Hum. Reprod. Advance Access published online on March 25, 2004
Human Reproduction, doi:10.1093/humrep/deh222
© 2004 by European Society of Human Reproduction and Embryology
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1 INSERM U459, Faculté de Médecine, 1 Place Verdun, F-59045 Lille Cedex, France; Laboratoire de Biologie de la Reproduction, Hôpital Jeanne de Flandres et Laboratoire d’Histologie, Faculté de Médecine, F-59037 Lille Cedex, France
* To whom correspondence should be addressed. 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:
Key words: cell death/flow cytometry/IVF/spermatozoa
Accepted November 22, 2003
Article
Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters
2 INSERM U459, Faculté de Médecine, 1 Place Verdun, F-59045 Lille Cedex, France
3 Laboratoire de Biologie de la Reproduction, Hôpital Jeanne de Flandres et Laboratoire d’Histologie, Faculté de Médecine, F-59037 Lille Cedex, France
4 INSERM U459, Faculté de Médecine, 1 Place Verdun, F-59045 Lille Cedex, France; INSERM U 459, Faculté de Médecine, 1 Place Verdun, F-59045 Lille Cedex, France
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