Stability of fluorochrome based assays to measure subcellular sperm functions.
OBJECTIVE
To evaluate the long-term stability of the fluorescence signals of new fluorescence-based semen analysis assays for clinical application.


METHODS
Semen samples from 87 unselected infertile patients were used to perform the following assays: (i) detection of active caspase-3 (n=17); (ii) integrity of the mitochondrial membrane potential (MMP) (n=17); (iii) externalization of phosphatidylserine (EPS) (n=16); and (iv) detection of intact acrosomes via CD46 (n=37). After the assays, 4% paraformaldehyde was added to all aliquots. The fluorescence intensity of each sample was evaluated by flow cytometry on days 0, 3, 7, 10 and 14.


RESULTS
Differences of up to +/-5% positive spermatozoa from the value measured at day 0 were estimated as acceptable deviation. The Caspase-3 FLICA showed mean differences<5% at day 3, 7 and 10. At day 14 the mean difference was 7.6%. In contrast, the disrupted MMP and the EPS detection showed differences>5% at day 3. The CD46-FITC labeling displayed absolute differences<5% CD46-positive spermatozoa at days 3, 7, 10 and 14.


CONCLUSIONS
Although immediate analysis of the fluorescence signals is recommended, it is possible to evaluate caspase-3 activation up to 10 days and CD46 up to 14 days after staining of sperm. The FACS evaluation of MMP and EPS detection should be conducted on the same day.
Grunewald
Sonja
S
Department of Dermatology/Andrology Unit, European Academy of Andrology (EAA) Training Center, University of Leipzig, Ph.-Rosenthal-Str. 23-25, Leipzig 04103, Germany.
Rasch
Manja
M
Reinhardt
Martin
M
Baumann
Thomas
T
Paasch
Uwe
U
Glander
Hans-Juergen
HJ
D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D013094:Spermatozoa / Q000502:physiology*
D005456:Fluorescent Dyes