A new multiparameter flow cytometric method for human semen analysis

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.