in any of the following outcomes: 
1. Death 
2. Life-threatening AE (Life-threatening means that the study participant was, in the  
  opinion of the investigator or sponsor, at immediate risk of death from the reaction as  
  it occurred.)  
3. Inpatient hospitalization or prolongation of existing hospitalization  
4. Persistent or significant incapacity or substantial disruption of the ability to conduct 
 
normal life functions  
5. Congenital abnormality or birth defect 
6. Important medical event that may not result in one of the above outcomes, but may 
 
jeopardize the health of the study participant or require medical or surgical 
 
intervention to prevent one of the outcomes listed in the above definition of serious 
 
event.  
 
Unexpected Adverse Event 
Any adverse event, the specificity or severity of which is not consistent with the symptoms 
reported with the use of the study supplement or the informed consent. 
AEs that can be associated with folic acid supplementation: 
• Fever  
• Weakness 
• Reddened skin 
• Shortness of breath 
• Skin rash 
• Itching 
 
AEs that can be associated with zinc supplementation: 
• Nausea 
• Vomiting 
• Upset stomach 
• Fever  


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• Weakness 
• Sore throat 
• Reddened skin 
• Shortness of breath 
• Skin rash 
• Itching 
• Tightness in chest 
• Trouble breathing 
• Wheezing 
 
Guidelines for assessing intensity of an adverse event 
The Investigator should use the following definitions when assessing intensity of an AE: 
• Mild: Transient (< 48 hours) or mild discomforts, no or minimal medical therapy or 
intervention required, hospitalization not necessary, no or little limitation in normal 
activities, nonprescription or single-use prescription therapy may be employed to relieve 
symptoms (e.g., aspirin for simple headache, acetaminophen with codeine for post-surgical 
pain).  Mild adverse events may be listed as expected consequences of the therapy for any 
given protocol, and standard supportive measures for such an expected event do not 
necessarily elevate the event to a higher intensity. 
• Moderate: Mild to moderate limitation in activity, some assistance may be needed; possibly 
none but usually minimal intervention/therapy required, hospitalization possible.  
• Severe: Marked limitation in activity, some assistance usually required; medical 
intervention/therapy required; hospitalization possible or likely. 
• Life-Threatening: Extreme limitation in activity, significant and immediate assistance 
required; significant medical/therapy intervention required to prevent loss of life; 
hospitalization, emergency treatment or hospice care probable. This grade is used when the 
participant was, in the view of the Investigator, at substantial risk of dying at the time of 
the adverse event, or it was suspected that use or continued use of the study supplements 
would have resulted in the participant’s death.  (This does not include a reaction that, had 
it occurred in a more serious form, might have caused death.  For example, drug-induced 
hepatitis that resolved without evidence of hepatic failure would not be considered life-
threatening even though drug-induced hepatitis can be fatal). 
 
 
Guidelines for determining causality of an adverse event 
The Investigator will use the following question when assessing causality of an adverse event to 
study supplement:  Is there a reasonable possibility that the study supplement caused the event? 
Reasonable possibility implies that there is evidence that the specific adverse event observed in 
the specific subject is caused by the study supplement. 
 
An affirmative answer designates the event as a suspected adverse reaction. 
 
 


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11.0 
REFERENCES 
 
(1)  Wagner C. Biochemical Role of Folate in Cellular Metabolism. In: Bailey LB, editor. Folate in 
Health and Disease.New York: Marcel Dekker; 1995. p. 23-42. 
 
(2)  Brody T. Folate in Nutritional Biochemistry. San Diego: Academic Press; 1999. 
 
(3)  Forges T, Monnier-Barbarino P, Alberto JM, Gueant-Rodriguez RM, Daval JL, Gueant JL. Impact 
of folate and homocysteine metabolism on human reproductive health. Hum Reprod Update 2007 
May;13(3):225-38. 
 
(4)  Hamdi SA, Nassif OI, Ardawi MS. Effect of marginal or severe dietary zinc deficiency on testicular 
development and functions of the rat. Arch Androl 1997 May;38(3):243-53. 
 
