Effects of different cryopreservation methods on DNA integrity and sperm chromatin quality in men.

BACKGROUND: Cryopreserved human sperm are used in assisted reproductive technology. However, the effect of cryopreservation on sperm DNA integrity is unclear. OBJECTIVES: The objectives of this study were to: (i) determine the impact of semen cryopreservation on human sperm DNA integrity and chromatin structure; (ii) test if parameters obtained from TUNEL and SCSA® correlate; and (iii) verify correlation between sperm motility, morphology and viability with TUNEL and SCSA® parameters. MATERIALS AND METHODS: Men attending a fertility clinic were recruited and grouped according to their sperm parameters (n = 9/group): normozoospermia, oligoasthenoteratozoospermia and teratozoospermia. Each semen sample was processed as follow: (i) directly frozen at -80 °C; (ii) diluted in Sperm Maintenance Medium, cooled for 30 min at 4 °C and frozen at -80 °C; (iii) diluted in Sperm Maintenance Medium; or (iv) in SpermFreeze. Each mixture from method (iii) and (iv) was then suspended for 30 min in liquid nitrogen vapor and plunged into liquid nitrogen. After at least two months of storage, samples were thawed at room temperature and analyzed for motility and viability, TUNEL and SCSA® assays. RESULTS: Progressive motility and viability decreased after freeze-thawing. TUNEL scores increased significantly in all samples after freezing-thawing while no significant change in the DNA fragmentation index (DFI) from SCSA® was observed. No change in the percentage high DNA stainability (HDS) was observed in normozoospermic samples; however it was significantly increased in all the methods in oligoasthenoteratozoospermic and in the methods (ii)-(iv) in teratozoospermic samples. The DFI and TUNEL scores correlated significantly with each other and inversely with sperm motility, viability and morphology. DISCUSSION AND CONCLUSION: Cryopreservation seems to be deleterious for the integrity of human sperm DNA and compaction. However, the sperm DFI was not affected during cryopreservation under the various methods of storage tested. Clinicians and investigators should take this information into consideration when using cryopreserved sperm for assisted reproduction.

The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin-V magnetic activated cell sorting.

Sperm chromatin integrity may affect the outcomes of assisted reproductive technology (ART). Developing a clinically reliable strategy to enrich sperm samples with high chromatin quality spermatozoa prior to sperm banking or use in ART would thus be advantageous. The objectives of this study were to: (i) assess the sperm chromatin quality in men with different categories of semen parameters; and (ii) evaluate the extents of Annexin-V magnetic-activated cell sorting (MACS) technology coupled with differential density gradient centrifugation (DGC) in improving sperm chromatin quality. Three categories of men from couples attending a university-based fertility clinic were recruited based on their semen parameters: normozoospermic (n = 13), asthenoteratozoospermic (n = 17) and teratozoospermic (n = 12). For each patient, spermatozoa in semen samples were processed first by DGC to enrich the motility and further by MACS to remove spermatozoa showing apoptotic features. The yield and enrichment of sperm quality was evaluated at each step with conventional semen parameters in conjunction with a combination of five complementary assays, to assess sperm maturity, chromatin structure, compaction and DNA integrity (Hyaluronic Binding Assay, SCSA, chromomycine A3 staining and TUNEL and COMET assays). Our results demonstrated that, compared with normozoospermic samples, raw asthenoteratozoospermic and teratozoospermic samples had a higher proportion of spermatozoa containing DNA breaks, but only asthenoteratozoospermic exhibited altered chromatin structure and decreased binding to hyaluronic acid. Interestingly, the DGC appeared to select for more mature spermatozoa with high DNA compaction. More importantly, in all categories of semen samples, Annexin-V MACS allows enrichment of spermatozoa with good chromatin quality as measured by the TUNEL and SCSA. Because effective treatment modalities to improve sperm DNA damage are limited, our results suggest a potential clinical value of MACS as a mean to enhance sperm quality that may improve assisted reproductive outcomes.