Seren CAN ÇELİK, Ümit ÖZEKİCİ, Mehmet CINCIK, Belgin SELAM, Yaprak DÖNMEZ ÇAKIL

Effects of antioxidants on motility and DNA integrity in frozen-thawed sperm

Öz Aims: This study aims to investigate whether the addition of the antioxidant complex including taurine, ascorbic acid, and glutathione into the cryopreservation medium affects the damage to sperm during the freezing process. Methods: Ejaculate samples of patients who applied for semen analysis to the Assisted Reproduction Unit of Private Adatip Hospital were used. Fresh samples were analyzed for standard semen quality parameters according to the World Health Organization guidelines. Samples within the normal range were evaluated for sperm DNA fragmentation using the Halosperm technique. Remaining ejaculates were washed with the gradient method before cryopreservation. Sperm samples of each patient were divided equally for freezing in a cryopreservation medium with or without the antioxidant supplementation. One month later, the samples were thawed. Post-thaw total motility and DNA fragmentation were determined for each sample. Results: Semen samples of 40 patients were analyzed. We observed decreased total motility (34.8 ±5.32 % vs. 65.5 ±6.42 %, P= 0.002) and increased sperm DNA fragmentation (52.3±5.42 % vs. 26.4±3.12 %, P= 0.002) in post-thaw semen samples following cryopreservation in comparison to fresh samples. The addition of antioxidants to the freezing medium did not have a statistically significant effect on sperm motility (38.3± 6.22 % vs. 34.8 ±5.32 %, P =0.07) and DNA damage (47.5±4.7 % vs. 52.3±5.42, P =0.08) when compared to control samples following the freezing process. Conclusions: We observed increased sperm DNA fragmentation and decreased total motility following cryopreservation. No significant improvement in sperm motility or DNA integrity was obtained after the addition of 5 µM of the antioxidants taurine, ascorbic acid, and glutathione to the freezing media.

Anahtar Kelimeler:

Halosperm, DNA fragmentation, antioxidants, sperm freezing, motility, cryopreservation

PDF

___

1. Sherman JK. Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertility and sterility. 1973 May;24(5):397-412.

2. Desrosiers P, Legare C, Leclerc P, Sullivan R. Membranous and structural damage that occur during cryopreservation of human sperm may be time-related events. Fertility and sterility. 2006 Jun;85(6):1744-52.

3. Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Karahuseyinoglu SJJoAR, Genetics. Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. 2008 August 01;25(8):403-11.

4. Oktay K, Harvey BE, Partridge AH, Quinn GP, Reinecke J, Taylor HS, et al. Fertility Preservation in Patients With Cancer: ASCO Clinical Practice Guideline Update. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2018 Jul 1;36(19):1994-2001.

5. Henkel R, Maaß G, Bödeker R-H, Scheibelhut C, Stalf T, Mehnert C, et al. Sperm function and assisted reproduction technology. Reprod Med Biol. 2005;4(1):7-30.

6. Horne G, Atkinson AD, Pease EH, Logue JP, Brison DR, Lieberman BA. Live birth with sperm cryopreserved for 21 years prior to cancer treatment: case report. Human reproduction (Oxford, England). 2004 Jun;19(6):1448-9.

7. Szell AZ, Bierbaum RC, Hazelrigg WB, Chetkowski RJ. Live births from frozen human semen stored for 40 years. Journal of assisted reproduction and genetics. 2013 Jun;30(6):743-4.

8. Di Santo M, Tarozzi N, Nadalini M, Borini A. Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART. Advances in urology. 2012;2012:854837.

9. Rozati H, Handley T, Jayasena CN. Process and Pitfalls of Sperm Cryopreservation. Journal of clinical medicine. 2017 Sep 19;6(9).

10. Hezavehei M, Sharafi M, Kouchesfahani HM, Henkel R, Agarwal A, Esmaeili V, et al. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reproductive biomedicine online. 2018 Sep;37(3):327-39.

11. Meamar M, Zribi N, Cambi M, Tamburrino L, Marchiani S, Filimberti E, et al. Sperm DNA fragmentation induced by cryopreservation: new insights and effect of a natural extract from Opuntia ficus-indica. Fertility and sterility. 2012 Aug;98(2):326-33.

12. Cankut S, Dinc T, Cincik M, Ozturk G, Selam B. Evaluation of Sperm DNA Fragmentation via Halosperm Technique and TUNEL Assay Before and After Cryopreservation. Reproductive sciences (Thousand Oaks, Calif). 2019 Dec;26(12):1575-81.

13. Le MT, Nguyen TTT, Nguyen TT, Nguyen TV, Nguyen TAT, Nguyen QHV, et al. Does conventional freezing affect sperm DNA fragmentation? Clinical and experimental reproductive medicine. 2019 Jun;46(2):67-75.

14. Thomson LK, Fleming SD, Aitken RJ, De Iuliis GN, Zieschang JA, Clark AM. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Human reproduction (Oxford, England). 2009 Sep;24(9):2061-70.

15. Said TM, Gaglani A, Agarwal A. Implication of apoptosis in sperm cryoinjury. Reproductive biomedicine online. 2010 Oct;21(4):456-62.

16. Donnelly ET, McClure N, Lewis SE. Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity. Fertility and sterility. 2001 Nov;76(5):892-900.

17. Agarwal A, Nallella KP, Allamaneni SS, Said TM. Role of antioxidants in treatment of male infertility: an overview of the literature. Reproductive biomedicine online. 2004 Jun;8(6):616-27.

