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Byline: W.Edward Richards, Sheila M. Dobin, Vicki Malone, Alfred B. Knight, Thomas J. Kuehl Keywords: Fluorescence in situ hybridization; sex selection; X chromosome; Y chromosome; human; spermatozoa; sperm cryopreservation Abstract: OBJECTIVE: Although most methods for selecting the sex of offspring by sorting spermatozoa are ineffective at shifting the ratio of Y- to X-containing cells, some commercial sources continue to offer such services. Our objective was to evaluate commercially 'sorted' samples with use of dual-color fluorescence in situ hybridization and to identify variations in assessment by comparing motile and total sperm populations, donors, observers, and fluorescence in situ hybridization probes. STUDY DESIGN: Cryopreserved sperm from seven anonymous donors were processed as for insemination. Sperm cells from each total sample or motile subfraction were prepared for fluorescence in situ hybridization by incubation with disulfide-reducing agents to expand sperm nuclei. Two sets of X and Y chromosome-specific, fluorophore-labeled deoxyribonucleic acid probes were used. At least 400 nuclei from each preparation were classified independently by three blinded observers. Hybridization efficiency, aneuploidy, and sex chromosome content were evaluated in subsets of five unsorted, five female-oriented, and five male-oriented samples. Total and motile subfractions were compared with eight samples. Fluorescence in situ hybridization probes were compared in five paired unsorted samples. RESULTS: No differences were detected between washed samples and paired motile subfractions. No differences in hybridization and aneuploidy were detected between groups of sorted samples. The Y/X ratio was significantly different between the sorted groups. However, male-oriented samples had a lower Y/X ratio than female-oriented samples did. Observer and probe choice accounted for small but significant variations that did not alter conclusions about the Y/X ratio for sorted samples. CONCLUSION: In a series of 10 sorted samples from one commercial source, dual-color fluorescence in situ hybridization demonstrated a small but significant shift in the sex chromosome ratios among samples. However, this shift was opposite to that expected by the orientation of the sorted samples. (Am J Obstet Gynecol 1997;176:1172-80.) Author Affiliation: Temple, Texas Article Note: (footnote) [star] From the Departments of Obstetrics and Gynecology,a Pathology,b Medical Biochemistry and Genetics,c and Pediatrics,d Scott & White Clinic and Memorial Hospital, Texas A&M University Health Science Center College of Medicine., [star][star] Supported by the Scott, Sherwood, and Brindley Foundation., a Reprint requests: Thomas J. Kuehl, PhD, Scott & White Clinic, 2401 S. 31st St., Temple, TX 76508., aa 0002-9378/97 $5.00 + 0 6/6/81338