Human Reproduction, Vol. 17, No. 10, 2665-2672,
October 2002
© 2002 European Society of Human Reproduction and Embryology
Apoptosis in human sperm: its correlation with semen quality and the presence of leukocytes
1 UCO di Ginecologia e Ostetricia, Dipartimento di Scienze della Riproduzione e dello Sviluppo, Università di Trieste, Istituto per l’Infanzia ‘Burlo Garofolo’, I.R.C.C.S., Trieste and 2 Laboratorio Analisi, Istituto per l’Infanzia ‘Burlo Garofolo’, I.R.C.C.S., Trieste, Italy
BACKGROUND: Apoptosis plays an important role in regulating spermatogenesis. However, the biological significance of apoptosis in ejaculated sperm is not yet clear. This study set out to investigate how apoptosis correlates with semen quality and the presence of seminal leukocytes. METHODS: Fifty-seven semen samples from the male partners of infertile couples were classified as normal or abnormal according to World Health Organization guidelines. Flow cytometry was used to evaluate sperm populations and seminal leukocytes. Preliminary flow cytometry analysis using 6-carboxyfluoresceindiacetate (6-CFDA), which identifies live cells, and propidium iodide (PI), which stains only dead cells, was performed in order to pinpoint the sperm region accurately. Having thus gated the sperm population, bivariate Annexin V/PI analysis was then carried out in order to measure the percentage of apoptotic and necrotic sperm and the apoptotic index (the ratio between apoptotic:live sperm). Leukocytes were counted by the standard peroxidase test and by flow cytometry using monoclonal antibody (mAb) anti-CD45 or anti-CD53. RESULTS: No significant differences in the apoptotic index and the percentage of live and apoptotic sperm were detected between the subjects with normal and abnormal semen. A significant inverse correlation between sperm concentration and the apoptotic index was observed only in the normal sperm group. There was no correlation between the concentration of leukocytes, detected either by peroxidase or by mAb anti-CD45 or anti-CD53, either with the percentage of apoptotic sperm or with the apoptotic index. In contrast, the ratio between CD45 positive leukocytes and sperm showed a significant correlation with the apoptotic index. A weaker correlation was found when leukocytes were counted by peroxidase, while no correlation was observed using mAb anti-CD53. CONCLUSIONS: Sperm apoptosis did not seem to be correlated with semen quality. In the absence of genito-urinary infection, one of the main functions of seminal leukocytes is probably to provide for the removal of apoptotic sperm.
Key words: apoptosis/flow cytometry/membrane phosphatidylserine/seminal leukocytes/sperm
3 To whom correspondence should be addressed at: UCO di Ginecologia e Ostetricia, Dipartimento di Scienze della Riproduzione e dello Sviluppo, Università di Trieste, Istituto per l’Infanzia ‘Burlo Garofolo’, I.R.C.C.S., Via dell’Istria 65/1, 34137 Trieste, Italy. E-mail: ricci{at}burlo.trieste.it
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