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 (R) correlate; and (iii) verify correlation between sperm motility, morphology and viability with TUNEL and SCSA (R) 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 degrees C; (ii) diluted in Sperm Maintenance Medium, cooled for 30 min at 4 degrees C and frozen at -80 degrees 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 (R) 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 (R) 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.