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

AbstractBackgroundCryopreserved human sperm are used in assisted reproductive technology. However, the effect of cryopreservation on spermDNAintegrity is unclear.ObjectivesThe objectives of this study were to: (i) determine the impact of semen cryopreservation on human spermDNAintegrity and chromatin structure; (ii) test if parameters obtained fromTUNELandSCSA® correlate; and (iii) verify correlation between sperm motility, morphology and viability withTUNELandSCSA® parameters.Materials and methodsMen 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,TUNELandSCSA® assays.ResultsProgressive motility and viability decreased after freeze‐thawing.TUNELscores increased significantly in all samples after freezing‐thawing while no significant change in theDNAfragmentation index (DFI) fromSCSA® was observed. No change in the percentage highDNAstainability (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. TheDFIandTUNELscores correlated significantly with each other and inversely with sperm motility, viability and morphology.Discussion and conclusionCryopreservation seems to be deleterious for the integrity of human spermDNAand compaction. However, the spermDFIwas 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.