Technologies for Cryoprotectant-Free Vitrification of Human Spermatozoa: Asepticity as a Criterion for Effectiveness

  • Vladimir Isachenko
  • Gohar Rahimi
  • Peter Mallmann
  • Raul Sanchez
  • Evgenia Isachenko


This review describes the 120-year history of technology for cryoprotectant-free cryopreservation of human spermatozoa by direct plunging into liquid nitrogen (vitrification). It explains why cryoprotectant-free vitrification of some human ejaculate samples is better than conventional freezing and vitrification with the presence of cryoprotectants. Special attention is given to the extremely high viability of viruses, bacteria, and mycoplasmas after cryoprotectant-free cryopreservation in culture medium and even in distilled water. This increases the potential risk of disease transmission through liquid nitrogen. The concept of asepticity is concretized as an obvious parameter for any medical assisted reproduction technology that includes cooling of cells in liquid nitrogen. The roles of nonpermeating compounds in media for cytoprotectant-free vitrification—carbohydrates, proteins, lipoproteins, antioxidants—are described. This review summarizes relevant data regarding two groups of different current technologies for cryoprotectant-free vitrification of human spermatozoa: those involving direct contact of spermatozoa with liquid nitrogen and those involving full isolation of these cells from liquid nitrogen (aseptic technologies).


Human Spermatozoa Vitrification Cryoprotectant-free Viruses Bacteria Mycoplasmas Asepticity Liquid nitrogen 


Competing interests

The authors declare that they have no competing interests.


  1. 1.
    Parkes AS. Preservation of human spermatozoa at low temperatures. Brit Med J. 1945;2:212–3.PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Jahnel F. Ueber die Widerstandsfehigkeit von menschlichen Spermatozoonen gegenueber starker Kaelte. Klin Wochenschr. 1938;17:1273–4.CrossRefGoogle Scholar
  3. 3.
    Hoagland H, Pincus G. Revival of mammalian sperm after immersion in liquid nitrogen. J Gener Physiol. 1942;25:337–44.CrossRefGoogle Scholar
  4. 4.
    Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949;164:666.PubMedCrossRefGoogle Scholar
  5. 5.
    Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, Mallmann P, Dattena M, Capobianco G, Peters D, Orth I, Isachenko E. Vitrification of human spermatozoa without cryoprotectants. Cryo Letters. 2002;23:93–102.PubMedGoogle Scholar
  6. 6.
    Talbot P, Chacon RS. A triple-stain technique for evaluating normal acrosome reactions of human sperm. J Exp Zool. 1981;215:201–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A. Birth following vitrification of a small number of human oocytes: case report. Hum Reprod. 1999;14:3077–9.PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Gao DY, Liu C, McGann LE, Watson PF, Kleinhans FW, Mazur P, Critser ES, Critser JK. Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum Reprod. 1995;10:1109–22.PubMedCrossRefGoogle Scholar
  9. 9.
    Gao D, Mazur P, Critser J. Fundamental cryobiology of mammalian spermatozoa. In: Karow AM, Critser JK, editors. Reproductive tissue banking. London: Academic; 1997. p. 263–328.CrossRefGoogle Scholar
  10. 10.
    Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their postthawing function. Reprod Fertil Dev. 1995;7:871–91.PubMedCrossRefGoogle Scholar
  11. 11.
    Critser JK, Huse-Benda AR, Aaker DV, Arnenson BW, Ball GD. Cryopreservation of human spermatozoa. 1. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction. Fertil Steril. 1987a;47:656–63.PubMedCrossRefGoogle Scholar
  12. 12.
    Critser JK, Arnenson BW, Aaker DV, Huse-Benda AR, Ball GD. Cryopreservation of human spermatozoa. 2. Post-thaw chronology of motility and zona-free hamster ova penetration. Fertil Steril. 1987b;47:980–4.PubMedCrossRefGoogle Scholar
  13. 13.
    Keel BA, Webster BW, Roberts DK. Effects of cryopreservation on the motility characteristics of human spermatozoa. J Reprod Fertil. 1987;81:213–20.PubMedCrossRefGoogle Scholar
  14. 14.
