Folic Acid and Zinc Supplementation Trial (FAZST)

Study Overview
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Brief Summary
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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: 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. 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. 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. 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. 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
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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.

Official Title
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Folic Acid and Zinc Supplementation Trial: A Multi-center, Double-blind, Block-randomized, Placebo-controlled Trial

Conditions
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Pregnancy, Live Birth, Spontaneous Abortion

Intervention / Treatment
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* Dietary Supplement: 5 mg folic acid and 30 mg elemental zinc
* Drug: Placebo Comparator: Placebo


Participation Criteria
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Eligibility Criteria
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Couples Inclusion Criteria: 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. Couples actively trying to conceive. 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: Female partner unwilling to participate (e.g., no abstraction of her assisted fertility treatment record or unwilling to complete baseline visit). Couples using donor, cryopreserved sperm, or sperm obtained via microsurgical or percutaneous epididymal sperm aspiration. Couples attempting to conceive with a gestational carrier (surrogate). Positive urine pregnancy test at screening. Male Inclusion Criteria: Willing to provide semen samples according to the proposed schedule at baseline, 2, 4, and 6 months of follow-up. 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: Age <18 years. Unwilling to abstain from use of non-study approved dietary supplements or medications containing folic acid or oral preparations containing zinc throughout the study. Unwilling to abstain from use of testosterone supplementation throughout the study. Diagnosis of Vitamin B12 deficiency or pernicious anemia. Consuming a vegan diet. A known genetic cause of male factor subfertility, including chromosomal disorders related to subfertility (e.g., Y chromosome deletions). 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. Dihydrofolate reductase inhibitors: Trimethoprim, Triamterene, Bactrim, Iclaprim Sulfonamides: Hydrochlorothiazide (HCTZ), Metolazone, Indapamide, Lasix, Bumex, Torsemide, Chlorthalidone, Acetazolamide, Mefruside, Xipamide Sulfonylureas: Glipizide, Glyburide Cox-2 inhibitors: Celecoxib Others: Valproic acid, Probenecid, Sulfasalazine, Sumatriptan, Mafenide, Ethoxzolamide, Sulfiram, Zonisamide, Dorzolamide (optic), Dichlorphenamide, Fluorouracil, Capecitabine, Methotrexate History of organ transplantation. Physician diagnosed: 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. 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. History of alcohol dependency disorder and/or other drug/substance dependency in the past 180 days. 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. History of vasectomy without reversal, obstructive azoospermia such as Congenital Bilateral Aplasia of Vas Deferens (CBAVD), or ejaculatory duct obstruction. Known allergy to folic acid or zinc dietary supplements. Female Exclusion Criteria: Age <18 or >45 years.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 45 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Folic acid and zinc supplementation<br>5 mg folic acid and 30 mg elemental zinc, taken orally, daily for 6 months. | Dietary Supplement: 5 mg folic acid and 30 mg elemental zinc<br> <br> |
| Placebo Comparator: Placebo<br>Matching placebo, taken orally daily for 6 months. | Drug: Placebo Comparator: Placebo<br> <br> |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Live Birth | Based on hospital delivery records | At delivery | 
| Semen Volume | Volume of the ejaculate, mL Assessed utilizing the World Health Organization (WHO) semen analysis procedure 5th edition World Health Organization. WHO laboratory manual for the Examination and processing of human semen. 5th Edition ed. Switzerland: 2010. | 6 months | 
| Sperm Concentration | Number of spermatozoa per unit of volume of semen Assessed utilizing the World Health Organization (WHO) semen analysis procedure 5th edition World Health Organization. WHO laboratory manual for the Examination and processing of human semen. 5th Edition ed. Switzerland: 2010. | 6 months | 
| Sperm Motility | % motile (including percentage of progressive motile sperm and percentage of nonprogressive motile sperm) Assessed utilizing the World Health Organization (WHO) semen analysis procedure 5th edition World Health Organization. WHO laboratory manual for the Examination and processing of human semen. 5th Edition ed. Switzerland: 2010. | 6 months | 
| Sperm Morphology | % normal morphology Assessed utilizing the World Health Organization (WHO) semen analysis procedure 5th edition World Health Organization. WHO laboratory manual for the Examination and processing of human semen. 5th Edition ed. Switzerland: 2010. | 6 months | 
| DNA Fragmentation Index | Comet assay used to measure sperm DNA integrity based on excess DNA strand breaks Assessed utilizing the World Health Organization (WHO) semen analysis procedure 5th edition World Health Organization. WHO laboratory manual for the Examination and processing of human semen. 5th Edition ed. Switzerland: 2010. | 6 months | 
