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Hum. Reprod. Advance Access originally published online on November 1, 2006
Human Reproduction 2007 22(2):485-494; doi:10.1093/humrep/del415
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A new multiparameter flow cytometric method for human semen analysis

S. Perticarari1, G. Ricci2,3, M. Granzotto1, R. Boscolo2, C. Pozzobon2, S. Guarnieri2, A. Sartore2 and G. Presani1

1 Clinical Analysis Unit, Department of Laboratory Medicine, Institute of Child Health IRCCS Burlo Garofolo and 2 Assisted Reproduction Unit, Department of Obstetrics and Gynaecology, Institute of Child Health IRCCS Burlo Garofolo and University of Trieste, Trieste, Italy

3 To whom correspondence should be addressed at: Assisted Reproduction Unit, Department of Obstetrics and Gynaecology, Institute of Child Health IRCCS Burlo Garofolo and University of Trieste, Via dell’Istria, 65/1, 34137 Trieste, Italy. E-mail: ricci{at}burlo.trieste.it

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.

Key words: apoptosis/flow cytometry/fluorospheres/sperm/Syto 16


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S. Perticarari, G. Ricci, R. Boscolo, M. De Santis, G. Pagnini, M. Martinelli, and G. Presani
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