The Effect of Folate and Folate Plus Zinc Supplementation on Endocrine Parameters and Sperm Characteristics in Sub-Fertile Men: A Systematic Review and Meta-Analysis

Morvarid Irani, Malihe Amirian, Ramin Sadeghi, Justine Le Lez, Robab Latifnejad Roudsari



Purpose: To evaluate the effect of folate and folate plus zinc supplementation on endocrine parameters and sperm characteristics in sub fertile men.
Materials and Methods: We conducted a systematic review and meta-analysis. Electronic databases of Medline, Scopus , Google scholar and Persian databases (SID, Iran medex, Magiran, Medlib, Iran doc) were searched from 1966 to December 2016 using a set of relevant keywords including “folate or folic acid AND (infertility, infertile, sterility)”.All available randomized controlled trials (RCTs), conducted on a sample of sub fertile men with semen analyses, who took oral folic acid or folate plus zinc, were included. Data collected included endocrine parameters and sperm characteristics. Statistical analyses were done by Comprehensive Meta-analysis Version 2.
Results: In total, seven studies were included. Six studies had sufficient data for meta-analysis. “Sperm concentration was statistically higher in men supplemented with folate than with placebo (P < .001)”. However, folate supplementation alone did not seem to be more effective than the placebo on the morphology (P = .056) and motility of the sperms (P = .652). Folate plus zinc supplementation did not show any statistically different effect on serum testosterone (P = .86), inhibin B (P = .84), FSH (P = .054), and sperm motility (P = .169) as compared to the placebo. Yet, folate plus zinc showed statistically higher effect on the sperm concentration (P < .001), morphology (P < .001), and serum folate level (P < .001) as compared to placebo.
Conclusion: Folate plus zinc supplementation has a positive effect on sperm characteristics in sub fertile men. However, these results should be interpreted with caution due to the important heterogeneity of the studies included in this meta-analysis. Further trials are still needed to confirm the current findings.

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Mosher WD. Fecundity and infertility in the United States. Am J Public Health. 1988;78:181-2.

Hamada A, Esteves SC, Agarwal A. Unexplained male infertility: potential causes and management. Human Andrology. 2011;1:2-16.

Showell MG, Brown J, Yazdani A, Stankiewicz MT, Hart RJ. Antioxidants for male subfertility. The Cochrane Library. 2014;12: CD007411

Pasqualotto FF. Investigação e reprodução assistida no tratamento da infertilidade masculina. Rev Bras Ginecol Obstet. 2007;29:103-12.

J.Downey, Yingling S, M.McKinney, N.Husami, .Jewelewicz R, J.Maidman. Mood disorders, psychiatric symptoms, and distress in women presenting for infertility evaluation. Fertil Steri. 1989;l 52:425–32.

Whiteford LM, Gonzalez L. Stigma: the hidden burden of infertility. Soc Sci Med . 1995;40:27–36.

Isidori AM, Pozza C, Gianfrilli D, Isidori A. Medical treatment to improve sperm quality. Reprod Biomed Online. 2006;12:704-14.

Mendiola J, Torres-Cantero AM, Agarwal A. Lifestyle factors and male infertility: an evidence-based review. Arch Med Sci. 2009;5:S3-12.

Tremellen K. Oxidative stress and male infertility—a clinical perspective. Hum Reprod Update. 2008;14:243-58.

Lucock M. Folic acid: nutritional biochemistry, molecular biology, and role in disease processes. Mol Genet Metab. 2000;71:121-38.

Joshi R, Adhikari S, Patro B, Chattopadhyay S, Mukherjee T. Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity. Free Radic Biol Med. 2001;30:1390-9.

Nematollahi‐Mahani S, Azizollahi G, Baneshi M, Safari Z, Azizollahi S. Effect of folic acid and zinc sulphate on endocrine parameters and seminal antioxidant level after varicocelectomy. Andrologia. 2014;46:240-5.

Joshi R, Adhikari S, Patro BS, Chattopadhyay S, Mukherjee T. Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity. Free Radic Biol Med .2001;30:1390–9.

Ebisch I, Thomas C, Peters W, Braat D, Steegers-Theunissen R. The importance of folate, zinc and antioxidants in the pathogenesis and prevention of subfertility. Hum Reprod Update. 2007;13:163-74.

Favier AE. The role of zinc in reproduction. Biol Trace Elem Res. 1992;32:363-82.

Zago MP, Oteiza PI. The antioxidant properties of zinc: interactions with iron and antioxidants. Free Radic Biol Med. 2001;31:266-74.

Chimienti F, Aouffen M, Favier A, Seve M. Zinc homeostasis-regulating proteins: new drug targets for triggering cell fate. Curr Drug Targets. 2003;4:323-38.