(5)  Favier AE. The role of zinc in reproduction. Hormonal mechanisms. Biol Trace Elem Res 1992 
January;32:363-82. 
 
(6)  Chimienti F, Aouffen M, Favier A, Seve M. Zinc homeostasis-regulating proteins: new drug targets 
for triggering cell fate. Curr Drug Targets 2003 May;4(4):323-38. 
 
(7)  Zago MP, Oteiza PI. The antioxidant properties of zinc: interactions with iron and antioxidants. 
Free Radic Biol Med 2001 July 15;31(2):266-74. 
 
(8)  Bray TM, Bettger WJ. The physiological role of zinc as an antioxidant. Free Radic Biol Med 
1990;8(3):281-91. 
 
(9)  Quinn PB, Cremin FM, O'Sullivan VR, Hewedi FM, Bond RJ. The influence of dietary folate 
supplementation on the incidence of teratogenesis in zinc-deficient rats. Br J Nutr 1990 
July;64(1):233-43. 
 (10)  Favier M, Faure P, Roussel AM, Coudray C, Blache D, Favier A. Zinc deficiency and dietary folate 
metabolism in pregnant rats. J Trace Elem Electrolytes Health Dis 1993 March;7(1):19-24. 
 (11)  Tamura T, Kaiser LL, Watson JE, Halsted CH, Hurley LS, Stokstad EL. Increased methionine 
synthetase activity in zinc-deficient rat liver. Arch Biochem Biophys 1987 July;256(1):311-6. 
 (12)  Wong WY, Merkus HM, Thomas CM, Menkveld R, Zielhuis GA, Steegers-Theunissen RP. Effects 
of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-
controlled trial. Fertil Steril 2002 March;77(3):491-8. 
 (13)  Boxmeer JC, Smit M, Utomo E et al. Low folate in seminal plasma is associated with increased 
sperm DNA damage. Fertil Steril 2008 August 21. 
 (14)  Vujkovic M, de Vries JH, Dohle GR et al. Associations between dietary patterns and semen quality 
in men undergoing IVF/ICSI treatment. Hum Reprod 2009 February 19. 
 (15)  Merviel P, Heraud MH, Grenier N, Lourdel E, Sanguinet P, Copin H. Predictive factors for 
pregnancy after intrauterine insemination (IUI): an analysis of 1038 cycles and a review of the 
literature. Fertil Steril 2010 January;93(1):79-88. 
 (16)  Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin 
heterogeneity to fertility. Science 1980 December 5;210(4474):1131-3. 


Folic Acid and Zinc Supplementation Trial Protocol 
 
November 6, 2018 
FAZST Version 11.0  
 
48 
 
 
 (17)  Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human 
spermatozoa: relationships with semen quality. J Androl 2000 January;21(1):33-44. 
 (18)  Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage 
impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril 2000 
January;73(1):43-50. 
 (19)  Larson KL, DeJonge CJ, Barnes AM, Jost LK, Evenson DP. Sperm chromatin structure assay 
parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum 
Reprod 2000 August;15(8):1717-22. 
 (20)  Saleh RA, Agarwal A, Nelson DR et al. Increased sperm nuclear DNA damage in 
normozoospermic infertile men: a prospective study. Fertil Steril 2002 August;78(2):313-8. 
 (21)  Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in 
vitro fertilization? Fertil Steril 2008 April;89(4):823-31. 
 (22)  Tarozzi N, Bizzaro D, Flamigni C, Borini A. Clinical relevance of sperm DNA damage in assisted 
reproduction. Reprod Biomed Online 2007 June;14(6):746-57. 
 (23)  Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA 
integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril 2001 
April;75(4):674-7. 
 (24)  Evenson DP, Jost LK, Marshall D et al. Utility of the sperm chromatin structure assay as a 
diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999 April;14(4):1039-
49. 
 (25)  Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in 
vitro fertilization? Fertil Steril 2008 April;89(4):823-31. 
 (26)  Rubio C, Rodrigo L, Mir P et al. Use of array comparative genomic hybridization (array-CGH) for 
embryo assessment: clinical results. Fertil Steril 2013 March 15;99(4):1044-8. 
 (27)  World Health Organization. WHO laboratory manual for the Examination and processing of 
human semen. 5th Edition ed. Switzerland: 2010. 
 (28)  Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting 
sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 2002 
January;23(1):25-43. 
 (29)  Veeck LL. Oocyte assessment and biological performance. Ann N Y Acad Sci 1988;541:259-
74.:259-74. 
 (30)  Wilcox AJ, Weinberg CR, O'Connor JF et al. Incidence of early loss of pregnancy. N Engl J Med 
1988 July 28;319(4):189-94. 
 (31)  Granger DA, Kivlighan KT, El-Sheikh M, Gordis EB, Stroud LR. Salivary alpha-amylase in 
biobehavioral research: recent developments and applications. Ann N Y Acad Sci 2007 
March;1098:122-44. 
 (32)  Mistry HD, Broughton Pipkin F, Redman CWG, Poston L. Selenium in reproductive health. 
American Journal of Obstetrics and Gynecology 2012 January;206(1):21-30. 


