• human spermatozoa;
  • apoptosis assays;
  • caspase-3;
  • mitochondrial membrane potential integrity;
  • CD46;
  • semen analysis


Aim: 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. Conclusion: 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.