Stability of fluorochrome based assays to measure subcellular sperm functions

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^TM 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.