Down-Regulated Expression of Cystathionine β-Synthase and Cystathionine γ-Lyase in Varicocele, and Infertile Men: A Case-Control Study

Document Type : Original Article


1 Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

2 R&D Department, Parthenogen, Piazza Indipendenza 11, Lugano 6900, Switzerland

3 Department of Cell and Molecular Biology, Cell Sciences Research Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran


Objective: Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are two important enzymes involved in 
One-Carbon metabolism. These enzymes play important roles in modulating oxidative stress and inflammation in male 
factor infertility through participating in the synthesis of glutathione (GSH) antioxidants in the trans-sulfuration pathway. 
Besides, the direct release of hydrogen sulfide (H2S) has anti-inflammatory and antioxidant effects. Therefore, the 
expression of CBS and CSE genes at mRNA levels in infertile and varicocele men was evaluated and compared to the 
healthy counterparts to clarify their possible role in the pathology of male infertility. 
Materials and Methods: In this case-control study, semen parameter assessment (concentration, morphology, and motility of sperms) was performed on 28 men with varicocele, 43 infertile men with abnormal sperm parameters, and 19 fertile men. RNA was extracted from sperm samples followed by cDNA synthesis and real-time polymerase chain reaction (PCR) using CBS, CSE, and GAPDH primers. 
Results: Sperm concentration and motility in infertile and varicocele groups were significantly lower (P=0.001), while 
spermatoza normal morphology was higher than fertile group (P=0.05). The expression levels of both CBS and CSE 
genes in infertile (P=0.04 and P=0.037 respectively) and varicocele (P=0.01 and P=0.046 respectively) groups were 
significantly lower than fertile group. Additionally, CBS gene expression indicated a positive correlation with expression 
of CSE gene (r=0.296, P=0.025) and sperm parameters. 
Conclusion: In light of our findings, there is a valid rationale to consider the primary role of CBS and CSE enzymes 
impairment in male factor infertility which specifically may point to a deficit in the release of essential antioxidants 
including the H2S as a molecular basis of infertility and warrants further investigation. 


