Evaluating the Impact of Hyaluronic Acid on Sperm Quality Before and After Cryopreservation in A Moroccan Cohort: A Pilot Study
-
Mehdi EL Mokaddem
,
Zeineb Zian
,
Amr Kchikich
,
Noureddine Louanjli
,
Naima Ghailani Nourouti
,
Amina Barakat
,
Moudou M Mbaye
,
Mohcine Bennani Mechita
Abstract
Objective: This study aimed to evaluate the effects of hyaluronic acid (HA) supplementation on motility, vitality, and DNA integrity in Moroccan fresh and cryopreserved sperm samples.
Materials and methods: A cohort of 60 male participants was divided into three groups, including normozoospermic, asthenozoospermic, and cryopreserved normozoospermic samples. Advanced microscopy and computer-assisted semen analysis (CASA) were used to analyze the sperm parameters before and after HA supplementation. DNA fragmentation was analyzed using TUNEL assay, while chromatin condensation was evaluated by aniline blue staining. Statistical analysis was performed using ANOVA and Duncan's test.
Results: HA supplementation improved sperm motility and vitality (p < 0.05) in the normozoospermic group without altering DNA fragmentation or chromatin condensation. In the asthenozoospermic group, motility and vitality were significantly improved (p < 0.05). In the cryopreserved normozoospermic group, HA improved motility and vitality after thawing (p < 0.05).
Conclusion: HA is a promising adjuvant for enhancing the quality of both fresh and cryopreserved sperm for ART without compromising DNA integrity. Studies need to be extended for further exploration of the mechanism of action and optimization of clinical protocols toward its use in routine practice, particularly for diverse populations and in the more difficult cases of infertility.
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2. Huang B, Wang Z, Kong Y, Jin M, Ma L. Global, regional and national burden of male infertility in 204 countries and territories between 1990 and 2019: an analysis of Global Burden of Disease Study. BMC Public Health. 2023;23(1).
3. Cavarocchi E, Whitfield M, Drouault M, Touré A, Ribeiro JC, Simon V. Human asthenozoospermia: Update on genetic causes, patient management, and clinical strategies. 2025:1–21.
4. Stavros S, Potiris A, Molopodi E, Mavrogianni D, Zikopoulos A, Louis K, et al. Sperm DNA fragmentation: Unraveling its imperative impact on male infertility based on recent evidence. Int J Mol Sci. 2024;25(18).
5. Agarwal A, Virk G, Ong C, du Plessis SS. Effect of oxidative stress on male reproduction. World J Mens Health. 2014;32(1):1.
6. Sharma R, Iovine C, Agarwal A, Henkel R. TUNEL assay—standardized method for testing sperm DNA fragmentation. Andrologia. 2021;53.
7. Agarwal A, Baskaran S, Parekh N, Cho CL, Henkel R, Vij S, et al. Male infertility. Lancet. 2021;397:319–33.
8. Hallam J, Burton P, Sanders K. Poor sperm chromatin condensation is associated with cryopreservation-induced DNA fragmentation and cell death in human spermatozoa. J Clin Med. 2024;13(14).
9. Fouladi-Nashta AA, Raheem KA, Marei WF, Ghafari F, Hartshorne GM. Regulation and roles of the hyaluronan system in mammalian reproduction. Reproduction. 2017;153(2):R43–58.
10. Simulescu V, Kalina M, Mondek J, Pekař M. Long-term degradation study of hyaluronic acid in aqueous solutions. Carbohydr Polym. 2016;137:664–8.
11. Sbracia M, Grasso J, Sayme N, Stronk J, Huszar G. Hyaluronic acid substantially increases retention of motility in cryopreserved human spermatozoa. Hum Reprod. 1997;12(9):1949–54.
12. Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MCG. Effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and lipid peroxidation. J Androl. 2000;21(6):895–902.
13. Qian L, Yu S, Zhou Y. Protective effect of hyaluronic acid on cryopreserved boar sperm. Int J Biol Macromol. 2016;87:287–9.
14. Bansal AK. Antioxidants and other potent strategies to reduce oxidative stress in semen. In: Free Radicals in Human Health and Disease. Springer India; 2015. p. 381–95.
15. Marchlewska K, Erkiert-Kusiak M, Walczak-Jędrzejowska R, Słowikowska-Hilczer J. Sperm migration and hyaluronic acid binding: implications for male fertility evaluation. Int J Mol Sci. 2024;25(18).