Folic Acid and Zinc Supplementation Trial Protocol 
 
November 6, 2018 
FAZST Version 11.0  
 
49 
 
 
 (33)  Apostoli P, Catalani S. Metal ions affecting reproduction and development. Met Ions Life Sci 
2011;8:263-303. 
 (34)  Eftekhar B, Mohammad K, Ardebili HE, Ghodsi M, Ketabchi E. Comparison of artificial neural 
network and logistic regression models for prediction of mortality in head trauma based on initial 
clinical data. BMC Med Inform Decis Mak 2005;5:3. 
 (35)  Ennis M, Hinton G, Naylor D, Revow M, Tibshirani R. A comparison of statistical learning 
methods on the Gusto database. Stat Med 1998 November 15;17(21):2501-8. 
 (36)  Zhou XH, Obuchowski NA, McClish DK. Statistical methods in diagnostic medicine. New York: 
Wiley; 2002. 
 (37)  Fluss R, Faraggi D, Reiser B. Estimation of the Youden Index and its associated cutoff point. Biom 
J 2005 August;47(4):458-72. 
 (38)  Greiner M, Pfeiffer D, Smith RD. Principles and practical application of the receiver-operating 
characteristic analysis for diagnostic tests. Prev Vet Med 2000 May 30;45(1-2):23-41. 
 (39)  Hernan MA, Brumback BA, Robins JM. Estimating the causal effect of zidovudine on CD4 count 
with a marginal structural model for repeated measures. Stat Med 2002 June 30;21(12):1689-709. 
 (40)  Hernan MA, Brumback B, Robins JM. Marginal structural models to estimate the causal effect of 
zidovudine on the survival of HIV-positive men. Epidemiology 2000 September;11(5):561-70. 
 (41)  Robins JM, Hernan MA, Brumback B. Marginal structural models and causal inference in 
epidemiology. Epidemiology 2000 September;11(5):550-60. 
 (42)  Wang C, Vlahov D, Galai N et al. The effect of HIV infection on overdose mortality. AIDS 2005 
June 10;19(9):935-42. 
 (43)  Cole SR, Hernan MA, Robins JM et al. Effect of highly active antiretroviral therapy on time to 
acquired immunodeficiency syndrome or death using marginal structural models. Am J Epidemiol 
2003 October 1;158(7):687-94. 
 (44)  Pearl J. Causality: models, reasoning, and inference. New York: Cambridge University Press; 
2000. 
 (45)  Hogan JW, Lancaster T. Instrumental variables and inverse probability weighting for causal 
inference from longitudinal observational studies. Stat Methods Med Res 2004 February;13(1):17-
48. 
 (46)  Lallas CD, Preminger GM, Pearle MS et al. Internet based multi-institutional clinical research: a 
convenient and secure option. J Urol 2004 May;171(5):1880-5. 
 (47)  Kuchenbecker J, Dick HB, Schmitz K, Behrens-Baumann W. Use of Internet Technologies for 
Data Acquisition in Large Clinical Trials. Telemedicine Journal and e-Health 2001 March 
1;7(1):73-6. 
 (48)  Wald. Folic acid, pernicious anaemia, and prevention of neural tube defects. Lancet 
1994;343(8893):307. 
 


