Comparison between Sperm Preparation before Cryopreservation and Post-Thawing Sperm Preparation using Density Gradient Centrifugation in Normozoospermic Semen Samples
Sperm cryopreservation has been considered as an efficient procedure for management of male fertility. Different freezing protocols have been developed to maximize the post freeze quality. The current study investigates the differences between sperm preparation before and after freeze. 120 Semen analysis reports from 18 sperm donors were analysed. 60 semen analysis reports were from samples that were frozen then washed (Group 1: Post-wash) while the other 60 semen analysis reports were obtained from samples that were washed then frozen (Group 2: Pre-wash). Sperm concentration and progressive motility were evaluated for each group and compared. Sperm concentration was higher in the pre-wash group compared to post-wash (33.05 ±12.9 vs 26.13 ±13.36, respectively). Progressive motility was higher in the post-wash group (82.3 ±14.9 vs 51.6 ±8.2). There was a significant decrease in sperm concentration between fresh samples and both (post- and pre-wash) results. Significant decreases in progressive motility were found in both methods. Sperm freeze using fresh semen samples and washed samples resulted in significant decrease in post freeze progressive motility. However, fresh samples had a higher post freeze recovery rate compared to washed samples (72.15% vs 56.32%). Semen preparation by density gradient centrifugation before freezing resulted in better sperm concentration, while the preparation of spermatozoa after freezing had a higher progressive motility. Therefore, sperm freezing methods should be adapted according to semen samples quality.
Mocé E, Fajardo AJ, Graham JK. Human sperm cryopreservation. European Medical Journal. 2016;1(1):86-91.
Agarwal A, Tvrda E. Slow Freezing of Human Sperm. Methods in molecular biology. 2017;1568:67-78. doi:10.1007/978-1-4939-6828-25.
Ngamwuttiwong T, Kunathikom S. Evaluation of cryoinjury of sperm chromatin according to liquid nitrogen vapour method (I). Journal of the medical association of Thailand. 2007;90(2):224-8. PMID: 17375624.
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. 2009;24:2061-2070. https://doi.org/10.1093/humrep/dep214.
Glander HJ, Schaller J. Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. MHR: Basic Science of Reproductive Medicine. 1999;5(2):109-115. doi: 10.1093/molehr/5.2.109.
Schuffner A, Karavani G, Levitas E, et al. Cryopreservation of fractionated, highly motile human spermatozoa: effect on membrane phosphatidylserine externalization and lipid peroxidation. Human Reproduction. 2001;16(10):2148-2153. https://doi.org/10.1093/humrep/16.10.2148.
Petyim S, Choavaratana R. Cryodamage on sperm chromatin according to different freezing methods, assessed by AO test. Journal of the Medical Association of Thailand. 2006;89(3):306-13. PMID: 16696413.
O'connell M, McClure N, Lewis SEM. The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Human Reproduction. 2002;17(3):704–709. doi: 10.1093/humrep/17.3.704.
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. Fertility and Sterility. 1987;47(6):980-4. doi:10.1016/s0015-0282(16)59233-4.
Raad G, Lteif L, Lahoud R, Azoury J, Azoury J, Tanios J, Hazzouri M, Azoury J. Cryopreservation media differentially affect sperm motility, morphology and DNA integrity. Andrology. 2018;6(6):836-845. doi: 10.1111/andr.12531.
Hammadeh ME, Askari AS, 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. International Journal of Andrology. 1999;22(3):155–62. doi: 10.1046/j.1365-2605.1999.00162.x.
Gómez-Torres MJ, Medrano L, Romero A, Fernández-Colom PJ, Aizpurúa J. Effectiveness of human spermatozoa biomarkers as indicators of structural damage during cryopreservation. Cryobiology. 2017;78:90-94. doi:10.1016/j.cryobiol.2017.06.008.
Hallak J, Sharma RK, Wellstead C, Agarwal A. Cryopreservation of human spermatozoa: comparison of TEST-yolk buffer and glycerol. International Journal of Fertility and Women's Medicine. 2000;45(1):38–42. PMID: 10721743.
Hammadeh ME, Greiner S, Rosenbaum P, Schmidt W. Comparison between human sperm preservation medium and TEST-Yolk buffer on protecting chromatin and morphology integrity of human spermatozoa in fertile and subfertile men after freeze-thawing procedure. Journal Andrology. 2001;22(6):1012-1018. doi: 10.1002/j.1939-4640.2001.tb03442.
