Volume 10, Issue 5 (10-2012)                   IJRM 2012, 10(5): 435-0 | Back to browse issues page

PMCID: 0

XML Persian Abstract Print


1- Department of Anatomical Sciences, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran , esmaeilt@sums.ac.ir
2- Department of Anatomical Sciences, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran
3- Islamic Azad University, Larestan, Iran
4- Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran
5- Shiraz Infertility Center, Shiraz, Iran
Abstract:   (537 Views)
Background: It has been claimed that by using different washing methods, the sperms can be separated according to size, motility, density, chromosomal content and surface markings and charge. These methods also reduce sperm chromatin deficiencies and screen the sperms before applying in assisted reproduction techniques.
Objective: This study compared simple density gradient methods and a combined method with albumin density gradient and PureSperm separation (alb/PureSperm) for sex preselection by double fluorescence in situ hybridization (FISH) versus chromomycin A3 staining to determine chromatin integrity. Materials and Methods: 30 normal semen samples were prepared with PureSperm, albumin gradients and alb/PureSperm. All samples were then stained by FISH and chromomycin A3. The results were compared with SPSS 11.5 and the Kruskal-Wallis test.
Results: The proportion of X-bearing spermatozoa by PureSperm separation (47.58±5.67) and Y-bearing spermatozoa by albumin gradient (46.13±3.83) methods were slightly higher than in putative normal sperm samples (1:1), but there were no significant differences in the X- or Y- bearing spermatozoa counts among the three methods. Albumin gradient separation tended to underestimate abnormal spermatozoa compared to PureSperm and combined alb/PureSperm.
Conclusion: Routine separation methods slightly enriched X- or Y- bearing spermatozoa, but the differences were not significant for clinical purposes. The combined alb/PureSperm method had no advantages for assessing sex ratio or chromatin integrity compared to simpler gradient methods.
Full-Text [PDF 520 kb]   (114 Downloads) |   |   Full-Text (HTML)  (39 Views)  
Type of Study: Original Article |