Folic Acid and Zinc Supplementation Trial Protocol 
 
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  (49)  Lan KKG, DeMets DL. Discrete sequential boundaries for clinical trials. Biometrika 1983; 
70(3):659-663 
 
(50)   O'Brien PC, Fleming TR.  A multiple testing procedure for clinical trials.  Biometrics, 
1979;35:549-556. 
 
 
 
 


 
1 of 2 
 
Folic Acid and Zinc Supplementation Trial 
Amendment 5.0 
Summary of Changes 
March 19, 2013 
 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
Minor formatting, grammatical and spelling errors 
replaced. 
All applicable sections 
Clarified the Primary Objective of the study by adding 
“attempting to conceive” and replacing “fertility 
treatment” with “assisted reproduction.” 
All applicable sections 
Added “dietary” in front of “supplements.” 
All applicable sections 
Replaced “treatment arms” with “cohorts.” 
All applicable sections 
Clarified secondary study endpoints. 
Study Synopsis 
Updated Study Background. 
2.0  Background 
Modified: 
Couples Inclusion Criteria: clarified all criteria  
Couples exclusion Criteria: 
Added: 
a. Couples attempting to conceive with a gestational 
carrier (surrogate) or a known egg donor. 
b. Positive urine pregnancy test at baseline. 
Male Exclusion Criteria: 
1. Deleted “vitamin B-12 deficiency” from criteria k. 
2. Added “Diagnosis of Vitamin B12 deficiency or 
pernicious anemia.” 
3. Added “Any other diagnosis of anemia within the 
last 5 years that has not been successfully 
treated.”  
3.2  Eligibility Criteria 
Added “minimum, range 30-40 mg”  
3.3  Study Supplement Formulation 
Changed “Fertilization and Embryo Quality for 
IVF/ISCI 
Procedures 
to 
“Early 
Embryonic 
Development Procedures.”  
 
Added the statement “When available, information 
from preimplantation genetic screening will be 
collected.” 
 
Replace 
“OI 
and/or 
IUI” 
with 
“other 
assisted 
reproduction techniques.” 
4.0  Outcome Measures, 7.2 Study 
 Endpoints and ITT Analysis.  
 
Added a urine pregnancy test for women at baseline.  
 
Added blood pressure and pulse rate measurements 
for men and women at baseline. 
5.2. Screening Process, 5.3 Baseline 
 
Study Visit, 5.6 Female Partner 
 
Follow Up, Table 1 and Table 2. 
Clarified randomization process. 
5.4  Randomization. 
Clarified follow up procedures. 
5.5  Active Follow Up & 5.6 Female  
 
 Follow Up. 


 
2 of 2 
 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
Clarified blood collection in males and females. 
5.7  Specimen Collection 
Clarified compensation information. 
5.8  Retention Plan and Compensation 
Deleted the statement “Adverse events will only be 
collected in the males receiving study supplements 
though important female associated study endpoints, 
including adverse pregnancy outcomes, will be 
collected throughout the study in a timely manner.” 
Added “Adverse events will be collected in the males 
receiving study supplements though important female 
associated 
study 
endpoints, 
including 
adverse 
pregnancy outcomes and pregnancy loss, will be 
collected throughout the study and reported as 
adverse events.” 
7.5  Safety Analyses 
Added section about Safety Analyses 
7.5   Safety Analyses 
Clarified biospecimen tracking 
8.3  Questionnaires and Other Data 
 
