Sperm DNA damage and cryopreservation: an oxidation mechanism

Abstract

ObjectiveTo compare levels of apoptosis, DNA damage and oxidative stress in semen samples before-after cryopreservation in order to identify the principal pathway of sperm damage. Compare the effect of male age over DNA damage and oxidative stress.DesignProspective and comparative studyMaterials and MethodsSixty semen samples from men who performed in vitro fertilization and 20 normozoospermic men (control group-sperm bank) were included. Semen analysis was performed as established by WHO-2010. An aliquot (500uL) of semen was cryopreserved with TYB, accordingly to manufacturer. Motile sperm (fresh and post-thawed) were selected by swim-up (HTF). Before-after cryopreservation and post swim-up procedure, it was assessed the following variables: lipid peroxidation-BODIPY, phosphatidylserine externalization by Annexin-V Kit, DNA fragmentation by TUNEL, DNA oxidative damage by 8OHDG (western blot) and vitality by Eosin. Patients were grouped according to their age: less than 40, 40-45, older than 45 y/o.ResultsLevels of Annexin-V, TUNEL, BODIPY and 8OH-DG increased after cryopreservation in patients and donors samples. However, it was observed that BODIPY and 8OH-DG prevail over Annexin-V. All variables were lower in donors group in compare to patient samples. After swim-up (fresh and post-thawed), variable values decreased, but swim-up samples after thawed had higher values in compare to swim-up (fresh). Vitality decreased after cryopreservation, contrary the control group samples presented more tolerance to cryoinjury. It was determined that these parameters significantly increase in men >45 y/o. All the comparisons performed on these analysis were statistically significant (p<0.05).ConclusionsTabled 1TUNEL PATIENTSTUNEL DONORSANNEXIN V PATIENTSANNEXIN V DONORSBODIPY PATIENTSBODIPY DONORS8OH-dG PATIENTSFRESH-BASAL20.1±12.814.7±4.28.9±3.26.7±2.812.4±4.89.6±3.165.3±13.6POST THAWED36.6±13.625.7±5.118.3±5.111.5±2.727.1±8.517.5±3.3112.6 ± 22.1 Open table in a new tab ObjectiveTo compare levels of apoptosis, DNA damage and oxidative stress in semen samples before-after cryopreservation in order to identify the principal pathway of sperm damage. Compare the effect of male age over DNA damage and oxidative stress. To compare levels of apoptosis, DNA damage and oxidative stress in semen samples before-after cryopreservation in order to identify the principal pathway of sperm damage. Compare the effect of male age over DNA damage and oxidative stress. DesignProspective and comparative study Prospective and comparative study Materials and MethodsSixty semen samples from men who performed in vitro fertilization and 20 normozoospermic men (control group-sperm bank) were included. Semen analysis was performed as established by WHO-2010. An aliquot (500uL) of semen was cryopreserved with TYB, accordingly to manufacturer. Motile sperm (fresh and post-thawed) were selected by swim-up (HTF). Before-after cryopreservation and post swim-up procedure, it was assessed the following variables: lipid peroxidation-BODIPY, phosphatidylserine externalization by Annexin-V Kit, DNA fragmentation by TUNEL, DNA oxidative damage by 8OHDG (western blot) and vitality by Eosin. Patients were grouped according to their age: less than 40, 40-45, older than 45 y/o. Sixty semen samples from men who performed in vitro fertilization and 20 normozoospermic men (control group-sperm bank) were included. Semen analysis was performed as established by WHO-2010. An aliquot (500uL) of semen was cryopreserved with TYB, accordingly to manufacturer. Motile sperm (fresh and post-thawed) were selected by swim-up (HTF). Before-after cryopreservation and post swim-up procedure, it was assessed the following variables: lipid peroxidation-BODIPY, phosphatidylserine externalization by Annexin-V Kit, DNA fragmentation by TUNEL, DNA oxidative damage by 8OHDG (western blot) and vitality by Eosin. Patients were grouped according to their age: less than 40, 40-45, older than 45 y/o. ResultsLevels of Annexin-V, TUNEL, BODIPY and 8OH-DG increased after cryopreservation in patients and donors samples. However, it was observed that BODIPY and 8OH-DG prevail over Annexin-V. All variables were lower in donors group in compare to patient samples. After swim-up (fresh and post-thawed), variable values decreased, but swim-up samples after thawed had higher values in compare to swim-up (fresh). Vitality decreased after cryopreservation, contrary the control group samples presented more tolerance to cryoinjury. It was determined that these parameters significantly increase in men >45 y/o. All the comparisons performed on these analysis were statistically significant (p<0.05). Levels of Annexin-V, TUNEL, BODIPY and 8OH-DG increased after cryopreservation in patients and donors samples. However, it was observed that BODIPY and 8OH-DG prevail over Annexin-V. All variables were lower in donors group in compare to patient samples. After swim-up (fresh and post-thawed), variable values decreased, but swim-up samples after thawed had higher values in compare to swim-up (fresh). Vitality decreased after cryopreservation, contrary the control group samples presented more tolerance to cryoinjury. It was determined that these parameters significantly increase in men >45 y/o. All the comparisons performed on these analysis were statistically significant (p<0.05). ConclusionsTabled 1TUNEL PATIENTSTUNEL DONORSANNEXIN V PATIENTSANNEXIN V DONORSBODIPY PATIENTSBODIPY DONORS8OH-dG PATIENTSFRESH-BASAL20.1±12.814.7±4.28.9±3.26.7±2.812.4±4.89.6±3.165.3±13.6POST THAWED36.6±13.625.7±5.118.3±5.111.5±2.727.1±8.517.5±3.3112.6 ± 22.1 Open table in a new tab