EVALUATING SEX-CHROMOSOME CONTENT OF SORTED HUMAN SPERM SAMPLES WITH USE OF DUAL-COLOR FLUORESCENCE IN-SITU HYBRIDIZATION

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 servi ces. Our objective was to evaluate commercially ''sorted'' samples wit h use of dual-color fluorescence in situ hybridization and to identify variations in assessment by comparing motile and total sperm populati ons, donors, observers, and fluorescence in situ hybridization probes. STUDY DESIGN: Cryopreserved sperm from seven anonymous donors were pr ocessed as for insemination. Sperm cells from each total sample or mot ile subfraction were prepared for fluorescence in situ hybridization b y incubation with disulfide-reducing agents to expand sperm nuclei. Tw o sets of X and Y chromosome-specific, fluorophore-labeled deoxyribonu cleic acid probes were used. At least 400 nuclei from each preparation were classified independently by three blinded observers. Hybridizati on efficiency, aneuploidy, and sex chromosome content were evaluated i n subsets of five unsorted, five female-oriented, and five male-orient ed samples. Total and motile subfractions were compared with eight sam ples. 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 hybr idization and aneuploidy were detected between groups of sorted sample s. The Y/X ratio was significantly different between the sorted groups . However, male-oriented samples had a lower Y/X ratio than female-ori ented samples did. Observer and probe choice accounted for small but s ignificant variations that did not alter conclusions about the Y/X rat io for sorted samples. CONCLUSION: In a series of 10 sorted samples fr om one commercial source, dual-color fluorescence in situ hybridizatio n demonstrated a small but significant shift in the sex chromosome rat ios among samples. However, this shift was opposite to that expected b y the orientation of the sorted samples.