Volume : 2, Issue : 11, NOV 2016

CRYOPRESERVATION OF TESTICULAR HUMAN SPERMATOZOA BY TWO TECHNIQUES: COMPARISONS OF MSOME, DNA FRAGMENTATION AND MITOCHONDRIAL MEMBRANE POTENTIAL

Sahabeh Etebary, Mohammad Ali Khalili, Nahid Yari, Seyed Mehdi Kalantar, Morteza Anvari

Abstract

Purpose To evaluate the efficacy of two testicular spermatozoa cryopreservation techniques in Azoospermic patients with MSOME, DNA fragmentation and mitochondrial assays. Methods Testicular sperm extraction samples were obtained from 15 men, aged 20-40 years old with obstructive azoospermia. The prepared samples were divided into three groups. Group I was assessed freshly. Group II was cryopreserved with vitrification method, and Group III was cryopreserved with cooling in liquid nitrogen vapor using droplet. After warming, the samples were examined in terms of motility, morphology with MSOME, DNA fragmentation and mitochondrial membrane potential. For morphologic assessment spermatozoa were classified as class 1 (High-quality), class 2 (Medium-quality) and class 3 (Low-quality). Results There was significant reduction in motility, viability and the mean percentage of class 1 spermatozoa after cryopreservation by both techniques (p<0.001). There was no significant difference between groups II and III regarding morphologic class 1, 2 and 3 (p>0.05). The DNA fragmentation was significantly increased to 29.48 % and 29.72 % respectively; also the number of sperms with functional mitochondria significantly decreased to 31.81 % and 31.98 % after vitrification and freezing in liquid nitrogen vapor (p<0.001). A correlation was found between morphologic class1 and 3 spermatozoa; mitochondrial membrane potential and DNA fragmentation. Conclusions cryopreservation had adverse effects on spermatozoa parameters. The best method of testicular sperm cryopreservation should yet to be determined, but selecting spermatozoa with minimum abnormality is an important issue in infertility treatment. To reach this goal assessment of frozen-thawed testicular spermatozoa with MSOME will be of particular value.

Keywords

Cryopreservation, Human spermatozoa, Vitrification, DNA fragmentation, Mitochondrial membrane potential, Azoospermia.

