ASSESSMENT OF HUMAN SPERM ACROSOME REACTION BY FLOW-CYTOMETRY - VALIDATION AND EVALUATION OF THE METHOD BY FLUORESCENCE-ACTIVATED CELL SORTING

Objective: To determine the applicability of flow cytometry to assess human sperm acrosome reaction. Design: Prospective evaluation of semen samples incubated overnight for the development of spontaneous acroso me reaction or exposed to the calcium ionophore A23187 (5 mu M) for 3 hours for induction of the acrosome reaction. Setting: University-affi liated tertiary care center. Patients: Normal healthy volunteers. Inte rventions: The spermatozoa were stained with fluorescein isothiocyanat e (FITC)-labeled pea agglutinin. The labeled samples were assessed vis ually and also subjected to analytic flow cytometry and fluorescence-a ctivated cell sorting. Main Outcome Measures: Acrosome reaction assess ed visually and by flow cytometry. Results: Flow cytometric analysis s howed a single peak of FITC fluorescence in the washed semen samples. A second peak of lower FITC fluorescence intensity was noted after ove rnight incubation or exposure to A23187, suggesting loss of fluorescen ce, which indicated the occurrence of the acrosome reaction. There was a statistically significant correlation between the assessment by the two methods (n = 41). However, although the mean difference between t he methods was small (2.49%), the difference between the two methods f or each individual sample can vary between -24% to +29%. When the sper m cells were subjected to cell sorting based on green fluorescence int ensity, reanalysis and visual scoring verified that the low intensity peak contained a majority of acrosome-reacted spermatozoa (77.52% +/- 2.39%). Conclusion: These results validated the flow cytometric method for assessment of acrosome-reacted spermatozoa. Although flow cytomet ry is more objective and less time consuming when many samples are ass essed at the same time, the visual method remains a useful and practic al procedure in the clinical andrology laboratory.