    Mossad H, Morshedi M, Torner JP, Oehninger S. Impact of cryopreservation on spermatozoa from infertile men—implication for artificial insemination. Arch Androl. 1994;33:51–7.PubMedCrossRefGoogle Scholar
  15. 15.
    Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu FC. Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligospermia. J Androl. 1989;10:214–20.PubMedCrossRefGoogle Scholar
  16. 16.
    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;13:232–41.PubMedGoogle Scholar
  17. 17.
    O’Connell M, McClure N, Lewis SEM. The effect of cryopreservation on sperm morphology, motility and mitochondrial function. Hum Reprod. 2002;17:704–9.PubMedCrossRefGoogle Scholar
  18. 18.
    Alvarez JG, Storey BT. Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant. J Androl. 1993;14:199–209.PubMedGoogle Scholar
  19. 19.
    Chatterjee S, Gagnon C. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Mol Reprod Dev. 2001;59:451–8.CrossRefGoogle Scholar
  20. 20.
    Isachenko E, Isachenko V, Katkov II, Nawroth F. Vitrification of human spermatozoa without cryoprotectants: review of problem and practical success. Reprod Biomed Online. 2003;6:191–200.PubMedCrossRefGoogle Scholar
  21. 21.
    Holt WV. Alternative strategies for the long-term preservation of spermatozoa. Reprod Fertil Dev. 1997;9:309–19.PubMedCrossRefGoogle Scholar
  22. 22.
    Thurston LM, Siggins K, Mileham AJ, Watson PF, Holt WV. Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation. Biol Reprod. 2002;66:545–54.PubMedCrossRefGoogle Scholar
  23. 23.
    Sakkas D, Tomlinson M. Assessment of sperm competence. Sem Reprod Med. 2000;18:133–9.CrossRefGoogle Scholar
  24. 24.
    Fraga CG, Motchnik P, Shigenaga MK, Helbock HJ, Jacon RA, Ames BN. Ascorbic acid protects against endogenous oxidative DNA damage in human sperm. Proc Natl Acad Sci USA. 1991;88:11033–66.CrossRefGoogle Scholar
  25. 25.
    Donnelly ET, Steele EK, McClure N, Lewis SEM. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod. 2001a;16:1191–9.PubMedCrossRefGoogle Scholar
  26. 26.
    Donnelly ET, McClure N, Lewis SE. Cryopreservation of human semen and prepared sperm: effect on motility parameters and DNA integrity. Fertil Steril. 2001b;76:892–900.PubMedCrossRefGoogle Scholar
  27. 27.
    Hammadeh ME, Askari AS, Georg T, Rosenbaum P, Schmidt W. Effect of freezing–thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Intern J Androl. 1999;22:155–62.CrossRefGoogle Scholar
  28. 28.
    Royere D, Hamamah S, Nicolle JC, Barthelemy C, Lansac J. Freezing and thawing alter chromatin stability of ejaculated human spermatozoa: fluorescence acridine orange staining and Fuelgen DNA cytophotometric studies. Gam Res. 1988;21:51–7.CrossRefGoogle Scholar
  29. 29.
    Manicardi GC, Bianchi PG, Pantano S, Azzoni P, Bizzaro D, Bianchi U, Sakkas D. Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromatin a(3) accessibility. Biol Reprod. 1995;52:64–867.CrossRefGoogle Scholar
  30. 30.
    Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Experiment Zool. 1999;284:696–704.CrossRefGoogle Scholar
  31. 31.
    Hunter JD, Bodner AJ, Hatch FT, Balhorn RL, Mazrimas JA, Mc-Queen AP, Gledhill BL. Single-strand nuclease action no heat-denatured spermiogenic chromatin. J Histochem Cytochem. 1976;24:901–7.PubMedCrossRefGoogle Scholar
  32. 32.
    Lopes S, Sun JG, Juriscova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlated with failed fertilization in intracytoplasmic sperm injection. Fertil Steril. 1998;69:528–32.PubMedCrossRefGoogle Scholar
  33. 33.