Forms,  Biospecimen Tracking 
Added the statement “Adverse events will be collected 
in the males receiving study supplements.  Important 
female associated study endpoints, including adverse 
pregnancy outcomes and pregnancy loss, will be 
collected throughout the study and reported as 
adverse events.” 
Added the fax number for SAE reporting. 
Added “Recommendations for reporting AEs for 
dietary supplements are described in the FDA 
“Guidance for Industry: Questions and Answers 
Regarding 
Adverse 
Event 
Reporting 
and 
Recordkeeping for Dietary Supplements as Required 
by the Dietary Supplement and Nonprescription Drug 
Consumer Protection Act” (2007) and if applicable, 
may be covered in the FDA regulations regarding 
drug products. 
Added the address for reporting supplement adverse 
events, if applicable. 
8.4  Adverse Event Reporting 
Replaced “SAS/STAT® data management programs” 
with “AdvantageEDC Suite (Integrity).” 
8.6  Quality Control Procedures  
Clarified investigational product procedures. 
8.8  Investigational Product 
Updated record retention. 
9.2  Retention of Records 
Added risks related to pulse and blood pressure 
measurement. 
Deleted “Since there are no proven medications to 
improve 
semen 
quality 
and 
fertility 
treatment 
outcomes, there are no alternative procedures to offer 
to participants.” 
9.3  Risks and Benefits 
 


 
1 of 3 
Folic Acid and Zinc Supplementation Trial 
Amendment 6.0 
Summary of Changes 
January 22, 2014 
 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
Minor formatting, grammatical and spelling errors 
replaced. 
All applicable sections 
Clarified abstinence criteria for males at all study 
visits, “the males will be asked to abstain from 
ejaculation for 2-5 days prior to presentation for their 
clinic visit” 
All applicable sections 
Updated the protocol synopsis to reflect changes in 
analysis and stratification.  Added:  
1. “Randomization will be stratified by site and 
assisted reproduction technique (IVF, non-
IVF/receiving fertility treatment at a study site, 
non-IVF/receiving fertility treatment at a non-
study site)”,  
2. “The semen parameters will also be assessed 
using analysis of covariance (ANCOVA) 
models with each semen parameter as the 
dependent variable, clinical center, IVF strata 
and treatment group as factors.  The outcome 
of live birth will also be assessed using a 
Mantel-Haenszel analysis to evaluate effects 
across fertility treatment strata”.  
3. Replaced “live birth” with “pregnancy loss”. 
4. “A stratified log-rank test will be used to 
assess treatment effects across IVF strata”. 
5. “Stratified analyses will also be performed for 
secondary outcomes. Specifically, continuous 
secondary outcomes stratified analyses will be 
conducted using ANCOVA models with IVF 
strata and clinical center as factors for the 
comparison of the two cohorts.  If a non-
parametric test is required, van Elteren tests 
will be used to compare the two cohorts, 
accounting for strata, to detect differences in 
the distributions of the secondary endpoints”. 
 
Protocol synopsis 


Amendment 6.0 - Summary of Changes 
January 22, 2014 
 
2 of 3 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
Added “Providing the DCC with de-identified data 
from the clinical questionnaire and SART databases 
for FAZST enrolled couples” as a responsibility of the 
TCC. 
Protocol synopsis 
Deleted “proportion of live births” from the secondary 
endpoints. 
Protocol Synopsis, 7.2 Study 
Endpoints and ITT Analysis 
Added “block” to describe randomization. 
All applicable sections 
Replaced “fertility” with “reproductive outcomes” 
3.1 Introduction 
Deleted “attempting pregnancy” and added “Women 
with regular periods may initiate their fertility therapy 
at the start of the woman’s menstrual cycle following 
randomization if randomization occurred within the 
first 10 days of the cycle, but must wait one menstrual 
cycle if the visit occurred after day 10 of the cycle). 
For women with irregular periods or amenorrhea, the 
male must be on the study supplement for 3 weeks 
prior to initiation of any ovulation induction medication 
(e.g., clomid, letrozole, gonadotropins)” to couples 
inclusion criteria. 
 
Deleted “or a known egg donor” and changed 
“baseline” to “screening” from couples exclusion 
criteria. 
 