Wong WY, Thomas CM, Merkus JM, Zielhuis GA, Steegers-Theunissen RP. Male factor subfertility: possible causes and the impact of nutritional factors. Fertil Steril. 2000;73:435-42.

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;77:491-8.

Nematollahi‐Mahani SN, Azizollahi G, Baneshi MR, Safari Z, Azizollahi S. Effect of folic acid and zinc sulphate on endocrine parameters and seminal antioxidant level after varicocelectomy. Andrologia. 2014;46:240-5.

Raigani M, Yaghmaei B, Amirjannti N, Lakpour N, Akhondi M, Zeraati H, et al. The micronutrient supplements, zinc sulphate and folic acid, did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men. Andrologia. 2014;46:956-62.

da Silva TM, Maia MCS, Arruda JT, Approbato FC, Mendonça CR, Approbato MS. Folic acid does not improve semen parametrs in sub fertile men: A double-blin, randomized, placebo-controlled study. JBRA Assisted Reproduction. 2013;17:152-7.

Busetto GM, Koverech A, Messano M, Antonini G, De Berardinis E, Gentile V. Prospective open-label study on the efficacy and tolerability of a combination of nutritional supplements in primary infertile patients with idiopathic astenoteratozoospermia. Arch Ital Urol Androl. 2012;84:137-40.

Ebisch I, Pierik F, De Jong F, Thomas C, STEEGERS‐THEUNISSEN R. Does folic acid and zinc sulphate intervention affect endocrine parameters and sperm characteristics in men? . Int J Androl. 2006;29:339-45.

Pierik FH, Vreeburg JT, Stijnen T, De Jong FH, Weber RF. Serum inhibin B as a marker of spermatogenesis. J Clin Endocrinol Metab. 1998;83:3110-4.

von Eckardstein S, Simoni M, Bergmann M, Weinbauer GF, Gassner P, Schepers AG, et al. Serum Inhibin B in Combination with Serum Follicle-Stimulating Hormone (FSH) Is a More Sensitive Marker Than Serum FSH Alone for Impaired Spermatogenesis in Men, But Cannot Predict the Presence of Sperm in Testicular Tissue Samples 1. J Clin Endocrinol Metab. 1999;84:2496-501.

Andersson A-M. Inhibin B in the assessment of seminiferous tubular function. Best Pract Res Clin Endocrinol Metab. 2000;14:389-97.

Anderson R, Sharpe R. Regulation of inhibin production in the human male and its clinical applications. Int J Androl. 2000;23:136-44.

Forges T, Monnier-Barbarino P, Alberto J, Gueant-Rodriguez R, Daval J, Gueant J. Impact of folate and homocysteine metabolism on human reproductive health. Hum Reprod Update. 2007;13:225-38.

Guzick DS, Overstreet JW, Factor-Litvak P, Brazil CK, Nakajima ST, Coutifaris C, et al. Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med. 2001;345:1388-93.

Higgins JP, Green S. Cochrane handbook for systematic reviews of interventions Version 5.1.0 . The Cochrane Collaboration. United Kingdom: John Wiley & Sons ; 2011.

Ebisch IM, van Heerde WL, Thomas CM, van der Put N, Wong WY, Steegers-Theunissen RP. C677T methylenetetrahydrofolate reductase polymorphism interferes with the effects of folic acid and zinc sulfate on sperm concentration. Fertil Steril. 2003;80:1190-4.

Azizollahi G, Azizollahi S, Babaei H, Kianinejad M, Baneshi MR, Nematollahi-mahani SN. Effects of supplement therapy on sperm parameters, protamine content and acrosomal integrity of varicocelectomized subjects. J Assist Reprod Genet. 2013;30:593-9.

Menkveld R, Wong WY, Lombard CJ, Wetzels AM, Thomas CM, Merkus HM, et al. Semen parameters, including WHO and strict criteria morphology, in a fertile and sub fertile population: an effort towards standardization of in-vivo thresholds . Hum Reprod. 2001;16:1165-71.

Ueland PM, Hustad S, Schneede J, Refsum H, Vollset SE. Biological and clinical implications of the MTHFR C677T polymorphism. Trends Pharmacol Sci. 2001;22:195-201.

van der Put NM, Trijbels F, van den Heuvel L, Blom H, Steegers-Theunissen R, Eskes T, et al. Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida. The Lancet. 1995;346:1070-1.

Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genetics 1995;10:111-3.

Malinow M, Nieto F, Kruger W, Duell P, Hess D, Gluckman R, et al. The effects of folic acid supplementation on plasma total homocysteine are modulated by multivitamin use and methylenetetrahydrofolate reductase genotypes. Arterioscler Thromb Vasc Biol. 1997;17:1157-62.

