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The use of flow cytometric sorting to separate sperm into enriched X- and Y chromosome bearing populations is the only preconception method of gender selection having a solid scientific basis and whose results can be validated by modern molecular techniques such as fluorescence in situ hybridization (FISH) or by birth results (phenotypic sex). With this method sperm DNA (located in the sperm head) is stained with a DNA-specific fluorescent dye (fluorochrome) and processed through a flow cytometer. The flow cytometer utilizes a laser to cause stained DNA to fluoresce and then measures differences in the amount of fluorescence emitted by the sperm. The differences between sperm fluorescence intensity allows for discrimination between the X- and Y bearing sperm such that the desired sperm may be recovered in a highly enriched population. Because the X chromosome is larger and contains more DNA than the Y chromosome in mammals, X-bearing sperm have more total DNA than Y-bearing sperm. Thus, when stained with a DNA-specific fluorochrome, the fluorescent signal emitted by an X-bearing sperm is brighter than the signal emitted by a Y-bearing sperm.
Using flow cytometry and the DNA-specific flurochrome Hoechst 33342, Dr. Lawrence Johnson and colleagues at the United States Department of Agricultures (USDA) Agricultural Research Service, Beltsville, MD, were the first to sort mammalian sperm into two enriched populations based on differing DNA content. The technology was patented by USDA in 1992. Reports using this procedure to produce normal births in different animal species soon followed. In the human there is a 2.8% difference in DNA content between X- and Y-bearing sperm. Dr. Johnson and colleagues reported the first successful flow cytometric separation of X and Y bearing human sperm into highly enriched populations. The first human pregnancy resulting from the use of flow cytometrically sorted sperm resulted from work done at the Genetics & IVF Institute (GIVF) a Fairfax, Virginia fertility program. In 1998, GIVF reported the birth of babies resulting from the use of flow cytometrically separated human sperm for intrauterine insemination (IUI), in vitro fertilization (IVF), and from intracytoplasmic sperm injection (ICSI).
In 1992 the USDA granted GIVF an exclusive license to apply the patented sorting technology for development and use in humans. Institutional review board (IRB) approval was received in 1994 to initiate clinical studies to use the technology for couples at risk for having children with X-linked disease. The clinical study was expanded in 1995 to offer family balancing. The United States Food and Drug Administration (FDA) approved an Investigational Device Exemption in 2000 for GIVF to continue to conduct a clinical trial of safety and efficacy of the MicroSort® Sperm Separation Technology. To date, over 900 babies have been born after using MicroSort® for sorting fresh or frozen sperm into enriched X- and Y bearing populations for use in IUI, IVF and ICSI. Flow cytometric sperm sorting in the livestock species and other animals was licensed by USDA to XY, Inc., a company in Colorado, and has resulted in the birth of tens of thousands of offspring in North and South America, Europe and Asia.
Successful flow cytometric sorting of sperm depends upon the accurate detection of differences in fluorescent signal intensity (a function of sperm DNA content) between the X and Y bearing sperm. The major factors affecting the fluorescent signals that result from the interaction of the sperm and the laser in the human are sperm head shape, degree of sperm-to-sperm differences in head shape, and the difference in DNA content between X-and Y-bearing sperm (2.8%). Human sperm heads (nuclei) are not symmetric about their axes; they have an oval appearance when lying flat but exhibit a narrow profile in longitudinal section. Because of sperm head shape and the nature of sperm nuclear chromatin packaging, the intensity of the fluorescent signal emitted by the edge of the sperm head differs from that emitted by the flat aspect, making sperm head orientation relative to the excitation light a critical factor in analyzing and sorting the sperm.
Sperm move through the flow cytometer in single file in a stream of cell culture medium. The stream is intersected by a laser beam which causes the stained sperm within the stream to fluoresce. Because orientation of the sperm relative to the laser is random ~10 to 20% of sperm are suitably oriented to achieve the most accurate analysis of DNA content and thus, collection of the fluorescent signal. This random orientation results in only a small percentage of sperm, perhaps 10%, being appropriately oriented for accurate sorting. The effects of sperm orientation, coupled with other factors, such as sperm-to-sperm variation within a specimen, the impact of sorting time on sperm survival, and the fact that only 50% of those sperm carry the desired sex chromosome, result in a very small proportion of available sperm actually being recovered by sorting.
The sorting of human sperm human sperm for insemination may be performed using freshly collected sperm or cryopreserved sperm. Semen is evaluation and processed using standard methods, then stained with Hoechst 33342 prior to sorting into separate X or Y sperm populations. At the conclusion of sorting, a sample of sorted sperm is evaluated for motility, progression, and the degree of enrichment in X- or Y-bearing sperm using fluorescence in situ hybridization (FISH). Sorted sperm are then utilized fresh for IUI or IVF/ICSI or are cryopreserved for future IVF/ICSI use.
[edit] Sources
Cran DG, Johnson LA, Miller NGA, et al. Production of bovine calves following separation of X- and Y-chromosome bearing sperm and in vitro fertilization. Vet Rec 1993;132:40-41.
Cran DG, Cochrane DJ, Johnson LA. et al. Separation of X- and Y-chromosome bearing bovine sperm by flow cytometry for use in IVF. Theriogenology 1994;41:183.
Fugger EF, Black SH, Keyvanfar K, Schulman JD. Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection. Hum Reprod 1998;30,2367-70.
Johnson LA, Flook JP, Look MV, et al. Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. 1987; Gamete Res 1987;16, 1-9.
Johnson LA, Flook J.P, Hawk HW. Sex preselection in rabbits: Live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 1989;41: 199-203.
Johnson LA. Sex preselection in swine: altered sex ratios in offspring following surgical insemination of flow sorted X- and Y-bearing sperm. Reprod. Domest Anim 1991;26, 309-314.
Johnson LA, Welch GR, Keyvanfar K, et al. 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.
Johnson LA, Pinkel D. Modification of a laser-based flow cytometer for high resolution DNA analysis of mammalian spermatozoa. Cytometry 1986;7, 268-273
Johnson, LA., Welch GR. Sex preselection: High speed flow cytometric sorting of X- and Y- sperm for maximum efficiency. Theriogenology 1999;52,1323-1341.
Johnson LA. Sexing mammalian sperm for production of offspring: the state-of-the-art. Animal Reprod Sci 2000;60-61:93-107.
Levinson G, Keyvanfar K, Wu JC, et al. DNA based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease. Mol Hum Reprod 1; Hum Reprod 1995;10, 979-982.
Moruzzi JF. Selecting a mamalian species for the separation of X- and Y-chromosome-bearing spermatozoa. J Reprod Fertil 1979;57:319-323.
Pinkel D, Gledhill BL, Van Dilla MA, et al. High resolution DNA measurements of mammalian sperm. Cytometry 1982;3:1-9.
Seidel GE, Garner DL. Current status of sexing mammalian spermatozoa. Reproduction 2002;124:733-743).
Sumner AT, Robinson JA. A difference in dry mass between the heads of X- and Y-bearing human spermatozoa. J Reprod Fert 1976;48, 9-15.
12.154.95.254 18:11, 6 April 2007 (UTC)
- Declined - this reads more like an essay or science-journal article than an encyclopedia entry. Please feel free to see Creating Your First Article for tips on how to write an article best for an encyclopedia. Patstuarttalk·edits 19:03, 6 April 2007 (UTC)