  1. Kumar N, Singh AK. Impact of environmental factors on human semen quality and male fertility: a narrative review. Environ Sci Eur. 2022; 34(6): 1-13.
  2. Jodar M, Soler-Ventura A, Oliva R; Molecular biology of reproduction and development research group. semen proteomics and male infertility. J Proteomics. 2017; 162: 125-134.
  3. Bisht S, Faiq M, Tolahunase M, Dada R. Oxidative stress and male infertility. Nat Rev Urol. 2017; 14(8): 470-485.
  4. Nishimura H, L’Hernault SW. Spermatogenesis. Curr Biol. 2017; 27(18): R988-R994.
  5. Menezo YJ, Silvestris E, Dale B, Elder K. Oxidative stress and alterations in DNA methylation: two sides of the same coin in reproduction. Reprod Biomed Online. 2016; 33(6): 668-683.
  6. Singh K, Jaiswal D. One-carbon metabolism, spermatogenesis, and male infertility. Reprod Sci. 2013; 20(6): 622-630.
  7. McBean GJ. The transsulfuration pathway: a source of cysteine for glutathione in astrocytes. Amino Acids. 2012; 42(1): 199-205.
  8. Xiao W, Loscalzo J. Metabolic responses to reductive stress. Antioxid Redox Signal. 2020; 32(18): 1330-1347.
  9. Renga B. Hydrogen sulfide generation in mammals: the molecular biology of cystathionine-β- synthase (CBS) and cystathionine-γ- lyase (CSE). Inflamm Allergy Drug Targets. 2011; 10(2): 85-91.
  10. Mishanina TV, Libiad M, Banerjee R. Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways. Nat Chem Biol. 2015; 11(7): 457-464.
  11. Han W, Dong Z, Dimitropoulou C, Su Y. Hydrogen sulfide ameliorates tobacco smoke-induced oxidative stress and emphysema in mice. Antioxid Redox Signal. 2011; 15(8): 2121-2134.
  12. Zhu XY, Gu H, Ni X. Hydrogen sulfide in the endocrine and reproductive systems. Expert Rev Clin Pharmacol. 2011; 4(1): 75 -82.
  13. Kimura H. Hydrogen sulfide: its production, release and functions. Amino Acids. 2011; 41(1): 113-121.
  14. Sugiura Y, Kashiba M, Maruyama K, Hoshikawa K, Sasaki R, Saito K, et al. Cadmium exposure alters metabolomics of sulfur-containing amino acids in rat testes. Antioxid Redox Signal. 2005; 7(5-6): 781-787.
  15. Corsello T, Komaravelli N, Casola A. Role of hydrogen sulfide in NRF2- and sirtuin-dependent maintenance of cellular redox balance. Antioxidants (Basel). 2018; 7(10): 129.
  16. Morales A, Garza M R G,Valdés O. A randomized clinical study assessing the effects of the antioxidants, resveratrol or SG1002, a hydrogen sulfide prodrug, on idiopathic oligoasthenozoospermia. Asian Pac J Reprod. 2015; 4(2): 106-111.
  17. Srilatha B, Muthulakshmi P, Adaikan PG, Moore PK. Endogenous hydrogen sulfide insufficiency as a predictor of sexual dysfunction in aging rats. Aging Male. 2012; 15(3): 153-158.
  18. Prudova A, Bauman Z, Braun A, Vitvitsky V, Lu SC, Banerjee R. S-adenosylmethionine stabilizes cystathionine beta-synthase and modulates redox capacity. Proc Natl Acad Sci USA. 2006; 103(17): 6489-6494.
  19. Toohey JI. Possible involvement of hydrosulfide in B12-dependent methyl group transfer. Molecules. 2017; 22(4): 582.
  20. Kabil O, Banerjee R. Enzymology of H2S biogenesis, decay and signaling. Antioxid Redox Signal. 2014; 20(5): 770-782.
  21. Wu Y, Ding R, Zhang X, Zhang J, Huang Q, Liu L, et al. Meet-in-metabolite analysis: a novel strategy to identify connections between arsenic exposure and male infertility. Environ Int. 2021;147:106360.
  22. Mohammadi P, Hassani-Bafrani H, Tavalaee M, Dattilo M, Nasr- Esfahani MH. One-carbon cycle support rescues sperm damage in experimentally induced varicocoele in rats. BJU Int. 2018; 122(3): 480-489.
  23. Wang J, Wang W, Li S, Han Y, Zhang P, Meng G, et al. Hydrogen sulfide as a potential target in preventing spermatogenic failure and testicular dysfunction. Antioxid Redox Signal. 2018; 28(16): 1447- 1462.
  24. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. 2010. Available from: (09 Sep 2020).
  25. Naher ZU, Biswas SK, Mollah FH, Ali M, Arslan MI. Role of glutathione in male infertility. Bangladesh J Med Biochem. 2011; 4(2): 20-25.
  26. Fafula RV, Onufrovych OK, Iefremova UP, Melnyk OV, Nakonechnyi IA, Vorobets DZ, et al. Glutathione content in sperm cells of infertile men. Regul Mech Biosyst. 2017; 2(8): 157-161.
  27. Stuppia L, Franzago M, Ballerini P, Gatta V, Antonucci I. Epigenetics and male reproduction: the consequences of paternal lifestyle on fertility, embryo development, and children lifetime health. Clin Epigenetics. 2015; 7: 120.
  28. Tavalaee M, Razavi S, Nasr-Esfahani MH. Influence of sperm chromatin anomalies on assisted reproductive technology outcome. Fertil Steril. 2009; 91(4): 1119-1126.
  29. Bahreinian M, Tavalaee M, Abbasi H, Kiani-Esfahani A, Shiravi AH, Nasr-Esfahani MH. DNA hypomethylation predisposes sperm to DNA damage in individuals with varicocele. Syst Biol Reprod Med. 2015; 61(4): 179-186.
  30. Tavalaee M, Bahreinian M, Barekat F, Abbasi H, Nasr-Esfahani MH. Effect of varicocelectomy on sperm functional characteristics and DNA methylation. Andrologia. 2015; 47(8): 904-909.
  31. Rashidi M, Tavalaee M, Abbasi H, Nomikos M, Nasr-Esfahani MH. Increased de novo DNA methylation enzymes in sperm of individuals with varicocele. Cell J. 2021; 23(4): 389-396.
  32. Nuño-Ayala M, Guillén N, Arnal C, Lou-Bonafonte JM, de Martino A, García-de-Jalón JA, et al. Cystathionine β-synthase deficiency causes infertility by impairing decidualization and gene expression networks in uterus implantation sites. Physiol Genomics. 2012; 44(14): 702-716.
  33. Li G, Xie ZZ, Chua JM, Wong PC, Bian J. Hydrogen sulfide protects testicular germ cells against heat-induced injury. Nitric Oxide. 2015; 46: 165-171.