Nallella K, Sharma R, Allamaneni S, Aziz N, Agarwal A. Cryopreservation of human spermatozoa: comparison of two cryopreservation methods and three cryoprotectants. Fertility and Sterility. 2004;82(4):913–918. doi: 10.1016/j.fertnstert.2004.02.126.
Nijs M, Ombelet W. Cryopreservation of human sperm, Human Fertility. 2001;4(3):158-163. doi: 10.1080/1464727012000199232.
Morris GJ. Rapidly cooled human sperm: no evidence of intracellular ice formation. Human Reproduction. 2006;21(8):2075-83. https://doi.org/10.1093/humrep/del116.
Sánchez R, Risopatrón J, Schulz M, Villegas JV, Isachenko V, Isachenko E. Vitrified sperm banks: the new aseptic technique for human spermatozoa allows cryopreservation at -86 °C. Andrologia. 2012;44(6):433-5. doi: 10.1111/j.1439-0272.2012.01314.
Isachenko V, Maettner R, Petrunkina AM, et al. Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary technology. Journal of Andrology. 2012;33(3):462-8. doi: 10.2164/jandrol.111.013789.
Esteves SC, Sharma RK, Thomas AJ Jr, Agarwal A. Effect of swim-up sperm washing and subsequent capacitation on acrosome status and functional membrane integrity of normal sperm. International journal of fertility and women's medicine. 2000;45(5):335-41. PMID: 11092705.
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. Human Reproduction. 2013;28(8):2045-57. doi: 10.1093/humrep/det253.
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. Journal of Assisted Reproduction and Genetics. 2014;31(12):1673-1680. doi: 10.1007/s10815-014-0332-y.
Palomar Rios A, Gascón A, Martínez JV, 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. Journal of Assisted Reproduction and Genetics. 2018;35(2):237-245. doi: 10.1007/s10815-017-1050-z.
Allamaneni SS, Agarwal A, Rama S, Ranganathan P, Sharma RK. Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa. Asian Journal of Andrology. 2005;7(1):86-92. doi: 10.1111/j.1745-7262.2005.00008.x.
Rahiminia T, Hosseini A, Anvari M, Ghasemi-Esmailabad S, Talebi AR. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity. Taiwan Journal of Obstetrics and Gynecology. 2017;56(4):472-476. doi: 10.1016/j.tjog.2017.02.004.
Counsel M, Bellinge R, Burton P. Vitality of oligozoospermic semen samples is improved by both swim-up and density gradient centrifugation before cryopreservation. Journal of Assisted Reproduction and Genetics. 2004;21(5):137–4. doi: 10.1023/b:jarg.0000031245.39921.2c.
Donnelly ET, McClure N, Lewis SEM. Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity. Fertility and Sterility. 2001;76(5):892-900. doi: 10.1016/S0015-0282(01)02834-5.
Kvitko, D, Azambuja, R, Reig, V, Martin, HD, Badalotti M, Petracco Á. Association between low motility and sperm DNA fragmentation despite sperm count in men. Fertility and Sterility, 2012;98(3):248-249. doi: 10.1016/j.fertnstert.2012.07.905.
Elbashir S, Magdi Y, Rashed A, Ibrahim MA, Edris Y, Abdelaziz, AM. Relationship between sperm progressive motility and DNA integrity in fertile and infertile men. Middle East Fertility Society Journal. 2018;23(3):195-198. https://doi.org/10.1016/j.mefs.2017.12.002.
Martínez-Soto JC, Landeras J, Gadea J. Spermatozoa and seminal plasma fatty acids as predictors of cryopreservation success. Andrology. 2013;1(3):365–375. doi: 10.1111/j.2047-2927.2012.00040.
Eini F, Kutenaei MA, Shirzeyli MH, Dastjerdi ZS, Omidi M, Novin MG. Normal seminal plasma could preserve human spermatozoa against cryopreservation damages in Oligozoospermic patients. BMC Molecular and Cell Biology. 2021;22(1):50. doi: 10.1186/s12860-021-00390-6.
Karabulut S, Demiroğlu-Zergeroğlu A, Yılmaz E, Kutlu P, Keskin I. Effects of human sperm cryopreservation on apoptotic markers in normozoospermic and non-normozoospermic patients. Zygote. 2018;26(4):308-313. doi: 10.1017/S0967199418000254.
Mitchell LA, De Iuliis GN, Aitken, RJ. The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology. International Journal of Andrology. 2011;34(1):2–13. doi: 10.1111/j.1365-2605.2009.01042.