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References

  1. Schoysman R, Vanderzwalmen P, Nijs M, Segal L, Segal-Bertin G, Geerts L et al. Pregnancy after fertilisation with human testicular spermatozoa. The Lancet. 1993;342(8881):1237-.
  2. Prince GS, Dolgina R, Studney P, Kaplan B, Ross L, Niederberger C. Quality of cryopreserved testicular sperm in patients with obstructive and nonobstructive azoospermia. The Journal of urology. 1999;161(5):1504-8.
  3. Schlegel P, Chang T. The testis, epididymis and ductus deferens. Campbell’s urology, 6th ed Philadelphia: Saunders. 1991;190.
  4. Schlegel PN, Su L-M. Physiological consequences of testicular sperm extraction. Hum Reprod. 1997;12(8):1688-92.
  5. Harrington TG, Schauer D, Gilbert BR. Percutaneous testis biopsy: an alternative to open testicular biopsy in the evaluation of the subfertile man. J Urol. 1996;156(5):1647-51.
  6. Verheyen G, Nagy Z, Joris H, De Croo I, Tournaye H, Van Steirteghem A. Quality of frozen-thawed testicular sperm and its preclinical use for intracytoplasmic sperm injection into in vitro-matured germinal-vesicle stage oocytes. Fertil Steril 1997;67(1):74-80.
  7. Romero J, Remohi J, Minguez Y, Rubio C, Pellicer A, Gil-Salom M. Fertilization after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Fertil Steril 1996;65(4):877-9.
  8. Gil-Salom M, Romero J, Ménguez Y, Rubio C, De los Santos MJ, Remohe J et al. Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Hum Reprod. 1996;11(6):1309-13.
  9. Prins GS, Dolgina R, Studney P, Kaplan B, Ross L, NIEDERBERGER C. Quality of cryopreserved testicular sperm in patients with obstructive and nonobstructive azoospermia. J Urol. 1999;161(5):1504-8.
  10. Ghanem M, Bakr NI, Elgayaar MA, El Mongy S, Fathy H, IBRAHIM AHA. Comparison of the outcome of intracytoplasmic sperm injection in obstructive and non?obstructive azoospermia in the first cycle: a report of case series and meta?analysis. Int J Androl. 2005;28(1):16-21.
  11. Schulze W, Salzbrunn A, Benson D. Pregnancy after intracytoplasmic sperm injection of spermatozoa extracted from frozen-thawed testicular biopsy. Hum Reprod. 1996;11(10):2197-9.
  12. Gangrade BK. Cryopreservation of testicular and epididymal sperm: techniques and clinical outcomes of assisted conception. Clinics. 2013;68:131-40.
  13. Habermann H, Seo R, Cieslak J, Niederberger C, Prins GS, Ross L. In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozen-thawed testicular spermatozoa. Fertil Steril 2000;73(5):955-60.
  14. Fukunaga N, Haigo K, Kyono K, Araki Y. Efficiency of using frozen-thawed testicular sperm for multiple intracytoplasmic sperm injections. Journal of assisted reproduction and genetics. 2001;18(12):634-7.
  15. Isachenko E, Isachenko V, Weiss J, Kreienberg R, Katkov I, Schulz M et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73.
  16. Vutyavanich T, Piromlertamorn W, Nunta S. Rapid freezing versus slow programmable freezing of human spermatozoa. Fertil Steril 2010;93(6):1921-8.
  17. Slabbert M, Du Plessis S, Huyser C. Large volume cryoprotectant?free vitrification: an alternative to conventional cryopreservation for human spermatozoa. Andrologia. 2015;47(5):594-9.
  18. Gil-Salom M, Romero J, Rubio C, Ruiz A, Remoh? J, Pellicer A. Intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Mol Cell Endocrinol 2000;169(1):15-9.
  19. O'connell M, McClure N, Lewis S. The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Hum Reprod. 2002;17(3):704-9.
  20. Royere D, Barthelemy C, Hamamah S, Lansac J. Cryopreservation of spermatozoa: a 1996 review. Hum Reprod Update. 1996;2(6):553-9.
  21. Hammadeh M, Askari A, Georg T, Rosenbaum P, Schmidt W. Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int J Androl. 1999;22(3):155-62.
  22. Bartoov B, Berkovitz A, Eltes F, Kogosowski A, Menezo Y, Barak Y. Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome. J Androl. 2002;23(1):1-8.
  23. Bartoov B, Berkovitz A, Eltes F. Selection of spermatozoa with normal nuclei to improve the pregnancy rate with intracytoplasmic sperm injection. N Engl J Med. 2001;345(14):1067-8.
  24. Balaban B, Yakin K, Alatas C, Oktem O, Isiklar A, Urman B. Clinical outcome of intracytoplasmic injection of spermatozoa morphologically selected under high magnification: a prospective randomized study. Reproductive biomedicine online. 2011;22(5):472-6.
  25. Garner DL, Thomas CA, Joerg HW, Dejarnette JM, Marshall CE. Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa. Biol Reprod 1997;57(6):1401-6.
  26. Kasai T, Ogawa K, Mizuno K, Nagai S, Uchida Y, Ohta S et al. Relationship between sperm mitochondrial membrane potential, sperm motility, and fertility potential. Asian journal of andrology. 2002;4(2):97-104.
  27. Thomson L, Fleming S, Aitken R, De Iuliis G, Zieschang J-A, Clark A. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum Reprod. 2009;24(9):2061-70.
  28. Lopes S, Sun J-G, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998;69(3):528-32.
