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We investigated the applicability of flow cytometric (FCM) sorting to select, with no deleterious effects, fractions of living, highly motile spermatozoa after staining with rhodamine 123 (Rh123) and propidium iodide (PI) for assessment of their mitochondrial activity and viability, respectively. Sperm cells were subjected to FCM sorting according to their Rh123 fluorescence intensity, and computer-aided sperm analysis (CASA) for percentage motility and movement characteristic measurements was carried out on the entire sperm populations and on the Rh123-positive (Rh123+) sorted fractions. A first experiment on five sperm samples from fertile donors pre-selected by either swim-up or simplified Percoll gradient indicated no detrimental effect of the FCM sorting procedure because: (a) the numbers of Rh123+ motile sperm were not decreased by FCM sorting; (b) data on the sorted fractions showed a tendency (not significant) for an increase in movement parameters rather than a drop; and (c) a significant decrease in the percentage of PI-positive (PI+) sperm (13% vs 3%; p &lt; 0.05) was measured. A second experiment was performed on sperm samples from four patients, only washed and re-suspended in B2 medium. This demonstrated a significant increase in some characteristics of movement quality related to a substantial and selective immobilization of the less motile sperm. The significant drop in the percentage of PI+ sperm after FCM sorting (p &lt; 0.01) was less pronounced than after FCM sorting of pre-selected sperm (12% vs 3%, respectively), indicating a lethal effect of FCM sorting on a small proportion of presumably moribund sperm. These preliminary data indicate a differential effect of FCM sorting on sperm according to their function characteristics and suggest the potential importance of these methods for the characterization in vitro of sperm subpopulations on the basis of functional criteria.

Flow cytometric sorting of living, highly motile human spermatozoa based on evaluation of their mitochondrial activity.