References
1. Schenker JG. Gender selection: cultural and religious perspective. J Assist Reprod Genet 2002; 19: 400-410. [DOI:10.1023/A:1016807605886]
2. Yan J, Feng HL, Chen ZJ, Hu J, Gao X, Qin Y. Influence of swim up time on the ratio of X- and Y-bearing spermatozoa. Eur J Obstet Gynecol Reprod Biol 2006; 129: 150-154. [DOI:10.1016/j.ejogrb.2006.02.020]
3. Sureau C. Gender selection: a crime against humanity or the exert fundamental right? Hum Repod 1999; 14: 867-868. [DOI:10.1093/humrep/14.4.867]
4. Classens OE, Oosthuizen CJ, Brusnicky J, Franken DR, Kruger TF. Fluorescent in situ hybridization evaluation of human Y-bearing spermatozoa separated by albumin density gradients. Fertil Steril 1995; 63: 417-418. [DOI:10.1016/S0015-0282(16)57381-6]
5. Flaherty SP, Michaliowsa J, Swann NJ, Dmowski WP, Mattews CD, Aitken RJ. Albumin gradients do not enrich Y-bearing human spermatozoa. Hum Reprod 1997; 12: 938-942. [DOI:10.1093/humrep/12.5.938]
6. Van Kooij RJ, Van Oost BA. Determination of sex ratio of spermatozoa with a deoxyribonucleic acid-probe and quinacrine staining: a comparison. Feril Steril 1992; 58: 384-386. [DOI:10.1016/S0015-0282(16)55226-1]
7. Johnson LA, Welch GR, Keyvanfar K, Dorfmann A, Fugger EF, Schulman JD. Gender pre selection in humans? Flow cytometric separation of X & Y sperm for the prevention of X-linked disease. Hum Reprod 1993; 8: 1733-1739. [DOI:10.1093/oxfordjournals.humrep.a137925]
8. Wang HX, Flaherty SP, Swann NJ, Matthews CD. Assessment of separation of X-bearing Y-bearing sperm on albumin gradients using double-label fluorescence in situ hybridization. Fertil Steril 1994; 61: 720-726. [DOI:10.1016/S0015-0282(16)56652-7]
9. Flaherty SP, Matthews CD. Application of modern molecular techniques to evaluate sperm sex selection methods. Mol Hum Reprod 1996; 2: 937-942. [DOI:10.1093/molehr/2.12.937]
10. Han TL, Ford JH, Webb GC, Flaherty SP, Correll A, Matthews CD. Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization. Mol Reprod Dev 1993; 34: 308-313. [DOI:10.1002/mrd.1080340311]
11. Karabinus DS. Flow cytometric sorting of human sperm microsort clinical trial update. Theriogenology 2009; 71: 74-79. [DOI:10.1016/j.theriogenology.2008.09.013]
12. Johnson LA, Schulman JD. The safety of sperm selection by flow cytometry. Hum Reprod 1994; 9: 758-759. [DOI:10.1093/oxfordjournals.humrep.a138591]
13. McNutt TL, Johnson LA. Flow cytometric sorting of sperm: Influence on fertilization and embryo/fetal development in the rabbit. Mol Reprod Dev 1996; 43: 261-267. https://doi.org/10.1002/(SICI)1098-2795(199602)43:2<261::AID-MRD16>3.0.CO;2-6 [DOI:10.1002/(SICI)1098-2795(199602)43:23.0.CO;2-6]
14. Merton JS, Haring RM, Stap J, Hoebe RA, Aten JA. Effect of flow cytometrically sorted frozen/thawed semen on success rate of in vitro bovine embryo production. IETS Annual Meeting, Nice, France; 1997.
15. Svalander PC, Lundin K, Holmes PV. Endotoxin level in percoll density gradient media used to prepare sperm for human IVF treatment. Hum Reprod 1995; 10 (Suuple.): 130.
16. Claaessens OE, Menkveld R, Harrison KL. Evaluation of three substitutes for Percoll in sperm isolation by density gradient centrifugation. Hum Reprod 1998; 13: 3139-3143. [DOI:10.1093/humrep/13.11.3139]
17. Mortimer D. Structured management as a basis for cost effective infertility care. In: The Male Gamete: From Basic Science to Clinical Applications. Gagnon C (ed). Cache River Press, Vienna, IL, USA; 1999.
18. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997; 56, 602-607. [DOI:10.1095/biolreprod56.3.602]
19. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor quality sperm samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998; 69: 528-532. [DOI:10.1016/S0015-0282(97)00536-0]
20. Bianchi PG, Manicardi G, Bizzaro D, Campana A, Bianchi U, Sakkas D. Use of the guanine-cytosine (GC) specific fluorochrome, chromomycin A3, as an indicator of poor sperm morphology. J Assist Reprod Genet 1996; 13: 246-250. [DOI:10.1007/BF02065944]
21. World Health Organization. laboratory manual for the Examination and processingof human semen. 5th Ed. Cambridge University Press; Cambridge; 2010.
22. Beernink FJ, Dmowski WP, Ericsson RJ. Sex preselection through albumin separation of sperm. Fertil Steril 1993; 59: 382-386. [DOI:10.1016/S0015-0282(16)55681-7]
23. Aleahmad F, Gourabi H, Zeinali B, Kazemi Ashtiani S, Baharvand H. Separation of X-and Y-bearing human spermatozoa by sperm isolation medium gradients evaluated by FISH. Reprod Biomed‌ Online 2009; 18: 475-478. [DOI:10.1016/S1472-6483(10)60122-8]
24. Chen MJ, Guu HF, Ho ES. Efficiency of sex pre-selection of spermatozoa by albumin separation method evaluated by double-labeled fluorescence in-situ hybridization. Hum Reprod 1997; 12: 1920-1926. [DOI:10.1093/humrep/12.9.1920]
25. Aribarg A, Ngeamvijawat J, Chanprasit Y, Sukcharoen N. Determination of the ratio of X and Y bearing spermatozoa after albumin gradient method using double-labeled fluorescence in situ hybridization (FISH). J Med Assoc Thai 1996; 79 (Suppl.): 88-95.
26. Andersen CY, Byskov AG. Enhanced separation of X and Y bearing sperm cells by a combined density gradient centrifugation evaluated by fluorescence in situ hybridization of the Y-chromosome. Acta Obstet Gynecol Scand 1997; 76: 131-134. [DOI:10.3109/00016349709050068]
27. Madrid-Bury N, Fernández R, Jiménez A, Pérez-Garnelo S, Moreira PN, Pintado B, et al. Effect of ejaculate, bull, and a double swim-up sperm processing method on sperm sex ratio. Zygote 2003; 11: 229-235. [DOI:10.1017/S0967199403002272]
28. Kobayashi J, Oguro H, Uchida H, Kohsaka T, Sasada H, Sato E. Assessment of bovine X- and Y-bearing spermatozoa in fractions by discontinuous percoll gradients with rapid fluorescence in situ hybridization. J Reprod Dev 2004; 50: 463-469. [DOI:10.1262/jrd.50.463]
29. Wolf CA, Brass KE, Rubin MIB, Pozzobon SE, Mozzaquatro FD, De La Corte FD. The effect of sperm selection by Percoll or swim-up on the sex ratio of in vitro produced bovine embryos. Anim Reprod 2008; 5: 110-115.
30. Hendriksen PJ. Do X and Y spermatozoa differ in proteins? Theriogenology 1999; 52: 1295-307. [DOI:10.1016/S0093-691X(99)00218-6]
31. Rose GA, Wong A. Experiences in Hong Kong with the though & practice of the albumin column method of sperm separation for sex selection. Hum Reprod 1998; 13: 146-149. [DOI:10.1093/humrep/13.1.146]
32. Sakkas D, Manicardi GC, Tomlinson M, Mandrioli M, Bizzaro, Bianchi PG, et al. The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear anomalies. Hum Reprod 2000; 15: 1112-1116. [DOI:10.1093/humrep/15.5.1112]
33. Kheirollahi-Kouhestani M, Razavi S, Tavalaee M, Deemeh MR, Mardani M, Moshtaghian J, et al. Selection of sperm based on combined density gradient and Zeta method may improve ICSI outcome. Hum Reprod 2009; 24: 2409-2416. [DOI:10.1093/humrep/dep088]