Cryopreservation of Limited Sperm Using A Combination of Sucrose and Taurine, Loaded on Two Different Devices, and Thawed at Two Different Temperatures

Document Type : Original Article


1 Department of Anatomy, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran

2 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran


Background: Cryopreservation of sperm is essential for patients with low sperm counts and couples undergoing infertility
treatment. The aim of this study was to compare the effects of Taurine (T) and Sucrose (S) in individual sperm
cryopreservation utilizing cryotop and petri dish and thawing at 37 and 42°C.
Materials and Methods: In this experimental study, 17 normospermic semen samples were processed using the
"Swim-up" procedure and progressively motile sperm were then isolated from these samples using an inverted microscope.
Sperm were added to droplets of "sucrose medium" with 25 mM Taurine antioxidant (S+T) and the commercial
cryoprotectant "Sperm Freeze" (CPA), loaded on a petri dish and cryotop. After rapid freezing of the samples, they
were thawed at two different temperatures (37°C and 42°C), and the sperm classical parameters, viability, and DNA
fragmentation were assessed.
Results: Statistical analysis displayed a significant increase in total and progressive motility in individual sperm
freezing on cryotop with CPA and thawing at 42°C (P<0.05). Other parameters did not show any differences between
the CPA and S+T groups and two thawing temperatures in either of the cryopreservation methods.
Conclusion: Although, both cryoprotectants (CPA and S+T) may preserve individual sperm effectively using cryotop,
the CPA and thawing at 42°C showed a better effect on the motility percentage of the small number of sperm.


