Abstract
Purpose:Double-label fluorescence in situ hybridization (FISH) was used to evaluate the efficiency of separating X-and Y-chromosome-bearing spermatozoa through 12-step discontinuous Percoll gradients.
Methods:Liquefied normal semen samples from 10 healthy donors were overlaid onto 25% Percoll and centrifuged. Parts of the sperm pellet were saved as control, while the remaining portion was separated by 12-step Percoll gradient. After centrifugation, the spermatozoa in the 80% Percoll layer were collected. The X:Y ratio of the control and separated spermatozoa was verified by double-label FISH (CEP SOX/SGYprobes) and scored blindly by one observer. Differences in the X: Y ratios between matched groups were analyzed by paired t testa.
Results:The overall average labeling efficiency was 99.2%. A significant enrichment (P = 0.02) of X-bearing spermatozoa was obtained in Percoll separated fractions (mean X:Y ratio = 52.2:46.4) compared with the control group (X:Y ratio = 49.5:48.4). Discontinuous Percoll gradients also decreased the proportion of aneuploid spermatozoa (from 1.0 to 0.8%), but the differences were nonsignificant.
Conclusions:Discontinuous Percoll separation did increase the X:Y ratio significantly, but the enrichment of X-bearing spermatozoa is insufficient for clinical use in preconceptional sex selection.
Keywords: fluorescence in situ hybridization, Percoll, X-sperm preselection
Full Text
The Full Text of this article is available as a PDF (433.8 KB).
REFERENCES
- 1.McKusick VA. Mendelian Inheritance in Man. 9th ed. Baltimore: Johns Hopkins University Press; 1990. [Google Scholar]
- 2.Reubinoff BE, Schenker JG. New advances in sex preselection. Fertil Steril. 1996;66:343–350. doi: 10.1016/s0015-0282(16)58498-2. [DOI] [PubMed] [Google Scholar]
- 3.Steeno O, Adimoelja A, Steeno J. Separation of X-and Y-bearing human spermatozoa with the Sephadex gel-filtration method. Andrologia. 1975;7:95–97. doi: 10.1111/j.1439-0272.1975.tb01234.x. [DOI] [PubMed] [Google Scholar]
- 4.Quinlivan WLG, Preciado K, Long TL, Sullivan H. Separation of human X and Y spermatozoa by albumin gradients and Sephadex chromatography. Fertil Steril. 1982;37:104–107. [PubMed] [Google Scholar]
- 5.Kaneko S, Yamaguchi J, Kobayashi T, Iizuka R. Separation of human X-and Y-bearing sperm using Percoll density gradient centrifugation. Fertil Steril. 1983;40:661–665. doi: 10.1016/s0015-0282(16)47427-3. [DOI] [PubMed] [Google Scholar]
- 6.Iizuka R, Kaneko S, Aoki R, Kobayashi T. Sexing of human sperm by discontinuous Percoll density gradient and its clinical application. Hum Reprod. 1987;2:573–575. doi: 10.1093/oxfordjournals.humrep.a136591. [DOI] [PubMed] [Google Scholar]
- 7.Johnson LA, Welch GR, Keyvanfar K, Dorfmann A, Fugger EF, Schulman JD. Gender preselection in humans? Flow cytometric separation of X and Y spermatozoa for the prevention of X-linked diseases. Hum Reprod. 1993;8:1733–1739. doi: 10.1093/oxfordjournals.humrep.a137925. [DOI] [PubMed] [Google Scholar]
- 8.Levinson G, Keyvanfar K, Wu JC, Fugger EF, Fields RA, Harton GL, Palmer FT, Sisson ME, Starr KM, Dennison-Lagos L, Calvo L, Sherins RJ, Bick D, Schulman JD, Black SH. DNA-based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease. Hum Reprod. 1995;10:979–982. doi: 10.1093/oxfordjournals.humrep.a136081. [DOI] [PubMed] [Google Scholar]
- 9.Barlow P, Vosa CG. The Y chromosome in human spermatozoa. Nature. 1970;226:961–962. doi: 10.1038/226961a0. [DOI] [PubMed] [Google Scholar]
- 10.Sumner AT, Robinson JA, Evans HJ. Distinguishing between X, Y and YY-bearing spermatozoa by fluorescence and DNA content. Nature. 1971;229:231–233. doi: 10.1038/newbio229231a0. [DOI] [PubMed] [Google Scholar]
- 11.Pearson PL, Bobrow M, Vosa CG. Technique for identifying Y chromosome in human interphase nuclei. Nature. 1970;226:78–80. doi: 10.1038/226078a0. [DOI] [PubMed] [Google Scholar]
- 12.Thomsen JL, Niebuhr E. The frequency of false-positive and false-negative results in the detection of Y-chromosomes in interphase nuclei. Hum Genet. 1986;73:27–30. doi: 10.1007/BF00292659. [DOI] [PubMed] [Google Scholar]
- 13.van Kooij RJ, van Oost BA. Determination of sex ratio of spermatozoa with a deoxyribonucleic acid-probe and quinacrine staining: a comparison. Fertil Steril. 1992;58:384–386. doi: 10.1016/s0015-0282(16)55226-1. [DOI] [PubMed] [Google Scholar]
- 14.Wang H-X, Flaherty SP, Swann NJ, Matthews CD. Discontinuous Percoll gradients enrich X-bearing human spermatozoa: A study using double-label fluorescence in-situ hybridization. Hum Reprod. 1994;9:1265–1270. doi: 10.1093/oxfordjournals.humrep.a138692. [DOI] [PubMed] [Google Scholar]
- 15.Tohyama Y, Oshio S, Yotsukura M, Umeda T, Mohri H. Percoll density gradients centrifugation can separate human X-bearing sperm. J Assist Reprod Genet. 1997;14(Suppl):194s. [Google Scholar]
- 16.Martini E, Speel EJ, Geraedts JP, Ramaekers FC, Hopman AH. Application of different in-situ hybridization detection methods for human sperm analysis. Hum Reprod. 1995;10:855–861. doi: 10.1093/oxfordjournals.humrep.a136050. [DOI] [PubMed] [Google Scholar]
- 17.Gledhill BL. Selection and separation of X-and Y-chromosome-bearing mammalian sperm. Gamete Res. 1988;20:377–395. doi: 10.1002/mrd.1120200312. [DOI] [PubMed] [Google Scholar]