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.

Keywords: human spermatozoa, apoptosis assays, caspase-3, mitochondrial membrane potential integrity, CD46, semen analysis

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