Vani G, Mukesh N, Rama Devi P, Usha Rani P, Reddy P. Methylenetetrahydrofolate reductase C677T polymorphism is not associated with male infertility in a South Indian population. Andrologia. 2012;44:252-9.

Chellat D, Rezgoune ML, Hamane D, Semmame O, Benlatrèche C, Abadi N, et al. Influence of methylenetetrahydrofolate reductase C677T gene polymorphisms in Algerian infertile men with azoospermia or severe oligozoospermia. Genet Test Mol Biomarkers. 2012;16:874-8.

Gava MM, Kayaki EA, Bianco B, Teles JS, Christofolini DM, Pompeo AC, et al. Polymorphisms in folate-related enzyme genes in idiopathic infertile Brazilian men. Reproductive Sciences. 2011;18:1267-72.

Gava MM, de Oliveira Chagas E, Bianco B, Christofolini DM, Pompeo ACL, Glina S, et al. Methylenetetrahydrofolate reductase polymorphisms are related to male infertility in Brazilian men. Genet Test Mol Biomarkers. 2011;15:153-7.

Safarinejad MR, Shafiei N, Safarinejad S. Relationship between genetic polymorphisms of methylenetetrahydrofolate reductase (C677T, A1298C, and G1793A) as risk factors for idiopathic male infertility. Reproductive Sciences. 2010;11: 304-15.

Yang Y, Zhang SZ, Li N, Zhang W. Single nucleotide polymorphism C677T in the methylenete‐trahydrofolate reductase gene might be a genetic risk factor for infertility for Chinese men with azoospermia or severe oligozoospermia. Asian J Androl. 2007;9:57-62.

Dhillon VS, Shahid M, Husain SA. Associations of MTHFR DNMT3b 4977 bp deletion in mtDNA and GSTM1 deletion, and aberrant CpG island hypermethylation of GSTM1 in non-obstructive infertility in Indian men. Mol Hum Reprod. 2007;13:213-22.

Paracchini V, Garte S, Taioli E. MTHFR C677T polymorphism, GSTM1 deletion and male infertility: a possible suggestion of a gene–gene interaction? Biomarkers. 2006;11:53-60.

Singh K, Singh S, Sah R, Singh I, Raman R. Mutation C677T in the methylenetetrahydrofolate reductase gene is associated with male infertility in an Indian population1 . Int J Androl. 2005;28:115-9.

Park JH, Lee HC, Jeong Y-M, Chung T-G, Kim H-J, Kim NK, et al. MTHFR C677T polymorphism associates with unexplained infertile male factors. J Assist Reprod Genet. 2005;22:361-8.

Stuppia L, Gatta V, Scarciolla O, Colosimo A, Guanciali-Franchi P, Calabrese G, et al. The methylenetethrahydrofolate reductase (MTHFR) C677T polymorphism and male infertility in Italy. J Endocrinol Invest. 2003;26:620-2.

Gupta N, Gupta S, Dama M, David A, Khanna G, Khanna A, et al. Strong association of 677 C> T substitution in the MTHFR gene with male infertility—a study on an indian population and a meta-analysis. PLoS One. 2011;6:e22277.

Lee H-C, Jeong Y-M, Lee SH, Cha KY, Song S-H, Kim NK, et al. Association study of four polymorphisms in three folate-related enzyme genes with non-obstructive male infertility. Hum Reprod. 2006;21:3162-70.

Bezold G, Lange M, Peter RU. Homozygous methylenetetrahydrofolate reductase C677T mutation and male infertility. N Engl J Med. 2001;344:1172-3.

Stern LL, Mason JB, Selhub J, Choi S-W. Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene. Cancer Epidemiol Biomarkers Prev. 2000;9:849-53.

Mfady DS, Sadiq MF, Khabour OF, Fararjeh AS, Abu-Awad A, Khader Y. Associations of variants in MTHFR and MTRR genes with male infertility in the Jordanian population. Gene. 2014;536:40-4.

Favier M, Faure P, Roussel A, Coudray C, Blache D, Favier A. Zinc deficiency and dietary folate metabolism in pregnant rats. J Trace Elem Electrolytes Health Dis. 1993;7:19-24.

Quinn PB, Cremin F, O'sullivan V, Hewedi F, Bond R. The influence of dietary folate supplementation on the incidence of teratogenesis in zinc-deficient rats. Br J Nutr. 1990;64:233-43.

Truong-Tran A, Ho L, Chai F, Zalewski P. Cellular zinc fluxes and the regulation of apoptosis/gene-directed cell death. J Nutr. 2000;130:1459S-66S.

Quinn PB, Cremin F, O’sullivan V, Hewedi F, Bond R. The influence of dietary folate supplementation on the incidence of teratogenesis in zinc-deficient rats. Br J Nutr. 1990;64:233-43.


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