    Fahy GM. The relevance of cryoprotectant “toxicity” to cryobiology. Cryobiology. 1986;23:1–13.PubMedCrossRefGoogle Scholar
  34. 34.
    Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrification as an approach to cryopreservation. Cryobiology. 1984;21:407–26.PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Pegg DE, Diaper MP. On the mechanism of injury to slowly frozen erythrocytes. Biophys J. 1988;54:471–88.PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Shaw J, Oranratnachai A, Trounson A. Fundamental cryobiology of mammalian oocytes and ovarian tissue. Theriogenology. 2000;53:59–72.PubMedCrossRefGoogle Scholar
  37. 37.
    Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl. 1987;8:338–48.PubMedCrossRefGoogle Scholar
  38. 38.
    Bauché F, Fouchard M, Jégou B. Antioxidant system in rat testicular cells. FEBS Lett. 1994;349:392–6.PubMedCrossRefGoogle Scholar
  39. 39.
    De Lamirande E, Leclerc P, Gagnon C. Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization. Mol Hum Reprod. 1997;3:175–94.PubMedCrossRefGoogle Scholar
  40. 40.
    Fahy GM, Rall WF. Vitrification: an overview. In: Tucker M, Liebermann J, editors. Vitrification in assisted reproduction: a user’s manual and trouble-shooting guide. London: CRC; 2007. p. 1–20.Google Scholar
  41. 41.
    Gu W, Hecht N. Developmental expression of glutathione peroxidase, catalase, and manganese superoxide dismutase mRNAs during spermatogenesis in the mouse. J Androl. 1996;17:256–62.PubMedGoogle Scholar
  42. 42.
    Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko A, Nawroth F, Dessole S, Katkov II, Van der Ven H. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online. 2005;10:350–4.PubMedCrossRefGoogle Scholar
  43. 43.
    Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat AG, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136:167–73.PubMedCrossRefGoogle Scholar
  44. 44.
    Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L. Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue. Proc Natl Acad Sci USA. 1995;92:8831–5.PubMedCrossRefGoogle Scholar
  45. 45.
    Muldrew K, McGann LE. Mechanisms of intracellular ice formation. Biophys J. 1990;57:525–33.PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Sasaki T, Wiedemann H, Matzner M, Chu ML, Timpl R. Expression of fibulin-2 by fibroblasts and deposition with fibronectin into a fibrillar matrix. J Cell Sci. 1996;109:2895–904.PubMedGoogle Scholar
  47. 47.
    Sikka C. Andrology lab corner: role of oxidative stress and antioxidants in andrology and assisted reproductive technology. J Androl. 2004;25:5–18.PubMedCrossRefGoogle Scholar
  48. 48.
    Steif P, Noday D, Rabin Y. Can thermal expansion differences between cryopreserved tissue and cryoprotective agents alone cause cracking? Cryo Letters. 2009;30:414–21.PubMedPubMedCentralGoogle Scholar
  49. 49.
    Tramer F, Rocco F, Micali F, Sandri G, Panfili E. Antioxidant systems in rat epididymal spermatozoa. Biol Reprod. 1998;59:753–8.PubMedCrossRefGoogle Scholar
  50. 50.
    Yavin S, Arav A. Measurement of essential physical properties of vitrification solutions. Theriogenology. 2007;67:81–9.PubMedCrossRefPubMedCentralGoogle Scholar
  51. 51.
    Zini A, De Lamirande E, Gagnon C. Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa. Int J Androl. 1993;16:183–8.PubMedCrossRefPubMedCentralGoogle Scholar
  52. 52.
    Koshimoto C, Mazur P. The effect of the osmolality of sugar-containing media, the type of sugar, and the mass and molar concentration of sugar on the survival of frozen-thawed mouse sperm. Cryobiology. 2002;45:80–90.PubMedCrossRefPubMedCentralGoogle Scholar
  53. 53.
    Wakayama T, Whittinhgam DG, Yanagimachi R. Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection. J Reprod Fertil. 1998;112:11–7.PubMedCrossRefPubMedCentralGoogle Scholar
  54. 54.
