Levels of DNA, Protein, Lipid Oxidation and Apoptosis Biomarkers in Semen of Men with Hyperviscous Semen: A Cross-Sectional Study

Document Type : Original Article


1 Department of Biology, Faculty of Sciences, Rasht Branch, Islamic Azad University, Rasht, Iran

2 Chemical Injuries Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran


Background: Semen hyperviscosity is a threatening cause of abnormal spermatozoa and infertility in men. We aimed
to evaluate oxidative stress, antioxidants depletion and sperm apoptosis as main reasons for poor quality of spermatozoa
in men with hyperviscous semen.
Materials and Methods: In this cross-sectional study, ejaculate specimens were collected from fertile (n=102) and
infertile men with hyperviscous semen (n=123) and without semen hyperviscosity (n=143). Total antioxidant capacity
(TAC), glutathione (GSH), malondialdehyde (MDA), protein carbonyl (PC), 8-hydroxydeoxyguanosine (8-OHdG),
and were measured in semen samples to estimate oxidative stress status. Gene expression pattern of BAX, CASPASE-9,
CASPASE-3, and BCL2 was assessed to estimate sperm apoptosis.
Results: The average of sperm count, normal morphology, normal motility, and sperm vitality in men with hyperviscous
semen was significantly lower than infertile subjects without hyperviscous semen (P<0.01). Men with hyperviscous semen
exhibited higher levels of PC (8.34 ± 1.03 nmol/mg vs. 6.01 ± 0.93 nmol/mg, P=0.008), MDA (1.14 ± 0.27 nmol/
ml vs. 0.89 ± 0.22 nmol/ml, P=0.031), 8-OHdG (259.71 ± 24.59 ng/ml vs. 197.13 ± 18.47 ng/ml, P=0.009), but lower
TAC contents (1250.44 ± 66.23 μM/L vs. 1784.31 ± 89.87 μM/L, P=0.018) and GSH (3.82 ± 1.05 μM vs. 5.89 ± 0.87
μM, P=0.021) than men with non-viscous semen. The expression of BAX, CASPASE-3 and CASPASE-9 genes in men
with hyperviscous semen was significantly increased by 1.39-fold (P=0.041), 1.47-fold (P=0.046), 1.29-fold (P=0.048),
respectively, as compared with the infertile subjects without hyperviscous semen. However, BCL2 expression in infertile
men without hyperviscous semen was higher compared to those with hyperviscous semen (1.36-fold, P=0.044).
Conclusion: Hyperviscous semen is markedly associated with depletion of seminal plasma antioxidants, sperm membrane
lipid peroxidation, DNA and protein oxidation, and sperm apoptosis. Antioxidant therapy might be considered as a
valuable strategy to protect sperm cells against oxidative damage in cases with seminal fluid hyperviscosity.


