Sex preselection by flow cytometric separation of X and Y chromosome-bearing sperm based on DNA difference: A review

Recent research on the flow cytometry of sperm for the purpose of predetermining gender of offspring has led to a validated method to separate X from Y chromosome-bearing sperm for use with in vitro fertilization and embryo transfer, intratubal insemination or intracytoplasmic sperm injection. The basis for the method is the sex chromosome-specific marker, DNA, which is present in greater amounts in X-bearing sperm than in Y-bearing sperm of mammals. Sperm are exposed to the vital dye Hoechst 33342 which binds to the minor groove of the DNA helix. Flow cytometric sorting of the sperm using a laser as the excitation source results in populations of Y- or X-bearing sperm that are 85-90% pure. Several hundred offspring have been produced from swine, rabbits, sheep and cattle that confirm the predicted sex. The method is currently being applied to the commercial embryo market. The method is not likely to be used in conjunction with standard cattle or swine artificial insemination practice in its current form since only about 4x10(5) sorted sperm can be produced per hour of sorting. The technology has also been applied to human sperm for use by couples that are at risk to sex-linked disease expression in their offspring. Populations of human sperm have been sorted with X and Y purities of about 80% as confirmed by DNA probe technology and fluorescence in situ hybridization.