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.
Perticarari
S
S
Clinical Analysis Unit, Department of Laboratory Medicine, Institute of Child Health IRCCS Burlo Garofolo, Italy.
Ricci
G
G
Granzotto
M
M
Boscolo
R
R
Pozzobon
C
C
Guarnieri
S
S
Sartore
A
A
Presani
G
G
D000328:Adult; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D003609:Dactinomycin / Q000031:analogs & derivatives; D004789:Enzyme Activation; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D017493:Leukocyte Common Antigens / Q000276:immunology; D007962:Leukocytes / Q000166:cytology; D008297:Male; D009336:Necrosis; D053978:Pentacyclic Triterpenes; D015203:Reproducibility of Results; D012661:Semen / Q000166:cytology* / Q000502:physiology*; D013094:Spermatozoa / Q000187:drug effects; D014315:Triterpenes / Q000494:pharmacology; D000094062:Betulinic Acid
D005456:Fluorescent Dyes; D053978:Pentacyclic Triterpenes; D014315:Triterpenes; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D017493:Leukocyte Common Antigens; D020169:Caspases; D000094062:Betulinic Acid