  1. Ashkani H, Akbari A, Heydari ST. Epidemiology of depression among infertile and fertile couples in Shiraz, southern Iran. Indian J Med Sci. 2006; 60(10): 399-406.
  2. Kumar N, Singh AK. Trends of male factor infertility, an important cause of infertility: a review of literature. J Hum Reprod Sci. 2015; 8(4): 191-196.
  3. Tahmasbpour Marzouni E, Stern C, Henrik Sinclair A, Tucker EJ. Stem cells and organs-on-chips: new promising technologies for human infertility treatment. Endocr Rev. 2022; 43 (5): 878-906.
  4. Tahmasbpour Marzouni E, Ilkhani H, Beigi Harchegani A, Shafaghatian H, Layali I, Shahriary A. Epigenetic modifications, a new approach to male infertility etiology: a review. Int J Fertil Steril. 2022; 16(1): 1-9.
  5. Tahmasbpour Marzouni E, Beigi Harchegani A, Layali I. Chromosomal aneuploidies and associated rare genetic syndromes involved in male infertility. J Men Health. 2021; 17(3): 7-17.
  6. Anamthathmakula P, Winuthayanon W. Mechanism of semen liquefaction and its potential for a novel non-hormonal contraception. Biol Reprod. 2020; 103(2): 411-426.
  7. Beigi Harchegani A, Rahmani H, Tahmasbpour E, Shahriary A. Hyperviscous semen causes poor sperm quality and male infertility through induction of oxidative stress. Curr Urol. 2019; 13(1): 1-6.
  8. Elia J, Delfino M, Imbrogno N, Capogreco F, Lucarelli M, Rossi T, et al. Human semen hyperviscosity: prevalence, pathogenesis and therapeutic aspects. Asian J Androl. 2009; 11(5): 609-615.
  9. El-Bahrawy K, Rateb S, Khalifa M, Monaco D, Lacalandra G. Physical and kinematic properties of cryopreserved camel sperm after elimination of semen viscosity by different techniques. Anim Reprod Sci. 2017; 187: 100-108.
  10. Kershaw CM, Evans G, Rodney R, Maxwell WMC. Papain and its inhibitor E-64 reduce camelid semen viscosity without impairing sperm function and improve post-thaw motility rates. Reprod Fertil Dev. 2017; 29(6): 1107-1114.
  11. Anamthathmakula P, Erickson JA, Winuthayanon W. Blocking serine protease activity prevents Semenogelin degradation leading to Hyperviscous semen in humans. Biol Reprod. 2022; 106(5): 879-887.
  12. Barbagallo F, La Vignera S, Cannarella R, Crafa A, Calogero AE, Condorelli RA. The relationship between seminal fluid hyperviscosity and oxidative stress: a systematic review. Antioxidants (Basel). 2021; 10(3): 356.
  13. Colagar AH, Marzony ET. Ascorbic Acid in human seminal plasma: determination and its relationship to sperm quality. J Clin Biochem Nutr. 2009; 45(2): 144-149.
  14. Colagar AH, Marzony ET, Chaichi MJ. Zinc levels in seminal plasma are associated with sperm quality in fertile and infertile men. Nutr Res. 2009; 29(2): 82-88.
  15. Jorsaraei SGA, Soleiman M, Gholinezhad-Chari M, Yousefreza Yousefnia P, Tahmasbpour E, Shafi H. DNA, protein and lipid peroxidation as oxidative stress biomarkers in seminal plasma of smoker and nonsmoker infertile men. Casp J Appl Sci Res. 2015; 4(9): 16-23.
  16. Hosseinzadeh Kolagar A, Pouramir M, Tahmasbpour Marzouni I. Seminal plasma total antioxidants capacity of the infertile smoker and nonsmoker men. Shahid Chamran University Journal of Science. 2008; NEW SERIES(19 (Section B)): 124- 131.
  17. Kefer JC, Agarwal A, Sabanegh E. Role of antioxidants in the treatment of male infertility. Int J Urol. 2009; 16(5): 449-457.
  18. Agarwal A, Prabakaran SA. Mechanism, measurement, and prevention of oxidative stress in male reproductive physiology. Indian J Exp Biol. 2005; 43(11): 963-974.
  19. Layali I, Tahmasbpour E, Joulaei M, Jorsaraei SG, Farzanegi P. Total antioxidant capacity and lipid peroxidation in semen of patient with hyperviscosity. Cell J. 2015; 16(4): 554-559.
  20. World Health Organization. Manuale di laboratorio della WHO per l'esame del liquido seminale umano e dell'interazione tra spermatozoi e muco cervicale [Laboratory manual of the WHO for the examination of human semen and sperm-cervical mucus interaction]. Ann Ist Super Sanita. 2001; 37(1): I-XII, 1-123.
  21. Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril. 1986; 46(6): 1118-1123.
  22. Jorsaraei SGA, Firoozjaee A, Yousofreza Yousofnia P, Tahmasbpour Marzony E, Sarabi E. Histopathological effects of single dose treatment of diazinon on testes structure in rat. Cell J. 2010; 12(1): 39-42.
  23. Benzie IF. Lipid peroxidation: a review of causes, consequences, measurement and dietary influences. Int J Food Sci Nutr. 1996; 47(3): 233-261.
  24. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969; 27(3): 502-522.
  25. Hosseinzadeh Colagar A, Pouramir M, Tahmasbpour Marzony E, Jorsaraei SGA. Relationship between seminal malondialdehyde levels and sperm quality in fertile and infertile men. Braz Arch Biol Technol. 2009; 52(6): 1387-1392.
  26. Gholinezhad M, Aliarab A, Abbaszadeh-Goudarzi G, Yousefnia-Pasha Y, Samadaian N, Rasolpour-Roshan K, et al. Nitric oxide, 8-hydroxydeoxyguanosine, and total antioxidant capacity in human seminal plasma of infertile men and their relationship with sperm parameters. Clin Exp Reprod Med. 2020; 47(1): 54-60.
  27. Levine RL, Williams JA, Stadtman ER, Shacter E. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol. 1994; 233: 346-357.
  28. Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, et al. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990; 186: 464-478.
  29. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25(4): 402-408.
  30. ELzanaty S, Malm J, Giwercman A. Visco-elasticity of seminal fluid in relation to the epididymal and accessory sex gland function and its impact on sperm motility. Int J Androl. 2004; 27(2): 94-100.
  31. Siciliano L, Tarantino P, Longobardi F, Rago V, De Stefano C, Carpino A. Impaired seminal antioxidant capacity in human semen with hyperviscosity or oligoasthenozoospermia. J Androl. 2001; 22(5): 798-803.
  32. Aydemir B, Onaran I, Kiziler AR, Alici B, Akyolcu MC. The influence of oxidative damage on viscosity of seminal fluid in infertile men. J Androl. 2008; 29(1): 41-46.
  33. Quayle AJ, Xu C, Mayer KH, Anderson DJ. T lymphocytes and macrophages, but not motile spermatozoa, are a significant source of human immunodeficiency virus in semen. J Infect Dis. 1997; 176(4): 960-968.
  34. Mahran Z, Saleh ME. Human semen hyperviscosity: prevalence and effects on physical and biochemical semen parameters in subfertile Egyptian men. Egypt J Dermatol Venerol . 2014; 34(2): 135-139.