Recent updates of sperm cryopreservation technique: a literature review

Aucky Hinting , Agustinus Agustinus

Aucky Hinting
Andrologist, Faculty of Medicine, Universitas Airlangga, Ferina-Center for Reproductive Medicine, Surabaya, Indonesia. Email:

Agustinus Agustinus
Andrologist, Faculty of Medicine, Universitas Airlangga, Ferina-Center for Reproductive Medicine, Surabaya, Indonesia
Online First: November 02, 2020 | Cite this Article
Hinting, A., Agustinus, A. 2020. Recent updates of sperm cryopreservation technique: a literature review. Indonesia Journal of Biomedical Science 14(2): 92-98. DOI:10.15562/ijbs.v14i2.230

Background: Nowadays, the decline in male fertility is a significant concern due to the mechanical, systems disturbances, genetic, diet, lifestyle, and environment. Currently, no strategies for medical protection of the germinal epithelium are available. Sperm cryopreservation technique has been established to overcome those issues. Accordingly, this study aims to informed the recent updates of sperm cryopreservation technique from kinds of literature. 

Methods: A review of relevant literature was performed to elaborate the recent updates in the sperm cryopreservation technique. A total of 45 qualified published literature of all years until 2019 were collected from several electronic database and manual search and included in this review.

Results: According to the literature, the first record of sperm cryopreservation was made by Lazzaro Spallanzani in 1776. He used to snow during winter and observed that the motility of human spermatozoa could be preserved after freezing and thawing. Sperm cryopreservation programs provide temporarily or permanently infertile men an opportunity to seek paternity. Sperm cryopreservation is the most effective and the most strongly recommended option for fertility preservation prior to any treatment or occupational gonadotoxic exposures. Sperm cryopreservation should be discussed and offered prior to therapy for malignant diseases. Freezing sperm before the initiation of treatment provides patients with future fertility potential.

Conclusion: Sperm cryopreservation could be offered to the patient before the vasectomy procedure in order to prevent reversal vasectomy or surgical sperm retrieval in the future.



Sengupta P, Borges E, Dutta S, Krajewska-Kulak E. Decline in sperm count in European men during the past 50 years. Hum Exp Toxicol. 2018;37(3):247–255.

Jacobs C, A KN, Tulp OL. An overview of the causes and consequences of male fertility decline. Perceptions Reprod Med. 2019;3(1):192–194.

Ragheb A, Sabanegh Jr. E. Male Fertility-Implications of Anticancer Treatment and Strategies to Mitigate Gonadotoxicity. Anticancer Agents Med Chem. 2012;10(1):92–102.

Vaziri MH, Gilani MAS, Firoozeh M, Kavousi A, Firoozeh M, Jazani RK, et al. The relationship between occupation and semen quality. Int J Fertil Steril. 2011;5(2):66–71.

Lambertini M, Del Mastro L, Pescio MC, Andersen CY, Azim HA, Peccatori FA, et al. Cancer and fertility preservation: International recommendations from an expert meeting. BMC Med. 2016;14(1):1–16.

Hezavehei M, Sharafi M, Kouchesfahani HM. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reprod Biomed Online. 2018;37(3):327–339.

Walters EM, Benson JD, Woods EJ, Critser JK. The history of sperm cryopreservation. In: Pacey AA, Tomlinson MJ, © P by CUP, editors. Sperm Banking: Theory and Practice. Cambridge University Press; 2009:1-17

Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949;164(4172):666.

Sherman JK. Research on Frozen Human Semen. Fertil Steril. 1964;15(5):485–500.

Perloff WH, Steinberger E, Sherman JK. Conception with Human Spermatozoa Frozen by Nitrogen Vapor Technic. Fertil Steril. 1964;15(5):501–504.

Patrizio P, Balmaceda TOJP, Asch RH. Successful fertilization, pregnancy, and birth using epididymal sperm frozen 24 hours after conventional oocyte insemination. Fertil Steril. 1995;64(4):863–865.

Podsiadly BT, Woolcott RJ, Stanger JD, Stevenson K. Pregnancy resulting from intracytoplasmic injection of cryopreserved spermatozoa recovered from testicular biopsy. Hum Reprod. 1996;11(6):1306–1308.

Gil-Salom M, Romero J, Mínguez Y, Rubio C, De Los Santos MJ, Remohí J, et al. Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Hum Reprod. 1996;11(6):1309–1313.

Fischer R, Baukloh V, Naether OGJ, Schulze W, Salzbrunn A, Benson DM. Pregnancy after intracytoplasmic sperm injection of spermatozoa extracted from frozen-thawed testicular biopsy. Hum Reprod. 1996;11(10):2197–2199.

Hovatta O, Foudila T, Siegberg R, Johansson K, Von Smitten K, Reima I. Pregnancy resulting from intracytoplasmic injection of spermatozoa from a frozen-thawed testicular biopsy specimen. Hum Reprod. 1996 Dec 1;11(11):2472–2473.

Behrman SJ, Sawada Y. Heterologous and Homologous Inseminations with Human Semen Frozen and Stored in a Liquid-Nitrogen Refrigerator. Fertil Steril. 1966;17(4):457–466.

Di Santo M, Tarozzi N, Nadalini M, Borini A. Human sperm cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART. Adv Urol. 2012;2012: 854837

Rios AP, Botella IM. Description and Outcomes of Current Clinical Techniques for Sperm Cryopreservation. EMJ Repro Health. 2019;7(1):79–92.

O’Neill HC, Nikoloska M, Ho HT, Doshi A, Maalouf W. Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage. J Assist Reprod Genet. 2019;36(8):1713–1720.