16. Chung E, Arafa M, Boitrelle F, Kandil H, Henkel R, Saleh R, et al. The new 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen. Asian J Androl. 2022;24:123–4.
17. Björndahl L, Söderlund I, Kvist U. Evaluation of one‐step eosin‐nigrosin staining technique for human sperm vitality assessment. Hum Reprod. 2003;18(4):813–6.
18. Sharma R, Biedenharn KR, Fedor JM, Agarwal A. Lifestyle factors and reproductive health: taking control of your fertility. Reprod Biol Endocrinol. 2013;11.
19. Tao Y, Sanger E, Saewu A, Leveille MC. Human sperm vitrification: state of the art. Reprod Biol Endocrinol. 2020;18.
20. Huang C, Tang YL, Hu JL, Zhou WJ, Huang ZH, Luo XF, et al. Update on techniques for cryopreservation of human spermatozoa. Asian J Androl. 2022;24:563–9.
21. Huszar G, Jakab A, Sakkas D, Ozenci CC, Cayli S, Delpiano E, et al. Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects. Reprod Biomed Online. 2007;14:650–63.
22. Roushandeh AM, Sohrabvand F, Amanpour S, Pasbakhsh P, Sobhani A, Nikzad H, et al. Effects of different doses of hyaloronan on human sperm motility, vitality and morphology. Acta Med Iran. 2009;47(5):369–72.
23. Parmegiani L, Cognigni GE, Bernardi S, Troilo E, Ciampaglia W, Filicori M. “Physiologic ICSI”: hyaluronic acid favors selection of spermatozoa without DNA fragmentation and with normal nucleus. Fertil Steril. 2010;93(2):598–604.
24. Huszar G, Ozenci CC, Cayli S, Zavaczki Z, Hansch E, Vigue L. Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status. Fertil Steril. 2003;79(Suppl 3):1616–24.
25. Saylan A, Abant B, Duman S. Efficacy of hyaluronic acid in the selection of human spermatozoa with intact DNA by the swim-up method. Cell J. 2016.
26. Jakab A, Sakkas D, Delpiano E, Cayli S, Kovanci E, Ward D, et al. Novel selection method for sperm with normal chromosomal aneuploidies during ICSI. Fertil Steril. 2005;84(6):1665–73.
27. Beck-Fruchter R, Shalev E, Weiss A. Clinical benefit of sperm hyaluronic acid binding in ICSI cycles: systematic review and meta-analysis. Reprod Biomed Online. 2016;32(3):286–98.
28. Venkatesh S, Singh A, Shamsi MB, Thilagavathi J, Kumar R, Mitra DK, et al. Clinical significance of sperm DNA damage threshold value in male infertility. Reprod Sci. 2011;18(10):1005–13.
29. Vozdova M, Oracova E, Kasikova K, Prinosilova P, Rybar R, Horinova V, et al. Balanced chromosomal translocations in men: relations among semen parameters, chromatin integrity, sperm meiotic segregation and aneuploidy. J Assist Reprod Genet. 2013;30(3):391–405.
30. Bucak MN, Ateşşahin A, Varişli Ö, Yüce A, Tekin N, Akçay A. Influence of trehalose, taurine, cysteamine and hyaluronan on ram semen after freeze-thawing. Theriogenology. 2007;67(5):1060–7.
31. Eidan SM. Effect of adding vitamin C with catalase or reduced glutathione to Tris extender on post-thaw semen of Holstein bulls. Anim Reprod Sci. 2016;167:1–7.
32. Salmani H, Nabi MM, Vaseghi-Dodaran H, Rahman MB, Mohammadi-Sangcheshmeh A, Shakeri M, et al. Effect of glutathione in soybean lecithin-based extender on goat semen quality after freeze-thawing. Small Rumin Res. 2013;112:123–7.
33. Peña FJ, Johannisson A, Wallgren M, Rodriguez-Martinez H. Antioxidant supplementation improves boar sperm motility and mitochondrial potential after cryopreservation. Anim Reprod Sci. 2003;78(1–2):85–98.
34. Singh A, Li P, Beachley V, McDonnell P, Elisseeff JH. Hyaluronic acid-binding contact lens with enhanced water retention. Contact Lens Anterior Eye. 2015;38(2):79–84.