    Rodgers W, Glaser M. Distributions of proteins and lipids in the erythrocyte membrane. Biochemistry. 1993;32:2591–12598.CrossRefGoogle Scholar
  55. 55.
    Jobim MIM, Oberst ER, Salbego CG, Souza DO, Wald VB, Tramontina F, Mattos RC. Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability. Theriogenology. 2004;61:255–66.PubMedCrossRefPubMedCentralGoogle Scholar
  56. 56.
    Speck RS, Jawetz E, Coleman VR. Studies on herpes simplex virus. I. The stability and preservation of egg-adapted herpes simplex virus. J Bacteriol. 1951;61:253–8.PubMedPubMedCentralGoogle Scholar
  57. 57.
    Melnick JL. Preservation of viruses by freezing. Federat Proc Suppl. 1965;15:280–3.Google Scholar
  58. 58.
    Tomlinson MR. Risk management in cryopreservation associated with assisted reproduction. Cryo Letters. 2008;29:165–74.PubMedGoogle Scholar
  59. 59.
    Hansen LJ, Daoussi R, Vervaet C, Remon JP, De Beer TR. Freeze-drying of live virus vaccines: a review. Vaccine. 2015;33:5507–19.PubMedCrossRefGoogle Scholar
  60. 60.
    Stringfellow DA, Wolfe DF, McGuire JA, Lauerman LH, Gray BW, Sparling PH. Effects of embryo-freezing and thawing techniques on the survivability of Brucella abortus. Theriogenology. 1986;26:553–9.PubMedCrossRefPubMedCentralGoogle Scholar
  61. 61.
    Polo L, Mañes-Lázaro R, Olmeda I, Cruz-Pio LE, Medina Á, Ferrer S, Pardo I. Influence of freezing temperatures prior to freeze-drying on viability of yeasts and lactic acid bacteria isolated from wine. J Appl Microbiol. 2017;122:1603. Scholar
  62. 62.
    Bermudez V, Miller RB, Johnson W, Rosendal S, Ruhnke L. Effect of sample freezing on the isolation of Mycoplasma spp. from the clitoral fossa of the mare. Can J Vet Res. 1988;52:147–8.PubMedPubMedCentralGoogle Scholar
  63. 63.
    Carlsen KH, Jensen JS. Mycoplasma genitalium PCR: does freezing of specimens affect sensitivity? J Clin Microbiol. 2010;48:3624–7.PubMedPubMedCentralCrossRefGoogle Scholar
  64. 64.
    Yugi H, Suzuki M, Sato S, Ozaki Y. Proceedings: freeze-drying of mycoplasma. Cryobiology. 1973;10:464–7.PubMedCrossRefPubMedCentralGoogle Scholar
  65. 65.
    Piasecka-Serafin M. The effect of the sediment accumulated in containers under experimental conditions on the infection of semen stored directly in liquid nitrogen (−196 °C). Bull Acad Pol Sci Biol. 1972;20:263–7.PubMedPubMedCentralGoogle Scholar
  66. 66.
    Benson EE. Cryopreservation of phytodiversity: a critical appraisal of theory and practice. Critic Rev Plant Sci. 2008;27:141–219.CrossRefGoogle Scholar
  67. 67.
    Bielanski A, Bergeron H, Lau PCK, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology. 2003;46:146–52.PubMedCrossRefPubMedCentralGoogle Scholar
  68. 68.
    Bielanski A. Experimental microbial contamination and disinfection of dry (vapour) shipper Dewars designed for short-term storage and transportation of cryopreserved germplasm and other biological specimens. Theriogenology. 2005a;63:1946–57.PubMedCrossRefPubMedCentralGoogle Scholar
  69. 69.
    Bielanski A. Non-transmission of bacterial and viral microbes to embryos and semen stored in the vapour phase of liquid nitrogen in dry shippers. Cryobiology. 2005b;50:206–10.PubMedCrossRefPubMedCentralGoogle Scholar
  70. 70.
    Bielanski A, Vajta G. Risk of contamination of germplasm during cryopreservation and cryobanking in IVF units. Hum Reprod. 2009;24:2457–67.PubMedCrossRefGoogle Scholar
  71. 71.