| Total Motile Sperm Count | Calculated as semen volume (mL) * sperm concentration (10^6 spermatozoa/mL) * motility (% motile) | 6 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Human Chorionic Gonadotropin (hCG) Detected Pregnancy (Implantation) | A quantitative hCG evaluation in serum > 5 milli-international units per milliliter (mIU/ml) | For IVF, 12 days post embryo transfer for day 5 embryo transfers, and 14 days post embryo transfer for day 3 embryo transfers; for couples undergoing OI/IUI, after self-report of positive pregnancy test | 
| Clinical Intrauterine Pregnancy | Visualized gestational sac in the uterus on ultrasound | approximately 6.5 weeks gestation | 
| 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. | approximately 6.5 weeks gestation | 
| Early Pregnancy Loss | hCG pregnancy loss will be defined as a serum hCG > 5 mIU/ml followed by a decline. Clinically recognized pregnancy losses will be defined as visualization of an intrauterine gestational sac followed by a loss prior to 20 weeks gestation. | hcG-detected pregnancy until 20 weeks of pregnancy | 
| Preeclampsia or Gestational Hypertension | Abstracted from hospital records and medical charts | Delivery | 
| Gestational Diabetes | Abstracted from hospital records and medical charts | Delivery | 
| Cesarean Delivery | Abstracted from hospital records and medical charts | Delivery | 
| Preterm Delivery | Abstracted from hospital records and medical charts | Delivery | 
| Small for Gestational Age | Abstracted from hospital records and medical charts | Delivery | 
| Gestational Age | Abstracted from hospital records and medical charts | Delivery | 
| Birth Weight | Abstracted from hospital records and medical charts | Delivery | 
| Stillbirth | Loss at or after 20 weeks gestation. Determined based on hospital records and medical chart abstraction. | Delivery | 
| Neonatal Mortality | Abstracted from hospital records and medical charts | Delivery | 
| Major Neonatal Complications | Abstracted from hospital records and medical charts: includes bronchopulmonary dysplasia, necrotizing enterocolitis, severe intraventricular hemorrhage, periventricular leukomalacia, and retinopathy of prematurity | Delivery | 
| Structural Malformations | Abstracted from birth record: includes major (n = 21; 6 with known genetic cause), minor (n = 6), and unclassified (n = 2) defects Structural birth defects: includes hydronephrosis/ureteropelvic junction obstruction, transposition of the great arteries, renal agenesis, cleft lip, club feet, multicystic/dysplastic kidney, tetralogy of fallot, gastroschisis, atrioventricular septal defects, other oral-facial defects, other cardiovascular defects, other CNS defects, other eye defects, other oral-facial defects, other anomalies, other syndromes | Delivery | 
| Severe Maternal Morbidity | Abstracted from delivery record: including postpartum hemorrhage, anemia requiring transfusion, sepsis, seizure, HELLP syndrome or preeclampsia with pulmonary edema | Delivery | 
| Fertilization Rate Per Cycle, % | Among participants in the IVF stratum Oocytes will be assessed 16-18 hours after insemination or microinjection to determine 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. | Up to 9 months of fertility treatment post-randomization | 
| Number of Good Quality Embryos on Day 5 Per Cycle | Among participants in the IVF stratum 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. | Up to 9 months of fertility treatment post-randomization | 
| Percentage of Good Quality Embryos on Day 5 Per Cycle | Among participants in the IVF stratum 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. | Up to 9 months of fertility treatment post-randomization | 
| Number of Embryos Transferred Per Cycle | Among participants in the IVF stratum | Up to 9 months of fertility treatment post-randomization | 
| Number of Embryos Cryopreserved Per Cycle | Among participants in the IVF stratum | Up to 9 months of fertility treatment post-randomization | 
| Sperm Penetration Per Cycle, % | Among participants in the IVF stratum | Up to 9 months of fertility treatment post-randomization | 
| Cells on Day 3 Per Embryo Per Cycle | Among participants in the IVF stratum | Up to 9 months of fertility treatment post-randomization | 
| Cells on Day 3 Per Embryo Per Cycle, Categorical | Number of cells per embryo among women in the IVF stratum | Up to 9 months of fertility treatment post-randomization | 
| Cells on Day 5 Per Embryo Per Cycle, Categorical | Among participants in the IVF stratum | Up to 9 months of fertility treatment post-randomization | 
| Embryo Morphology on Day 3 Per Cycle, Categorical | Among participants in the IVF stratum Embryos will be scored three days after fertilization according to the size and shape of blastomeres and to their degree of fragmentation. Veeck LL. Oocyte assessment and biological performance. Ann N Y Acad Sci 1988;541:259-74.:259-74. | Up to 9 months of fertility treatment post-randomization | 
| Embryo Morphology on Day 5 Per Cycle, Categorical | Among participants in the IVF stratum 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. | Up to 9 months of fertility treatment post-randomization | 
| Method of Fertilization Per Cycle | Among participants in the in vitro fertilization (IVF) stratum: method of fertilization classified into intracytoplasmic sperm injection (ICSI) and other | Up to 9 months of fertility treatment post-randomization | 
| Quality of Embryos Transferred Per Cycle, Categorical | Among participants in the IVF stratum Embryonic grading based on Society for Assisted Reproductive Technologies (SART) morphology criteria | Up to 9 months of fertility treatment post-randomization | 
| Chromosomal Complement of Embryo Per Cycle | Among participants in the IVF stratum Chromosomal complement in the embryo assessed using methodology cited by Rubio et al. 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. | Up to 9 months of fertility treatment post-randomization | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Folic Acid, Zinc, Semen, In vitro fertilization, Assisted reproductive technology, Ovulation induction, Intrauterine insemination, Pregnancy, Live Birth, Abortion, spontaneous