Li Y, Zhou L, Lv M, Ge P, Liu Y, Zhou D. Vitrification and conventional freezing methods in sperm cryopreservation: A systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2019;233:84–92.

Dupesh S, Rasappan P, Shila S, Gunasekaran K. Human Sperm Freezing: Mini Update. Adv Reprod Sci. 2018;06(3):59–69.

Kwon I-K, Park K-E, Niwa K. Activation, Pronuclear Formation, and Development In Vitro of Pig Oocytes Following Intracytoplasmic Injection of Freeze-Dried Spermatozoa. Biol Reprod. 2004;71(5):1430–1436.

Watanabe H, Asano T, Abe Y, Fukui Y, Suzuki H. Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes. J Assist Reprod Genet. 2009;26(9–10):531–536.

Kaneko T, Ito H, Sakamoto H, Onuma M, Inoue-Murayama M. Sperm Preservation by Freeze-Drying for the Conservation of Wild Animals. PLoS One. 2014;9(11):e113381.

Olaciregui M, Gil L. Freeze-dried spermatozoa: A future tool? Reprod Domest Anim. 2017;52(Suppl 2):248–254.

Kusakabe H, Yanagimachi R, Kamiguchi Y. Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes. Hum Reprod. 2008;23(2):233–239.

Gianaroli L, Magli MC, Stanghellini I, Crippa A, Crivello AM, Pescatori ES, et al. DNA integrity is maintained after freeze-drying of human spermatozoa. Fertil Steril. 2012;97(5):1067-1073.e1.

Esteves SC, Sharma RK, Thomas Jr AJ, Agarwal A. Improvement in motion characteristics and acrosome status in cryopreserved human spermatozoa by swim-up processing before freezing. Hum Reprod. 2000;15(10):2173–2179.

Petyim S, Neungton C, Thanaboonyawat I, Laokirkkiat P, Choavaratana R. Sperm preparation before freezing improves sperm motility and reduces apoptosis in post-freezing-thawing sperm compared with post-thawing sperm preparation. J Assist Reprod Genet. 2014;31(12):1673–1680.

Brugnon F, Ouchchane L, Pons-Rejraji H, Artonne C, Farigoule M, Janny L. Density gradient centrifugation prior to cryopreservation and hypotaurine supplementation improve post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia. Hum Reprod. 2013;28(8):2045–2057.

Suyono SS, Hinting A, Lunardhi H, I’tishom R. Density Gradient Centrifugation Pra-freezing Mengoptimalkan Persentase Morfologi Normal Spermatozoa Pasca-thawing. Maj Kedokt Bandung. 2018;50(3):133–139.

Fabozzi G, Starita MF, Rega E, Alteri A, Colicchia A, Piscitelli C, et al. Evaluation of the Efficiency of Two Different Freezing Media and Two Different Protocols to Preserve Human Spermatozoa from Cryoinjury. Int J Reprod Med. 2016;2016:1–6.

Palomar Rios A, Gascón A, Martínez J V., Balasch S, Molina Botella I. Sperm preparation after freezing improves motile sperm count, motility, and viability in frozen-thawed sperm compared with sperm preparation before freezing-thawing process. J Assist Reprod Genet. 2018;35(2):237–245.

Martínez-Soto JC, Landeras J, Gadea J. Spermatozoa and seminal plasma fatty acids as predictors of cryopreservation success. Andrology. 2013;1(3):365–375.

Barrios B, Pérez-Pé R, Gallego M, Tato A, Osada J, Muiño-Blanco T, et al. Seminal Plasma Proteins Revert the Cold-Shock Damage on Ram Sperm Membrane1. Biol Reprod. 2000;63(5):1531–1537.

Patel M, Gandotra VK, Cheema RS, Bansal AK, Kumar A. Seminal plasma heparin binding proteins improve semen quality by reducing oxidative stress during cryopreservation of cattle bull semen. Asian-Australasian J Anim Sci. 2016;29(9):1247–1255.

Cayan S, Lee D, Conaghan J, Givens CA, Ryan IP, Schriock ED, et al. A comparison of ICSI outcomes with fresh and cryopreserved epididymal spermatozoa from the same couples. Hum Reprod. 2001;16(3):495–499.

Kalsi J, Thum MY, Muneer A, Pryor J, Abdullah H, Minhas S. Analysis of the outcome of intracytoplasmic sperm injection using fresh or frozen sperm. BJU Int. 2011;107(7):1124–1128.

Dafopoulos K, Griesinger G, Schultze-Mosgau A, Orief Y, Schöpper B, Nikolettos N, et al. Factors affecting outcome after ICSI with spermatozoa retrieved from cryopreserved testicular tissue in non-obstructive azoospermia. Reprod Biomed Online. 2005;10(4):455–460.

Esteves S. Clinical management of infertile men with nonobstructive azoospermia. Asian J Androl. 2015;17(3):459–470.

Gangrade BK. Cryopreservation of testicular and epididymal sperm: Techniques and clinical outcomes of assisted conception. Clinics. 2013;68(Suppl 1):131–140.

Desai NN, Blackmon H, Goldfarb J, Foundation CC, Clinic C, Centre F, et al. Article Single sperm cryopreservation on cryoloops : an alternative to hamster zona for freezing individual spermatozoa. 2004;9(1):47–53.

Thurston LM, Holt WV, Watson PF. Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate freezers: a comparison. Theriogenology. 2003;60(1):101‐113.

Devi L, Goel S. Fertility preservation through gonadal cryopreservation. Reprod Med Biol. 2016;15(4):235–51.

Radford J. Restoration of Fertility after Treatment for Cancer. Horm Res Paediatr. 2003;59(Suppl 1):21–23.

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