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Hum. Reprod. Advance Access originally published online on March 25, 2004
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Human Reproduction, Vol. 19, No. 5, 1127-1134, May 2004
© 2004 European Society of Human Reproduction and Embryology

Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters

Carole Marchetti1,2, Miguel-Angel Gallego1, André Defossez2, Pierre Formstecher1 and Philippe Marchetti1,5

1 INSERM U459, Faculté de Médecine, 1 Place Verdun, F-59045 Lille Cedex and 2 Laboratoire de Biologie de la Reproduction, Hôpital Jeanne de Flandres et Laboratoire d’Histologie, Faculté de Médecine, F-59037 Lille Cedex, France

5 To whom correspondence should be addressed at: INSERM U 459, Faculté de Médecine, 1 Place Verdun, F-59045 Lille Cedex, France. e-mail: philippe.marchetti{at}lille.inserm.fr

BACKGROUND: Detection of apoptosis in sperm samples may help evaluate sperm quality. Recently, it has been suggested that in some ejaculated sperm populations, apoptosis is caspase dependent. The aim of this study was to investigate the presence of activated caspases and examine possible correlations with apoptosis and sperm parameters in semen samples prepared for IVF. METHODS: To detect activated caspases, neat semen from infertile patients and sperm prepared by PureSperm gradient were stained with the fluorescein isothyocyanate-Val-Ala-Asp-fluoromethylketone (FITC-VAD-fmk) and analysed by flow cytometry. Cell death was determined by DNA fragmentation (TUNEL) and mitochondrial membrane potential. Sperm parameters were studied by conventional microscopy. RESULTS: FITC-VAD-fmk stained sperm cells in situ and the subcellular labeling pattern was compatible with the known localization of caspases. A significant correlation was found between the frequency of FITC-VAD-fmk stained cells and cell death markers. In both prepared sperm and neat semen a negative correlation was found between the percentage of FITC-VAD-fmk positive cells and standard parameters (concentration/motility). FITC-VAD-fmk positive cells negatively correlated with high fertilization rates after IVF. CONCLUSIONS: Labelling of sperm cells with the activated caspases-reacting fluorochrome provides a sensitive assay for detection of sperm apoptosis. This cytometric assay can be helpful to test sperm before IVF.

Key words: cell death/flow cytometry/IVF/spermatozoa


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