    Bielanski A, Lalonde A. Effect of cryopreservation by slow cooling and vitrification on viral contamination of IVF embryos experimentally exposed to bovine viral diarrhea virus and bovine herpesvirus-1. Theriogenology. 2009;72:919–25.PubMedCrossRefPubMedCentralGoogle Scholar
  72. 72.
    Bielanski A. A review of the risk of contamination of semen and embryos during cryopreservation and measures to limit cross-contamination during banking to prevent disease transmission in ET practices. Theriogenology. 2012;77:467–82.PubMedCrossRefPubMedCentralGoogle Scholar
  73. 73.
    Bielanski A. Biosafety in embryos and semen cryopreservation, storage, management and transport. Adv Experiment Med Biolo. 2014;753:429–65.CrossRefGoogle Scholar
  74. 74.
    Charles GN, Sire DJ. Transmission of papovavirus by cryotherapy applicator. J Amer Med Assoc. 1971;218:1435.CrossRefGoogle Scholar
  75. 75.
    Hawkins AE, Zuckerman MA, Briggs M, Gilson RJ, Goldstone AH, Brink NS, Tedder RS. Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank. J Virol Meth. 1996;60:81–8.CrossRefGoogle Scholar
  76. 76.
    Jones SK, Darville JM. Transmission of virus-particles by cryotherapy and multi-use caustic pencils: a problem to a dermatologist? Br J Dermatol. 1989;121:481–6.PubMedCrossRefPubMedCentralGoogle Scholar
  77. 77.
    Mortimer D. Current and future concepts and practices in human sperm cryobanking. Reprod Biomed Online. 2004;9:134–51.PubMedCrossRefPubMedCentralGoogle Scholar
  78. 78.
    Pomeroy KO, Harris S, Conaghan J, Papadakis M, Centola G, Basuray R, Battaglia D. Storage of cryopreserved reproductive tissues: evidence that cross-contamination of infectious agents is a negligible risk. Fertil Steril. (2010;94:1181–8.PubMedCrossRefPubMedCentralGoogle Scholar
  79. 79.
    Rall WF. Avoidance of microbial cross-contamination of cryopreserved gametes, embryos, cells and tissues during storage in liquid nitrogen. Embryologists’ Newsletter. 2003;6:2–15.Google Scholar
  80. 80.
    Schaffer TW, Everen J, Silver GH, Came PE. Biohazard: virus contaminated liquid nitrogen. Science. 1976;192:25–6.Google Scholar
  81. 81.
    Tedder RS, Zuckerman MA, Goldstone AH, Hawkins AE, Fielding A, Briggs EM, Irwin D, Blair S, Gorman AM, Patterson KG, Linch DC, Heptstonstall J, Brinscs NS. Hepatitis-B transmission from contaminated cryopreservation tank. Lancet. 1995;346:137–40.PubMedCrossRefGoogle Scholar
  82. 82.
    Isachenko V. Response: efficacy of ultraviolet sterilization of liquid nitrogen. Reprod Biomed Online. 2011;22:502.PubMedCrossRefGoogle Scholar
  83. 83.
    Parmegiani L, Accorsi A, Cognigni GE, Bernardi S, Troilo E, Filicori M. Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos. Fertil Steril. 2010;94:1525–8.PubMedCrossRefGoogle Scholar
  84. 84.
    Wikipedia. Deinococcus radiodurans. Wikipedia, the free encyclopedia.
  85. 85.
    Aoki S, Ito S, Watanabe T. UV survival of human mycoplasmas: evidence of dark reactivation in Mycoplasma buccale. Microbiol Immunol. 1979;23:147–58.PubMedCrossRefGoogle Scholar
  86. 86.
    Hampar B, Aaronson SA, Derge JG, Chakrabarty M, Showalter SD, Dunn CY. Activation of an endogenous mouse type C virus by ultraviolet-irradiated herpes simplex virus types 1 and 2. Proc Natl Acad Sci USA. 1976;73:646–50.PubMedCrossRefGoogle Scholar
  87. 87.