Lee D, Jee BC. Evaluation of normal morphology, DNA fragmentation, and hyaluronic acid binding ability of human spermatozoa after using four different commercial media for density gradient centrifugation. Clinical and Experimental Reproductive Medicine. 2019;46(1):8-13. doi: 10.5653/cerm.2019.46.1.8.
Friberg J, Gemzell C. Sperm-freezing and donor insemination. International Journal of Fertility. 1977;22(3):148-54. PMID: 24008.
Critser JK, Huse Benda AR, Aaker DV, Arneson BW, Ball GD. Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility. Fertility Sterility. 1988;50(2):314-320. PMID: 3396701.
Amesse LS, Srivastava G, Uddin D, Pfaff-Amesse T. Comparison of cryopreserved sperm in vaporous and liquid nitrogen. Journal of Reproductive Medicine. 2003;48(5):319-24. PMID: 12815902.
Sharma R, Kattoor AJ, Ghulmiyyah J, Agarwal A. Effect of sperm storage and selection techniques on sperm parameters. Systems Biology in Reproductive Medicine. 2015;61(1):1-12. doi: 10.3109/19396368.2014.976720.
Kitporntheranunt M, Om-songkram S, Srichawana K. A Comparison of Glycerol Egg Yolk Citrate and Sperm Freeze Medium in Human Sperm Cryopreservation. Journal of the medical association of Thailand. 2017;100(8):142-147.
Li YX, Zhou L, Lv MQ, Ge P, Liu YC, Zhou DX. Vitrification and conventional freezing methods in sperm cryopreservation: A systematic review and meta-analysis. European Journal of Obstetrics & Gynecology and Reproductive Biology. 2019;233:84-92. doi: 10.1016/j.ejogrb.2018.11.028.
Check DJ, Katsoff D, Check JH. Effect of an intermediate hold with vapor freezing on subsequent hypoosmotic swelling in thawed sperm. Archives of Andrology. 1995;35(1):79-81. doi: 10.3109/01485019508987857.
Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT, Kleinhans FW, Critser JK. Effect of Cryoprotectant Solutes on Water Permeability of Human Spermatozoa, Biology of Reproduction. 1995;53(5):985–995. doi: 10.1095/biolreprod53.5.985.
Liu J, Tanrikut C, Wright DL, et al. Cryopreservation of human spermatozoa with minimal non-permeable cryoprotectant. Cryobiology. 2016;73(2):162-7. doi: 10.1016/j.cryobiol.2016.08.004.
Slabbert M, du Plessis SS, Huyser C. Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa. Andrologia. 2015;47(5):594-9. doi: 10.1111/and.12307.
Tao Y, Sanger E, Saewu, A. Human sperm vitrification: the state of the art. Reproductive Biology and Endocrinology. 2020;18(1):17. doi: 10.1186/s12958-020-00580-5.
Schulz M, Risopatrón J, Uribe P, Isachenko E, Isachenko V, Sánchez R. Human sperm vitrification: A scientific report. Andrology. 2020;8(6):1642-1650. doi: 10.1111/andr.12847.
Maleki B, Khalili MA, Gholizadeh L, Mangoli E, Agha-Rahimi A. Single sperm vitrification with permeable cryoprotectant-free medium is more effective in patients with severe oligozoospermia and azoospermia. Cryobiology. 2022;104:15-22. doi: 10.1016/j.cryobiol.2021.11.176.
Wang M, Todorov P, Wang W, et al. Cryoprotectants-Free Vitrification and Conventional Freezing of Human Spermatozoa: A Comparative Transcript Profiling. International Journal of Molecular Sciences. 2022;23(6):30-47. doi: 10.3390/ijms23063047.
Saritha KR, Bongso A. Comparative evaluation of fresh and washed human sperm cryopreserved in vapor and liquid phases of liquid nitrogen. Journal of Andrology. 2001;22(5):857-62. PMID: 11545300.
Thomson LK, Fleming SD, Barone K, Zieschang J, Clark AM. The effect of repeated freezing and thawing on human sperm DNA fragmentation. Fertility and Sterility. 2010;93(4):1147-1156. https://doi.org/10.1016/j.fertnstert.2008.11.023.
Le MT, Nguyen TTT, Nguyen TT, et al. Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects. The European Journal of Obstetrics & Gynaecology and Reproductive Biology. 2019;2(34):14-20. doi: 10.1016/j.ejogrb.2019.01.001.
Copyright (c) 2022 Journal of Infertility and Reproductive Biology
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.