Measuring Two Early Apoptotic Alterations In Human Sperm And Their Relationships With Conventional Semen Parameters And The Sperm DNA Damage

Section I :Measuring two early apoptotic alterations in human Sperm and their relationships with conventional semen parameters and thesperm DNA damageObjective: The aim of the present study was to test if the two early apoptotic alterations in ejaculated sperm can be reliable new biological assay for sperm quality.Methods: Two early apoptotic changes in the semen of 56 men were assessed using Annexin V / PI staining for phosphatidylserine (PS) externalization and JC-1 staining for mitochondrial membrane potential (MMP). The results were compared with conventional semen parameters and DNA fragmentation identified by the TUNEL assay.Results: The different labelling patterns in the bivariate Annexin V /PI analysis identified four distinctive spermatozoa populations. The percentage of 'AN-/ PI-' spermatozoa positively correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL (+) spermatozoa. The level of 'AN-/ PI+' spermatozoa negatively correlated with conventional semen parameters and MMP, but positively correlated with TUNEL (+) spermatozoa. The level of early apoptotic 'AN+/ PI-'spermatozoa negatively correlated with MMP and sperm motility. The level of late apoptotic 'AN+/ PI+' spermatozoa negatively correlated with conventional semen parameters and MMP, and positively correlated with TUNEL (+) spermatozoa. MMP negatively correlated with TUNEL (+) spermatozoa.Conclusions: In conclusion, although early apoptotic 'AN+/ PI-'spermatozoa only negatively correlated with sperm motility, the differences in proportion of each subpopulation of spermatozoa (according to Annexin V / PI staining), especially the percentage of 'AN-/ PI-' spermatozoa and decreased MMP may be significant markers for diagnosing male infertility. They possibly bring additional information to predict the outcome of IVF. Further studies are needed to determine their relationships with the occurrence of pregnancy or fertilization in vitro. Section II :SELECTING NON-APOPTOTIC SPERMATOZA IN THE SEMEN OF INFERTILE PATIENCEObjective: The aim of our study was to investigate the presence of phosphatidylserine (PS) externalization, mitochondrial membrane potential (MMP) and DNA fragmentation in purified sperm populations obtained from ejaculates of infertile male patients in comparison with healthy donors, and to evaluate the impact of combination Magnetic cell sorting (MACS) with Density gradient centrifugation (DGC) on the presence of PS externalization, MMP and DNA fragmentation with the spermatozoa of infertile male patients.Methods: Semen samples from 15 infertile male patients were analyzed. Another semen samples from 10 healthy donors were analyzed for comparison. Semen samples of infertile male patients were subjected to separation on a DGC followed by MACS. PS extemalization was detected using Annexin-V/PI staining. MMP was detected using JC-1 staining .DNA fragmentation was detected with a terminal deoxynucleotidyl transferase mediated dUTP nick end label (TUNEL) assay.Results: Higher PS extemalization (20.33+7.88% vs. 8.00±4. 32%), higher DNA fragmentation (18.87 + 9. 43% vs. 3.00 + 1. 33%) and lower MMP (68.53±10.32% vs. 81.90 + 7. 95%) were found in the purified sperm populations of infertile male patients in comparison with healthy donors. Annexin V-negative sperm displayed superior quality in terms of high motility, low PS extemalization, high MMP, and small extent of DNA fragmentation.Conclusions: DGC followed by MACS can be used to prepare samples from infertile male patients characterized by poor sperm quality to select spermatozoa with less apoptosis and DNA damage, which is expected to enhance the efficiency of assisted reproduction.