    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;28:352–8.PubMedCrossRefGoogle Scholar
  88. 88.
    Chen Y, Li L, Qian Y, Xu C, Zhu Y, Huang H, Jin F, Ye Y. Small-volume vitrification for human spermatozoa in the absence of cryoprotectants by using Cryotop. Andrologia. 2015;47:694–9.PubMedCrossRefGoogle Scholar
  89. 89.
    Agha-Rahimi A, Khalili MA, Nottola SA, Miglietta S, Moradi A. Cryoprotectant-free vitrification of human spermatozoa in new artificial seminal fluid. Andrology. 2016;4:1037–44.PubMedCrossRefGoogle Scholar
  90. 90.
    Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, Van der Ven H. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod. 2004;71:1167–73.PubMedCrossRefGoogle Scholar
  91. 91.
    Dinnyes A, Dai Y, Jiang S, Yang X. High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Biol Reprod. 2000;63:513–8.PubMedCrossRefGoogle Scholar
  92. 92.
    Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev. 1998;51:53–8.PubMedPubMedCentralCrossRefGoogle Scholar
  93. 93.
    Isachenko V, Maettner R, Petrunkina AM, Mallmann P, Rahimi G, Sterzik K, Sanchez R, Risopatron J, Damjanoski I, Isachenko E. Cryoprotectant-free vitrification of human spermatozoa in large (up to 0.5 mL) volume: a novel technology. Clin Lab. 2011a;57:643–50.PubMedGoogle Scholar
  94. 94.
    Isachenko V, Isachenko E, Petrunkina AM, Sanchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies. Reprod Fertil Dev. 2011b;24:323–6.CrossRefGoogle Scholar
  95. 95.
    Isachenko V, Katkov II, Yakovenko S, Lulat A, Ulug M, Arvas A, Isachenko E. Vitrification of human laser treated blastocysts within cut standard straws (CSS): novel aseptic packaging and reduced concentrations of cryoprotectants. Cryobiology. 2007;54:305–9.PubMedCrossRefGoogle Scholar
  96. 96.
    Sanchez R, Isachenko V, Petrunkina AM, Risopatron J, Schulz M, Isachenko E. Live birth after intrauterine insemination with spermatozoa from an oligoasthenozoospermic patient vitrified without permeable cryoprotectants. J Androl. 2012;33:559–62.PubMedCrossRefGoogle Scholar
  97. 97.
    Isachenko V, Maettner R, Petrunkina AM, Sterzik K, Mallmann P, Rahimi G, Sanchez R, Risopatron J, Damjanoski I, Isachenko E. Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary technology. J Androl. 2012;33:462–8.PubMedCrossRefGoogle Scholar
  98. 98.
    Khalili MA, Adib M, Halvaei I, Nabi A. Vitrification of neat semen alters sperm parameters and DNA integrity. Urol J. 2014;11:1465–70.PubMedGoogle Scholar
  99. 99.
    Kuznyetsov V, Moskovtsev SI, Crowe M, Lulat AG, Librach CL. Vitrification of a small number of spermatozoa in normozoospermic and severely oligozoospermic samples. Syst Biol Reprod Med. 2015;61:13–7.PubMedCrossRefGoogle Scholar
  100. 100.
    Slabbert M, Du Plessis SS, Huyser C. Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa. Andrologia. 2015;47:594–9.PubMedCrossRefGoogle Scholar
  101. 101.
    Ali Mohamed MS. Slow cryopreservation is not superior to vitrification in human spermatozoa; an experimental controlled study. Iran J Reprod Med. 2015;13:633–44.PubMedPubMedCentralGoogle Scholar
  102. 102.
    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;48:111–3.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Vladimir Isachenko
    • 1
  • Gohar Rahimi
    • 1
  • Peter Mallmann
    • 1
  • Raul Sanchez
    • 2
  • Evgenia Isachenko
    • 1
  1. 1.University Maternal Hospital, Department of Obstetrics and Gynecology, Cologne UniversityCologneGermany
  2. 2.Department of Preclinical Science, Faculty of MedicineUniversidad de La FronteraTemucoChile

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