Added “males < 18 years” from male exclusion 
criteria. 
3.2 Eligibility criteria, 5.1 Recruitment 
Added the stratification by site and assisted 
reproduction technique. 
3.3 Dietary Supplement Formulation 
Replaced “abortion” with “pregnancy loss” 
Deleted “Clinically recognized spontaneous abortions 
will be defined as those with a gestational sac 
detected from ultrasound scans followed by a loss 
before 20 weeks gestation”. 
Added “visualization” to clarify the review of ectopic 
pregnancies and deleted “ultimately requiring medical 
or surgical therapy”.  
Added “estimated” and “based on ovulation predictor 
kits or trigger shots” to clarify the estimated date of 
conception in IVF participants. 
4.0 Outcome measures 
Added additional recruitment techniques.  
5.1 Recruitment 
Clarified the screening process for the pregnancy test. 5.2. Screening Process 
Added “receiving fertility treatment at a study site; and 
non-IVF/ receiving fertility treatment at a non-study 
site” to clarify stratification at randomization.  
 
5.4 Randomization 


Amendment 6.0 - Summary of Changes 
January 22, 2014 
 
3 of 3 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
Updated the female schedule events to include the 
New Couples Questionnaire and to delete the 24 hour 
recall and women’s pregnancy questionnaire. 
Table 2: Schedule of Events: Female 
Partners.  
 
Updated the compensation to: 
1. Baseline visit $50  
2. 2nd visit $50  Participants will receive an 
additional $15 if 80% of the daily journals have 
been submitted since the previous visit 
3. 3rd visit $75  Participants will receive an 
additional $15 if 80% of the daily journals have 
been submitted since the previous visit 
4. 4th visit $125  Participants will receive an 
additional $20 if 80% of the daily journals have 
been submitted since the previous visit 
 
5.8 Retention Plan and Compensation 
Deleted “Proportion of live births -  IVF versus Non-
IVF ART”   
Added “The semen parameters will also be assessed 
using analysis of covariance (ANCOVA) models with 
each semen parameter as the dependent variable, 
clinical center, IVF strata and treatment group as 
factors.  The outcome of live birth will be assessed 
using a Mantel-Haenszel analysis to evaluate 
treatment effect across IVF strata” in the primary 
analysis section. 
In the secondary analysis section, added: “A stratified 
log-rank test will be used to assess treatment effect 
across IVF strata” and “For continuous secondary 
outcomes stratified analyses will be conducted using 
ANCOVA models with IVF strata and clinical center as 
factors for the comparison of the two cohorts.  If a 
non-parametric test is required, van Elteren tests will 
be used to compare the two cohorts, accounting for 
strata, to detect differences in the distributions of the 
secondary endpoints”. 
7.2 Study Endpoints and ITT Analysis 
Elaborated on the block analysis and the strata 
calculations. 
7.3 Interim Analysis 
Updated the investigational product section to include 
details on the current investigational product. 
Updated the storage and disposition of supplies. 
8.8 Investigational Product 
Updated the NICHD division to “Division of Intramural 
Population Health Research”.  Clarified HSP training. 
9.3 Risks and Benefits 
Added specifics of the Certificate of Confidentiality 
9.5 Confidentiality Procedures 
Updated references 
11.0 References 
 


 
Folic Acid and Zinc Supplementation Trial 
Amendment 7.0 
Summary of Changes 
February 21, 2014 
 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
Added the endpoint “Reproductive hormones and 
other measured biomarkers – Measured urinary, 
serum, and salivary concentrations of reproductive 
hormones, particularly androgens, proteomic analysis 
of human sperm and cardiometabolic risk factors and 
markers of oxidative stress, as well as measures of 
trace elements in toenails”.  
Protocol Synopsis 
Added the biospecimen outcome measures 
“Reproductive hormones and other biomarkers will be 
measured from stored urine, serum, and saliva 
samples for assessment of reproductive hormone 
concentrations, particularly androgens, 
cardiometabolic risk factors, markers of oxidative 
stress, and measures of trace metals in toenails. A 
proteomic analysis of human sperm will also provide 
valuable information regarding potential mechanisms” 
 
4.0 Outcome Measures, 7.2 Study 
Endpoints and ITT Analysis 
Updated the references to the effect sizes and to table 
3   
7.3 Interim Analysis 
 
Page 1 of 1 


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Page 1 of 1 
 
Folic Acid and Zinc Supplementation Trial 
Amendment 9.0 
Summary of Changes 
October 30, 2015 
 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
Removed the following exclusion criteria: 
 
“Diagnosis of anemia at screening. All participants will 
be screened at baseline for anemia and any men that 
screen positive (Hemoglobin <13 gm/dL) will be 
excluded.” 
 
