Relationship between nuclear DNA fragmentation, mitochondrial DNA damage and standard sperm parameters in spermatozoa of fertile and sub-fertile men before and after freeze-thawing procedure.

Abstract

The aim of this study was to assess the stability of nuclear and mitochondrial DNA (n-DNA and mt-DNA) of spermatozoa under freeze-thawing and to find out the correlation between them and their association with standard sperm parameters. Forty-three semen samples were collected from fertile (G.1; n=29) and sub-fertile (G.2; n=14). N-DNA fragmentation was determined by TUNEL assay and mt-DNA using caspase 3 staining. Each semen sample was frozen at -196°C by the programmed freezer. Freeze-thawing decrease vitality, total motility and membrane integrity from (43.02±22.74%; 31.63±18.15%; 51.5±24.82%) to (22.71±17.3%; 9.21±6.61%; 34.64±19.92% respectively [p<.001]). G.1 native spermatozoa stained positive with TUNEL and caspase 3 were (14.85±17.6% and 5.8±11.59%) and increased after freeze-thawing to 27.54±19.74% (p=.004) and 7.3±6.13% (p=.01) respectively. In G.2, TUNEL and caspase 3 were (19.84±17.52% and 7.53±8.56%) and increased to (29.48±16.97% [p=.03] and 10.21±11.73%). In conclusion, freeze-thawing process affects not only semen parameters but also n-DNA and mt-DNA. Therefore, n-DNA and mt-DNA could be used as sensitive parameters for assessment of the cryodamage of human spermatozoa.