Asian Journal of Andrology (2005) 7, 86–92; doi:10.1111/j.1008-862X.2005.00008.x
Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa
Shyam S R Allamaneni, Ashok Agarwal, Sreedhar Rama, Pavithra Ranganathan and Rakesh K Sharma
Center for Advanced Research in Human Reproduction, Infertility and Sexual Function, Glickman Urological Institute and Department of Obstetrics-Gynecology, Cleveland Clinic Foundation, Cleveland, Ohio, USA
Correspondence: Ashok Agarwal, Professor of Surgery; Director, Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function, Glickman Urological Institute and Department of Obstetrics-Gynecology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Desk A19.1, Cleveland, OH 44195, USA. Fax: +1-216-636-3118/445-6049. E-mail: agarwaa@ccf.org
Received 19 May 2004; Accepted 6 December 2004.
Top of pageAbstract
Aim:
To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate.
Methods:
This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels.
Results:
Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2
106 vs. 17.6
106), recovery rates (69.2 %vs. 50.0 %), and longevity at 4 hours (83.0 %vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 %vs. 19.7 %) than ISolate.
Conclusion:
Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART.
Keywords:
spermatozoa, cryopreservation, density gradient centrifugation, sperm motility
Top of pageReferences
- Saleh R, Agarwal A. Oxidative stress and male infertility: from research bench to clinical practice. J Androl 2002; 23: 737–52. | PubMed | ChemPort |
- Mortimer D. Sperm preparation methods. J Androl 2000; 21: 357–66. | PubMed | ChemPort |
- Rogers BJ, Perreault SD, Bentwood BJ, McCarville C, Hale RW, Soderdahl DW. Variability in the human-hamster in vitro assay for fertility evaluation. Fertil Steril 1983; 39: 204–11. | PubMed | ChemPort |
- Yamamoto Y, Maenosono S, Okada H, Miyagawa I, Sofikitis N. Comparisons of sperm quality, morphometry and function among human sperm populations recovered via SpermPrep II filtration, swim-up and Percoll density gradient methods. Andrologia 1997; 29: 303–10. | PubMed | ChemPort |
- Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl 1988; 9: 367–76. | PubMed | ChemPort |
- Yogev L, Gamzu R, Paz G, Kleiman S, Botchan A, Hauser R, et al. Pre-freezing sperm preparation does not impair thawed spermatozoa binding to the zona pellucida. Hum Reprod 1999; 14: 114–7. | Article | PubMed | ChemPort |
- Sharma RK, Agarwal A. Sperm quality improvement in cryopreserved human semen. J Urol 1996; 156: 1008–12. | Article | PubMed | ChemPort |
- Subak LL, Adamson GD, Boltz NL. Therapeutic donor insemination: a prospective randomized trial of fresh versus frozen sperm. Am J Obstet Gynecol 1992; 166: 1597–604; discussion 604–6. | PubMed | ChemPort |
- Sakkas D, Manicardi GC, Tomlinson M, Mandrioli M, Bizzaro D, Bianchi PG, et al. The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies. Hum Reprod 2000; 15: 1112–6. | Article | PubMed | ChemPort |
- Centola GM, Herko R, Andolina E, Weisensel S. Comparison of sperm separation methods: effect on recovery, motility, motion parameters, and hyperactivation. Fertil Steril 1998; 70: 1173–5. | Article | PubMed | ChemPort |
- Makkar G, Ng HY, Yeung SB, Ho PC. Comparison of two colloidal silica-based sperm separation media with a non-silicabased medium. Fertil Steril 1999; 72: 796–802. | Article | PubMed | ChemPort |
- WHO Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge: Cambridge University Press, 1992.
- Hallak J, Sharma RK, Wellstead C, Agarwal A. Cryopreservation of human spermatozoa: comparison of TEST-yolk buffer and glycerol. Int J Fertil Womens Med 2000; 45: 38–42. | PubMed | ChemPort |
- Byrd W, Drobnis EZ, Kutteh WH, Marshburn P, Carr BR. Intrauterine insemination with frozen donor sperm: a prospective randomized trial comparing three different sperm preparation techniques. Fertil Steril 1994; 62: 850–6. | PubMed | ChemPort |
- Claassens O, Kaskar K, Coetzee K, Lombard C, Franken D, Kruger T. Comparison of motility characteristics and normal sperm morphology of human semen samples separated by percoll density gradient centrifugation. Arch Androl 1996; 36: 127–32. | Article | PubMed | ChemPort |
- Arora M, Carver-Ward JA, Jaroudi KA, Sieck UV. Is Percoll safe for in vivo use? Fertil Steril 1994; 61: 979–81. | PubMed | ChemPort |
- Soderlund B, Lundin K. The use of silane-coated silica particles for density gradient centrifugation in in-vitro fertilization. Hum Reprod 2000; 15: 857–60. | Article | PubMed | ChemPort |
- Chen MJ, Bongso A. Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception. Hum Reprod 1999; 14: 759–64. | Article | PubMed | ChemPort |
- Chan CC, Chen IC, Liu JY, Huang YC, Wu GJ. Comparison of nitric oxide production motion characteristics of sperm after cryopreserved in three different preparations. Arch Androl 2004; 50: 1–3. | Article | PubMed | ChemPort |
- Hammadeh ME, Kuhnen A, Amer AS, Rosenbaum P, Schmidt W. Comparison of sperm preparation methods: effect on chromatin and morphology recovery rates and their consequences on the clinical outcome after in vitro fertilization embryo transfer. Int J Androl 2001; 24: 360–8. | Article | PubMed | ChemPort |
- Claassens OE, Menkveld R, Harrison KL. Evaluation of three substitutes for Percoll in sperm isolation by density gradient centrifugation. Hum Reprod 1998; 13: 3139–43. | Article | PubMed | ChemPort |