35. Suzuki K, Asano A, Eriksson B, Niwa K, Nagai T, Rodriguez-Martinez H. Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal plasma, COC-conditioned medium and hyaluronan. Int J Androl. 2002;25(2):84–93.
36. Erenpreiss J, Spano M, Erenpreisa J, Bungum M, Giwercman A. Sperm chromatin structure and male fertility: biological and clinical aspects. Asian J Androl. 2006;8:11–29.
37. Peris SI, Morrier A, Dufour M, Bailey JL. Cryopreservation of ram semen facilitates sperm DNA damage. J Androl. 2004;25(2):224–33.
2. Huang B, Wang Z, Kong Y, Jin M, Ma L. Global, regional and national burden of male infertility in 204 countries and territories between 1990 and 2019: an analysis of Global Burden of Disease Study. BMC Public Health. 2023;23(1).
3. Cavarocchi E, Whitfield M, Drouault M, Touré A, Ribeiro JC, Simon V. Human asthenozoospermia: Update on genetic causes, patient management, and clinical strategies. 2025:1–21.
4. Stavros S, Potiris A, Molopodi E, Mavrogianni D, Zikopoulos A, Louis K, et al. Sperm DNA fragmentation: Unraveling its imperative impact on male infertility based on recent evidence. Int J Mol Sci. 2024;25(18).
5. Agarwal A, Virk G, Ong C, du Plessis SS. Effect of oxidative stress on male reproduction. World J Mens Health. 2014;32(1):1.
6. Sharma R, Iovine C, Agarwal A, Henkel R. TUNEL assay—standardized method for testing sperm DNA fragmentation. Andrologia. 2021;53.
7. Agarwal A, Baskaran S, Parekh N, Cho CL, Henkel R, Vij S, et al. Male infertility. Lancet. 2021;397:319–33.
8. Hallam J, Burton P, Sanders K. Poor sperm chromatin condensation is associated with cryopreservation-induced DNA fragmentation and cell death in human spermatozoa. J Clin Med. 2024;13(14).
9. Fouladi-Nashta AA, Raheem KA, Marei WF, Ghafari F, Hartshorne GM. Regulation and roles of the hyaluronan system in mammalian reproduction. Reproduction. 2017;153(2):R43–58.
10. Simulescu V, Kalina M, Mondek J, Pekař M. Long-term degradation study of hyaluronic acid in aqueous solutions. Carbohydr Polym. 2016;137:664–8.
11. Sbracia M, Grasso J, Sayme N, Stronk J, Huszar G. Hyaluronic acid substantially increases retention of motility in cryopreserved human spermatozoa. Hum Reprod. 1997;12(9):1949–54.
12. Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MCG. Effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and lipid peroxidation. J Androl. 2000;21(6):895–902.
13. Qian L, Yu S, Zhou Y. Protective effect of hyaluronic acid on cryopreserved boar sperm. Int J Biol Macromol. 2016;87:287–9.
14. Bansal AK. Antioxidants and other potent strategies to reduce oxidative stress in semen. In: Free Radicals in Human Health and Disease. Springer India; 2015. p. 381–95.
15. Marchlewska K, Erkiert-Kusiak M, Walczak-Jędrzejowska R, Słowikowska-Hilczer J. Sperm migration and hyaluronic acid binding: implications for male fertility evaluation. Int J Mol Sci. 2024;25(18).
16. Chung E, Arafa M, Boitrelle F, Kandil H, Henkel R, Saleh R, et al. The new 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen. Asian J Androl. 2022;24:123–4.
17. Björndahl L, Söderlund I, Kvist U. Evaluation of one‐step eosin‐nigrosin staining technique for human sperm vitality assessment. Hum Reprod. 2003;18(4):813–6.
18. Sharma R, Biedenharn KR, Fedor JM, Agarwal A. Lifestyle factors and reproductive health: taking control of your fertility. Reprod Biol Endocrinol. 2013;11.
19. Tao Y, Sanger E, Saewu A, Leveille MC. Human sperm vitrification: state of the art. Reprod Biol Endocrinol. 2020;18.
20. Huang C, Tang YL, Hu JL, Zhou WJ, Huang ZH, Luo XF, et al. Update on techniques for cryopreservation of human spermatozoa. Asian J Androl. 2022;24:563–9.
21. Huszar G, Jakab A, Sakkas D, Ozenci CC, Cayli S, Delpiano E, et al. Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects. Reprod Biomed Online. 2007;14:650–63.