 
“Any other diagnosis of anemia within the last 5 years 
that has not been successfully treated.” 
 
Addition of the following exclusion criterion:  
 
“History of organ transplantation“ 
 
3.2 Eligibility Criteria 
Revised information regarding the updated anemia 
testing procedures “Testing for anemia (Hemoglobin 
<13 gm/dL) in males will be done using a point-of-
care, hemoglobin meter, as a preliminary screen for 
men who require additional evaluation to rule out B12 
deficiency.  Men who screen positive for anemia 
(hemoglobin <13) will have blood drawn for a serum 
B12 with reflex to methylmalonic acid (MMA), but may 
still be enrolled and randomized.  They will be advised 
of their abnormal lab result, and to discuss this with 
their primary care provider.” 
5.2 Screening Process  
 
 
 
 
 
 


 
Page 1 of 2 
 
Folic Acid and Zinc Supplementation Trial 
Amendment 10.0 
Summary of Changes 
March 09, 2016 
 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
Section 7.3 Interim Analysis was renamed Sample 
Size and Power Determinations. 
Language regarding interim analysis was removed 
and from this section.   
7.3 Interim Analysis 
Revised information regarding the updated interim 
analysis:   
The interim analyses will be performed based first on 
the sequential interim monitoring of the semen quality 
parameters (sperm concentration; total sperm motility; 
and sperm morphology (abnormal form)) after 50% of 
the FAZST participants have completed their six 
months follow-up.  The analyses will be unadjusted by 
any covariates, in line with the ITT approach.  The 
overall Type I error rate will be controlled at a pre-
specified level, accounting for both multiple semen 
outcomes and the interim analysis.  As the effects of 
the nutritional supplementation are not clearly 
understood, the study should be stopped early only if 
there is strong evidence of harm.  Given this a one-
sided alpha spending function, which approximates 
the O’Brien-Fleming50 boundary, will be developed.  
First, a Bonferroni adjustment will be used to 
distribute the (sometimes called experiment-wise) 
Type I error alpha among the multiple semen 
parameters.  A α=0.025 (one-sided) is to be assigned 
to the experiment-wide alpha level for the assessment 
of semen parameters, where α3=α/3 = 0.0083 will be 
used to compute the boundary for each individual test.  
For each semen parameter, the amount of α3 
available for the interim analysis performed at time t is 
α3(t) and will be obtained from the O’Brien-Fleming 
spending function.  Time t for the first stage of the 
FAZST interim analysis will be once 1200 (50% of the 
participants) have completed six months of follow-up.  
Using these assumptions the test statistic for lower 
bound of harm will be 3.5547 for each of the 3 
comparisons 
with 
α3(t) 
= 
0.00019 
where 
t 
represents50% of the participants completing 6 
months of follow-up.    The DSMB will consider the 
interim analysis results as a resource to evaluate the 
risk of study treatment.  When a stopping boundary is 
crossed, there will be an indication that the treatment 
has an increased risk of harm for at least one of the  
 


 
Page 2 of 2 
 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
semen parameters.  The DSMB will consider the 
consistency of all semen analysis data to determine 
whether the second stage of the interim analysis 
should be considered.   
If the DSMB determines that the second stage of the 
interim analysis is applicable, it will be conducted 
based on the available data and the outcome of live 
birth.  For this stage, the estimated information 
fraction will be defined as the total number of 
participants completing 6 months of follow-up at the 
time of database closure for the second stage interim 
analysis.   Similarly the alpha spending function 
approach will be applied to the sequential monitoring 
of live birth.  An overall alpha of 0.05 and a one-sided 
procedure in data monitoring will be used.  The 
hypothesis will be tested by a one-sided O’Brien-
Fleming alpha spending design.   Assuming PLB = 
0.63 in the placebo arm and a RR of 1.100, interim 
test boundaries using the O’Brien-Fleming alpha 
spending design based on various values of the 
information fraction are provided in the table below. 
When a stopping boundary is crossed, there will be an 
indication that the treatment has an increased risk of 
harm for live birth.  The DSMB will consider this result 
and all live birth data to determine whether a 
suspension of the trial intervention should be 
considered.   
Table 6. Examples of Critical Values of the 
Test Statistics for Live Birth 
Information 
Fraction 
Lower Bound 
0.20 
4.8769 
0.25 
4.3326 
0.30 
3.9286 
0.35 
3.6128 
0.40 
3.3569 
0.50 
2.9626 
 
