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A new multiparameter flow cytometric method for human semen analysis.

S Perticarari·G Ricci·M Granzotto·R Boscolo·C Pozzobon·S Guarnieri·A Sartore·G Presani
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Abstract

BACKGROUND: The objectives of this study were (i) to evaluate whether the combined use of Syto 16 and 7-amino-actinomycin-D (7-AAD) allows the detection of sperm apoptosis and (ii) to describe a new multiparameter flow cytometric method to assess simultaneously sperm concentration (SC), viability and apoptosis as well as leukocyte concentration. METHODS: Semen samples from 68 patients were evaluated according to World Health Organization (WHO) criteria (normal, n=26; abnormal, n=42). The detection of activated caspases before and after betulinic acid (BA) incubation was carried out in 13 semen samples by flow cytometry using fluorescein-labelled inhibitors of caspases (FLICA). A multiparameter flow cytometric analysis was performed in 55 semen samples. Fluorescent microspheres were used to assess SC. Sperm apoptosis was detected by staining sperm with Syto 16 and 7-AAD. Leukocytes were counted using monoclonal anti-CD45. RESULTS: A significant correlation between the percentage of the spermatozoa with low Syto 16 fluorescence and the percentage of spermatozoa containing activated caspases was found (r=0.68, P=0.0106; n=13). After incubation with BA, an increase of the percentage of apoptotic cells was observed in all samples, using both the Syto 16/7-AAD and the caspase activation methods. There was a good correlation between flow cytometry and optical microscopy for sperm (r=0.98, P < 0.0001) and leukocyte counting (r=0.64, P <0.0001). The percentage of apoptotic sperm was inversely correlated with both SC (r=-0.303, P=0.0246) and morphology (r=-0.384, P=0.0050) but not with motility. CONCLUSIONS: The combination of Syto 16/7-AAD provides a sensitive assay to detect sperm apoptosis. The multiparameter flow cytometric method described offers the possibility of a simultaneous, simple, rapid and accurate assessment of several semen parameters.

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AdultApoptosisCaspasesDactinomycinEnzyme ActivationFlow CytometryFluorescent DyesHumansLeukocyte Common AntigensLeukocytesMaleNecrosisPentacyclic TriterpenesReproducibility of ResultsSemenSpermatozoaTriterpenesBetulinic AcidJournal ArticleResearch Support, Non-U.S. Gov't

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S Perticarari, G Ricci, M Granzotto, R Boscolo, C Pozzobon, S Guarnieri, A Sartore, G Presani. 2006-11-01. A new multiparameter flow cytometric method for human semen analysis.. https://doi.org/10.1093/humrep%2Fdel415

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