  29. Nagy ZP, Verheyen G, Tournaye H, Devroey P, Van Steirteghem A. An improved treatment procedure for testicular biopsy specimens offers more efficient sperm recovery: case series. Fertil Steril 1997;68(2):376-9.
  30. Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko I, Nawroth F et al. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reproductive biomedicine online. 2005;10(3):350-4.
  31. Organization WH. WHO Laboratory Manual for the Examination and Processing of Human Semen. World Health Organization; 2010.
  32. Jeyendran R, Van der Ven H, Perez-Pelaez M, Crabo B, Zaneveld L. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984;70(1):219-28.
  33. Verheyen G, Joris H, Crits K, Nagy Z, Tournaye H, Van Steirteghem A. Comparison of different hypo-osmotic swelling solutions to select viable immotile spermatozoa for potential use in intracytoplasmic sperm injection. Hum Reprod Update. 1997;3(3):195-203.
  34. Cassuto NG, Bouret D, Plouchart JM, Jellad S, Vanderzwalmen P, Balet R et al. A new real-time morphology classification for human spermatozoa: a link for fertilization and improved embryo quality. Fertil Steril 2009;92(5):1616-25.
  35. Küpker W, Schlegel PN, Al-Hasani S, Fornara P, Johannisson R, Sandmann J et al. Use of frozen-thawed testicular sperm for intracytoplasmic sperm injection. Fertil Steril 2000;73(3):453-8.
  36. Weidel L, Prins GS. Cryosurvival of human spermatozoa frozen in eight different buffer systems. J Androl. 1987;8(1):41-7.
  37. Gao D, Liu J, Liu C, McGann L, Watson P, Kleinhans F et al. Andrology: Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum Reprod. 1995;10(5):1109-22.
  38. Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu F. Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J Androl. 1989;10(3):214-20.
  39. Karlsson JO. A theoretical model of intracellular devitrification. Cryobiology. 2001;42(3):154-69.
  40. Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F et al. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod 2004;71(4):1167-73.
  41. Celik-Ozenci C, Jakab A, Kovacs T, Catalanotti J, Demir R, Bray-Ward P et al. Sperm selection for ICSI: shape properties do notpredict the absence or presence of numericalchromosomal aberrations. Hum Reprod. 2004;19(9):2052-9.
  42. Wilding M, Coppola G, Di Matteo L, Palagiano A, Fusco E, Dale B. Intracytoplasmic injection of morphologically selected spermatozoa (IMSI) improves outcome after assisted reproduction by deselecting physiologically poor quality spermatozoa. J Assist Reprod Genet. 2011;28(3):253-62.
  43. Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod. 2001;16(6):1191-9.
  44. Howards S. The epididymis, sperm maturation, and capacitation. Infertility in the Male. 1983:121-34.
  45. Critser JK, Arneson BW, Aaker DV, Huse-Benda AR, Ball GD. Cryopreservation of human spermatozoa. II. Postthaw chronology of motility and of zona-free hamster ova penetration. Fertil Steril 1987;47(6):980-4.
  46. Alvarez JG, Storey BT. Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation. J Androl 1992;13(3):232-41.
  47. Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet JW, Crowe JH. Cold shock damage is due to lipid phase transitions in cell membranes: a demonstration using sperm as a model. Journal of Experimental Zoology. 1993;265(4):432-7.
  48. Sereni E, Bonu M, Fava L, Sciajno R, Serrao L, Preti S et al. Freezing spermatozoa obtained by testicular fine needle aspiration: a new technique. Reproductive biomedicine online. 2008;16(1):89-95.
  49. Meseguer M, Garrido N, Martínez-Conejero JA, Simón C, Pellicer A, Remohí J. Role of cholesterol, calcium, and mitochondrial activity in the susceptibility for cryodamage after a cycle of freezing and thawing. Fertil Steril 2004;81(3):588-94.
  50. Fraga CG, Motchnik PA, Shigenaga MK, Helbock HJ, Jacob RA, Ames BN. Ascorbic acid protects against endogenous oxidative DNA damage in human sperm. Proc Natl Acad Sci U S A. 1991;88(24):11003-6.
  51. Greco E, Scarselli F, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S et al. Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Hum Reprod. 2005;20(1):226-30.
  52. Franco Jr J, Mauri A, Petersen C, Massaro F, Silva L, Felipe V et al. Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa. Int J Androl. 2012;35(1):46-51.
  53. Dalzell LH, McVicar CM, McClure N, Lutton D, Lewis SE. Effects of short and long incubations on DNA fragmentation of testicular sperm. Fertil Steril 2004;82(5):1443-5.
  54. Hunter J, Bodner A, Hatch F, Balhorn R, Mazrimas J, McQueen A et al. Single-strand nuclease action on heat-denatured spermiogenic chromatin. J Histochem Cytochem. 1976;24(8):901-7.
  55. Knez K, Tomazevic T, Zorn B, Vrtacnik-Bokal E, Virant-Klun I. Intracytoplasmic morphologically selected sperm injection improves development and quality of preimplantation embryos in teratozoospermia patients. Reproductive biomedicine online. 2012;25(2):168-79.