18. Atig F, Raffa M, Habib B-A, Kerkeni A, Saad A, Ajina M. Impact of seminal trace element and glutathione levels on semen quality of Tunisian infertile men. BMC Urol. 2012;12:6-.

19. Donnelly ET, Mcclure N, Lewis SEM. Glutathione and hypotaurine in vitro: effects on human sperm motility, DNA integrity and production of reactive oxygen species. Mutagenesis. 2000;15(1):61-8.

20. World Health Organization. WHO Laboratory Manual for the examination and processing of human semen. Cambridge: Cambridge University Press; 2010.

21. Menkveld R, Stander FS, Kotze TJ, Kruger TF, van Zyl JA. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Human reproduction (Oxford, England). 1990 Jul;5(5):586-92.

22. Fernandez JL, Muriel L, Goyanes V, Segrelles E, Gosalvez J, Enciso M, et al. Halosperm is an easy, available, and cost-effective alternative for determining sperm DNA fragmentation. Fertility and sterility. 2005 Oct;84(4):860.

23. Lewis SE, John Aitken R, Conner SJ, Iuliis GD, Evenson DP, Henkel R, et al. The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment. Reproductive biomedicine online. 2013 Oct;27(4):325-37.

24. Martin-Hidalgo D, Bragado MJ, Batista AR, Oliveira PF, Alves MG. Antioxidants and Male Fertility: from Molecular Studies to Clinical Evidence. Antioxidants (Basel, Switzerland). 2019 Apr 5;8(4).

25. Kefer JC, Agarwal A, Sabanegh E. Role of antioxidants in the treatment of male infertility. International journal of urology : official journal of the Japanese Urological Association. 2009 May;16(5):449-57.

26. Showell MG, Mackenzie-Proctor R, Brown J, Yazdani A, Stankiewicz MT, Hart RJ. Antioxidants for male subfertility. The Cochrane database of systematic reviews. 2014 (12):Cd007411.

27. Figueroa E, Farias JG, Lee-Estevez M, Valdebenito I, Risopatrón J, Magnotti C, et al. Sperm cryopreservation with supplementation of α-tocopherol and ascorbic acid in freezing media increase sperm function and fertility rate in Atlantic salmon (Salmo salar). Aquaculture. 2018 2018/08/01/;493:1-8.

28. Zribi N, Feki Chakroun N, El Euch H, Gargouri J, Bahloul A, Ammar Keskes L. Effects of cryopreservation on human sperm deoxyribonucleic acid integrity. Fertility and sterility. 2010;93(1):159-66.

29. Agarwal A, Makker K, Sharma R. Clinical relevance of oxidative stress in male factor infertility: an update. American journal of reproductive immunology (New York, NY : 1989). 2008 Jan;59(1):2-11.

30. Kalthur G, Raj S, Thiyagarajan A, Kumar S, Kumar P, Adiga SK. Vitamin E supplementation in semen-freezing medium improves the motility and protects sperm from freeze-thaw-induced DNA damage. Fertility and sterility. 2011 Mar 1;95(3):1149-51.

31. Banihani S, Agarwal A, Sharma R, Bayachou M. Cryoprotective effect of L-carnitine on motility, vitality and DNA oxidation of human spermatozoa. Andrologia. 2014 Aug;46(6):637-41.

32. Ghorbani M, Vatannejad A, Khodadadi I, Amiri I, Tavilani H. PROTECTIVE EFFECTS OF GLUTATHIONE SUPPLEMENTATION AGAINST OXIDATIVE STRESS DURING CRYOPRESERVATION OF HUMAN SPERMATOZOA. Cryo letters. 2016 Jan-Feb;37(1):34-40.

33. Taylor K, Roberts P, Sanders K, Burton P. Effect of antioxidant supplementation of cryopreservation medium on post-thaw integrity of human spermatozoa. Reproductive biomedicine online. 2009 Feb;18(2):184-9.

34. Mangoli E, Talebi AR, Anvari M, Taheri F, Vatanparast M, Rahiminia T, et al. Vitamin C attenuates negative effects of vitrification on sperm parameters, chromatin quality, apoptosis and acrosome reaction in neat and prepared normozoospermic samples. Taiwanese Journal of Obstetrics and Gynecology. 2018 2018/04/01/;57(2):200-4.

35. Askari HA, Check JH, Peymer N, Bollendorf A. Effect of natural antioxidants tocopherol and ascorbic acids in maintenance of sperm activity during freeze-thaw process. Archives of andrology. 1994 Jul-Aug;33(1):11-5.

36. Verma A, Kanwar KC. Human sperm motility and lipid peroxidation in different ascorbic acid concentrations: an in vitro analysis. Andrologia. 1998 Nov;30(6):325-9.

37. Gadea J, Molla M, Selles E, Marco MA, Garcia-Vazquez FA, Gardon JC. Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology. 2011 2011/02/01/;62(1):40-6.

38. Partyka A, Rodak O, Bajzert J, Kochan J, Nizanski W. The Effect of L-Carnitine, Hypotaurine, and Taurine Supplementation on the Quality of Cryopreserved Chicken Semen. BioMed research international. 2017;2017:7279341.

39. Halotech DNA SL. Halosperm. Available from: https://www.halotechdna.com/wp-content/uploads/2015/09/Halosperm-web.pdf . Accessed July 06, 2020.