22. Roushandeh AM, Sohrabvand F, Amanpour S, Pasbakhsh P, Sobhani A, Nikzad H, et al. Effects of different doses of hyaloronan on human sperm motility, vitality and morphology. Acta Med Iran. 2009;47(5):369–72.
23. Parmegiani L, Cognigni GE, Bernardi S, Troilo E, Ciampaglia W, Filicori M. “Physiologic ICSI”: hyaluronic acid favors selection of spermatozoa without DNA fragmentation and with normal nucleus. Fertil Steril. 2010;93(2):598–604.
24. Huszar G, Ozenci CC, Cayli S, Zavaczki Z, Hansch E, Vigue L. Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status. Fertil Steril. 2003;79(Suppl 3):1616–24.
25. Saylan A, Abant B, Duman S. Efficacy of hyaluronic acid in the selection of human spermatozoa with intact DNA by the swim-up method. Cell J. 2016.
26. Jakab A, Sakkas D, Delpiano E, Cayli S, Kovanci E, Ward D, et al. Novel selection method for sperm with normal chromosomal aneuploidies during ICSI. Fertil Steril. 2005;84(6):1665–73.
27. Beck-Fruchter R, Shalev E, Weiss A. Clinical benefit of sperm hyaluronic acid binding in ICSI cycles: systematic review and meta-analysis. Reprod Biomed Online. 2016;32(3):286–98.
28. Venkatesh S, Singh A, Shamsi MB, Thilagavathi J, Kumar R, Mitra DK, et al. Clinical significance of sperm DNA damage threshold value in male infertility. Reprod Sci. 2011;18(10):1005–13.
29. Vozdova M, Oracova E, Kasikova K, Prinosilova P, Rybar R, Horinova V, et al. Balanced chromosomal translocations in men: relations among semen parameters, chromatin integrity, sperm meiotic segregation and aneuploidy. J Assist Reprod Genet. 2013;30(3):391–405.
30. Bucak MN, Ateşşahin A, Varişli Ö, Yüce A, Tekin N, Akçay A. Influence of trehalose, taurine, cysteamine and hyaluronan on ram semen after freeze-thawing. Theriogenology. 2007;67(5):1060–7.
31. Eidan SM. Effect of adding vitamin C with catalase or reduced glutathione to Tris extender on post-thaw semen of Holstein bulls. Anim Reprod Sci. 2016;167:1–7.
32. Salmani H, Nabi MM, Vaseghi-Dodaran H, Rahman MB, Mohammadi-Sangcheshmeh A, Shakeri M, et al. Effect of glutathione in soybean lecithin-based extender on goat semen quality after freeze-thawing. Small Rumin Res. 2013;112:123–7.
33. Peña FJ, Johannisson A, Wallgren M, Rodriguez-Martinez H. Antioxidant supplementation improves boar sperm motility and mitochondrial potential after cryopreservation. Anim Reprod Sci. 2003;78(1–2):85–98.
34. Singh A, Li P, Beachley V, McDonnell P, Elisseeff JH. Hyaluronic acid-binding contact lens with enhanced water retention. Contact Lens Anterior Eye. 2015;38(2):79–84.
35. Suzuki K, Asano A, Eriksson B, Niwa K, Nagai T, Rodriguez-Martinez H. Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal plasma, COC-conditioned medium and hyaluronan. Int J Androl. 2002;25(2):84–93.
36. Erenpreiss J, Spano M, Erenpreisa J, Bungum M, Giwercman A. Sperm chromatin structure and male fertility: biological and clinical aspects. Asian J Androl. 2006;8:11–29.
37. Peris SI, Morrier A, Dufour M, Bailey JL. Cryopreservation of ram semen facilitates sperm DNA damage. J Androl. 2004;25(2):224–33.
| Files | ||
| Issue | Vol 19, No 4 (December 2025) | |
| Section | Original Articles | |
| Keywords | ||
| Cryopreservation Fertility Hyaluronic Acid Male infertility Morocco | ||
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How to Cite
1.
Mokaddem M, Zian Z, Kchikich A, Louanjli N, Nourouti N, Barakat A, Mbaye M, Mechita M. Evaluating the Impact of Hyaluronic Acid on Sperm Quality Before and After Cryopreservation in A Moroccan Cohort: A Pilot Study. J Family Reprod Health. 2025;19(4):301-309.
