Folic Acid and Zinc Supplementation Trial (FAZST)

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.

Couples Inclusion Criteria:

1. 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.

2. Couples actively trying to conceive.

3. 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).

Couples Exclusion Criteria:

1. Female partner unwilling to participate (e.g., no abstraction of her assisted
fertility treatment record or unwilling to complete baseline visit).

2. Couples using donor, cryopreserved sperm, or sperm obtained via microsurgical or
percutaneous epididymal sperm aspiration.

3. Couples attempting to conceive with a gestational carrier (surrogate).

4. Positive urine pregnancy test at screening.

Male Inclusion Criteria:

1. Willing to provide semen samples according to the proposed schedule at baseline, 2, 4,
and 6 months of follow-up.

2. 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:

1. Age <18 years.

2. Unwilling to abstain from use of non-study approved dietary supplements or medications
containing folic acid or oral preparations containing zinc throughout the study.

3. Unwilling to abstain from use of testosterone supplementation throughout the study.

4. Diagnosis of Vitamin B12 deficiency or pernicious anemia.

5. Consuming a vegan diet.

6. A known genetic cause of male factor subfertility, including chromosomal disorders
related to subfertility (e.g., Y chromosome deletions).

7. Males currently using and unwilling (or unable) to discontinue the following drugs
known to interact with folic acid or interfere with the biosynthesis of folic acid
will be excluded.

1. Dihydrofolate reductase inhibitors: Trimethoprim, Triamterene, Bactrim, Iclaprim

2. Sulfonamides: Hydrochlorothiazide (HCTZ), Metolazone, Indapamide, Lasix, Bumex,
Torsemide, Chlorthalidone, Acetazolamide, Mefruside, Xipamide

3. Sulfonylureas: Glipizide, Glyburide

4. Cox-2 inhibitors: Celecoxib

5. Others: Valproic acid, Probenecid, Sulfasalazine, Sumatriptan, Mafenide,
Ethoxzolamide, Sulfiram, Zonisamide, Dorzolamide (optic), Dichlorphenamide,
Fluorouracil, Capecitabine, Methotrexate

8. History of organ transplantation.

9. Physician diagnosed:

1. 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.

2. Crohn's disease, celiac disease, ulcerative colitis, gastric bypass surgery, lap
band surgery or history of intestinal surgery to remove a portion of small bowel.
History of diseases/symptoms that require folic acid dietary supplementation,
such as megaloblastic anemia, homocystinemia, and homocystinuria.

3. History of alcohol dependency disorder and/or other drug/substance dependency in
the past 180 days.

4. 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.

10. History of vasectomy without reversal, obstructive azoospermia such as Congenital
Bilateral Aplasia of Vas Deferens (CBAVD), or ejaculatory duct obstruction.

11. Known allergy to folic acid or zinc dietary supplements.

Female Exclusion Criteria:

Age <18 or >45 years.