Main Subjects

  1. Diedrich K, Fauser BC, Devroey P; Evian Annual Reproduction (EVAR) Workshop Group 2009. Cancer and fertility: strategies to preserve fertility. Reprod Biomed Online. 2011; 22(3): 232-248.
  2. Liu S, Li F. Cryopreservation of single-sperm: where are we today? Reprod Biol Endocrinol. 2020; 18(1): 41.
  3. Zhu ZJ, Zhai J, Hu JL, Wang YZ, Chen W, Liu F, et al. Cryopiece, a novel carrier with faster cooling rate, high recovery rate and retrieval rate, for individual sperm cryopreservation. Transl Androl Urol. 2021; 10(3): 1121-1132.
  4. Herbemont C, Mnallah S, Grynberg M, Sifer C. Prospective comparison of different techniques for cryopreservation of small numbers of human spermatozoa. Gynecol Obstet Fertil Senol. 2019; 47(11): 797-801.
  5. Peng QP, Cao SF, Lyu QF, Xue SG, Jin W, Liu XY, et al. A novel method for cryopreservation of individual human spermatozoa. In Vitro Cell Dev Biol Anim. 2011; 47(8): 565-572.
  6. Sereni E, Bonu MA, Fava L, Sciajno R, Serrao L, Preti S, et al. Freezing spermatozoa obtained by testicular fine needle aspiration: a new technique. Reprod Biomed Online. 2008; 16(1): 89-95.
  7. Endo Y, Fujii Y, Shintani K, Seo M, Motoyama H, Funahashi H. Single spermatozoon freezing using Cryotop. J Mamm Ova Res. 2011; 28(1): 47-52.
  8. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology. 2007; 67(1): 73-80.
  9. Najafi A, Adutwum E, Yari A, Salehi E, Mikaeili S, Dashtestani F, et al. Melatonin affects membrane integrity, intracellular reactive oxygen species, caspase3 activity and AKT phosphorylation in frozen thawed human sperm. Cell Tissue Res. 2018; 372(1): 149-159.
  10. Chen Y, Li L, Qian Y, Xu C, Zhu Y, Huang H, et al. Small-volume vitrification for human spermatozoa in the absence of cryoprotectants by using Cryotop. Andrologia. 2015; 47(6): 694- 699.
  11. 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(6): 563-569.
  12. Surai PF, Earle-Payne K, Kidd MT. Taurine as a natural antioxidant: from direct antioxidant effects to protective action in various toxicological models. Antioxidants (Basel). 2021; 10(12): 1876.
  13. Li Y, Peng Q, Shang J, Dong W, Wu S, Guo X, et al. The role of taurine in male reproduction: Physiology, pathology and toxicology. Front Endocrinol (Lausanne). 2023; 14: 1017886.
  14. Seify M, Zarabadipour M, Ghaleno LR, Alizadeh A, Rezazadeh Valojerdi M. The anti-oxidant roles of Taurine and Hypotaurine on acrosome integrity, HBA and HSPA2 of the human sperm during vitrification and post warming in two different temperature. Cryobiology. 2019; 90: 89-95.
  15. Abdalkarim Salih S, Daghigh-Kia H, Mehdipour M, Najafi A. Does ergothioneine and thawing temperatures improve rooster semen post-thawed quality? Poult Sci. 2021; 100(10): 101405.
  16. Mansilla MA, Merino O, Risopatrón J, Isachenko V, Isachenko E, Sánchez R. High temperature is essential for preserved human sperm function during the devitrification process. Andrologia. 2016; 48(1): 111-113.
  17. AbdelHafez F, Bedaiwy M, El-Nashar SA, Sabanegh E, Desai N. Techniques for cryopreservation of individual or small numbers of human spermatozoa: a systematic review. Hum Reprod Update. 2009; 15(2): 153-164.
  18. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva; WHO Press; 2010.
  19. Ghaleno LR, Valojerdi MR, Janzamin E, Chehrazi M, Sharbatoghli M, Yazdi RS. Evaluation of conventional semen parameters, intracellular reactive oxygen species, DNA fragmentation and dysfunction of mitochondrial membrane potential after semen preparation techniques: a flow cytometric study. Arch Gynecol Obstet. 2014; 289(1): 173-180.
  20. Sereni E, Bonu MA, Fava L, Sciajno R, Serrao L, Preti S, et al. Freezing spermatozoa obtained by testicular fine needle aspiration: a new technique. Reprod Biomed Online. 2008; 16(1): 89-95.
  21. Agha-Rahimi A, Khalili MA, Nabi A, Ashourzadeh S. Vitrification is not superior to rapid freezing of normozoospermic spermatozoa: effects on sperm parameters, DNA fragmentation and hyaluronan binding. Reprod Biomed Online. 2014; 28(3): 352-358.
  22. Sharma R, Agarwal A. Spermatogenesis: an overview. In: Zini A, Agarwal A, editors. Sperm chromatin. New York: Springer; 2011: 19-44.
  23. Huang WJ, Zhang D, Hong ZW, Chen ZB, Dong LH, Zhang Y, et al. Sequential interval micro-droplet loading in closed hemi-straw carrier system: a convenient and efficient method for ultra-rapid cryopreservation in extreme oligozoospermia. Cryobiology. 2020; 93: 75-83.
  24. Bouamama N, Briot P, Testart J. Comparison of two methods of cryoconservation of sperm when in very small numbers. Gynecol Obstet Fertil. 2003; 31(2): 132-135.
  25. Pabón D, Meseguer M, Sevillano G, Cobo A, Romero JL, Remohí J, et al. A new system of sperm cryopreservation: evaluation of survival, motility, DNA oxidation, and mitochondrial activity. Andrology. 2019; 7(3): 293-301.
  26. Schulz M, Risopatrón J, Uribe P, Isachenko E, Isachenko V, Sánchez R. Human sperm vitrification: a scientific report. Andrology. 2020; 8(6): 1642-1650.
  27. Malo C, Elwing B, Soederstroem L, Lundeheim N, Morrell JM, Skidmore JA. Effect of different freezing rates and thawing temperatures on cryosurvival of dromedary camel spermatozoa. Theriogenology. 2019; 125: 43-48.
  28. El-Ahwany A, Samir H, Alahwany H. Using two different thawing temperatures and their effect on the motility recovery of human cryopreserved sperms in cancer patients. Middle East Fertil Soc J. 2018; 23(4): 331-334.
  29. Karimi Zarchi M, Maleki B, Dehghani Ashkezari M, Motamed Zadeh L, Agha-Rahimi A. The effects of in vitro incubation of asthenoteratozoospermic semen after density gradient centrifugation at room temperature and 37°c on sperm parameters, chromatin quality and DNA fragmentation in a short time period. J Reprod Infertil. 2020; 21(4): 275-282.
  30. Thijssen A, Klerkx E, Huyser C, Bosmans E, Campo R, Ombelet W. Influence of temperature and sperm preparation on the quality of spermatozoa. Reprod Biomed Online. 2014; 28(4): 436-442.