 
7.4 Interim Analysis   
 
 
 
 
 
 


Folic Acid and Zinc Supplementation Trial 
Amendment 11.0 
Summary of Changes 
November 6, 2018 
 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
Revised text regarding following pregnant couples who 
underwent IVF. 
Original text: Couples initiating an IVF cycle in the sixth 
or seventh month after randomization will continue to be 
followed if pregnancy occurs for pregnancy outcomes for 
9 months following the start of their IVF assisted 
conception cycle.  
 
Revised text: Couples initiating an IVF cycle in the sixth or 
seventh month after randomization will continue to be 
followed if pregnancy occurs for pregnancy outcomes 
during the initial 9-month period.  
5.6 Female Partner Follow-up 
Language regarding specific semen collection container 
was removed and from this section.   
5.7 Specimen Collection 
Language specifying ‘(Withdrawal)’ was added to this 
section’s subhead: “Exit Questionnaire”.  
5.8 Retention Plan and Compensation 
Revised text regarding safety analyses.  
Original text: Adverse events will be collected in the males 
receiving study supplements though important female 
associated study endpoints, including adverse pregnancy 
outcomes and pregnancy loss, will be collected throughout 
the study and reported as adverse events.  
 
Revised text: Adverse events will be collected in the males 
receiving study supplements though important female 
associated study endpoints, including adverse pregnancy 
outcomes and pregnancy loss, will be collected throughout 
the study and reported as adverse events via questionnaire 
and medical chart abstraction.  
7.6 Safety Analyses 
Revised text regarding data storage and quality control.   
Original text: Study records should be maintained for at 
least two years post completion of the final study report or 
publication of the primary endpoints. 
 
Revised text:  Study records should be maintained for at 
least three years post completion of the final study report 
or publication of the primary endpoints or per local site 
IRB requirements (whichever is longer). 
8.2 FAZST Web-based System 
Revised text regarding AE collection period and follow-up. 
Original text: Important female associated study endpoints, 
including adverse pregnancy outcomes and pregnancy loss, 
will be collected throughout the study and reported as 
adverse events. 
 
Revised text:  Important female associated study 
endpoints, including adverse pregnancy outcomes and 
pregnancy loss, will be collected throughout the study and 
reported as adverse events via questionnaires and 
medical record abstraction forms. 
8.4 Adverse Experience Reporting 


FAZST Amendment 11.0 Summary of Changes  
November 6, 2018 
 
AMENDMENT CHANGE 
SECTION(S) AFFECTED BY 
AMENDMENT CHANGE 
Revised text regarding IRB review and informed consent.  
Original text: Two IRBs will review this protocol: 1) 
University of Utah, and 2) The EMMES Corporation. 
NICHD will be covered under a reliance agreement with 
the University of Utah.   
 
Revised text: Original text: Four IRBs will review this 
protocol: 1) University of Utah, and 2) The Emmes 
Corporation 3) University of Iowa 4) Northwestern 
University. 
9.0 Human Subjects Protection and 
Confidentiality Procedures 
Revised text regarding participant confidentiality.  
Original text: No social security numbers will be collected.  
De-identified data will be submitted to a central database 
designed by the DCC.  
 
Revised text: Social security numbers may be collected for 
payment purposes only but will not be submitted to a 
central database.   
9.0 Human Subjects Protection and 
Confidentiality Procedures  
 
Language regarding maintaining regulatory binders was 
removed and from this section.  This information is located 
in the Manual of Procedures.   
9.1 Regulatory Binders