Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis.
BACKGROUND
Whereas studies have revealed that the cryopreservation of human semen increases sperm DNA fragmentation, the mechanisms involved in this type of cryo-injury are largely unknown. Elucidation of these mechanisms may provide insight into preventing such injury.


METHODS
We obtained 60 semen samples from 60 men and conducted experiments to determine the cause of cryopreservation-induced DNA fragmentation using 8-oxo-7,8-dihydro-2'deoxyguanosine (8OHdG) as a biomarker of oxidative stress, percentage caspase positive cells as an indicator of apoptosis, the potential antioxidant genistein and the caspase inhibitor Z-VAD(OMe)-FMK.


RESULTS
Cryopreservation led to a significant increase in percentage DNA fragmentation, percentage 8OHdG and percentage caspase positive cells (P < 0.001). Percentage DNA fragmentation was positively correlated with percentage 8OHdG before (r = 0.756, P < 0.001) and after cryopreservation (r = 0.528, P = 0.017). The addition of 50 and 100 microM genistein to the cryoprotectant had a significant protective effect on sperm DNA (P < 0.001) although the caspase inhibitor demonstrated no difference to the control.


CONCLUSIONS
Human sperm DNA fragmentation is associated with an increase in oxidative stress during cryopreservation, rather than the activation of caspases and apoptosis. The estrogenic compound genistein may be useful in reducing this effect but larger trials are needed to confirm this.
Thomson
L K
LK
Fertility First, PO Box 807, Hurstville, NSW 2220, Australia. lthomson@fertilityfirst.com.au
Fleming
S D
SD
Aitken
R J
RJ
De Iuliis
G N
GN
Zieschang
J-A
JA
Clark
A M
AM
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000328:Adult; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D015925:Cryopreservation; D004249:DNA Damage / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D003849:Deoxyguanosine / Q000031:analogs & derivatives; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D008297:Male; D008875:Middle Aged; D018384:Oxidative Stress; D012662:Semen Preservation / Q000009:adverse effects*; D013094:Spermatozoa / Q000502:physiology*
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; C476093:z-Val-Ala-Asp(Ome)-fluoromethylketone; D000080242:8-Hydroxy-2'-Deoxyguanosine; D019833:Genistein; D020169:Caspases; D003849:Deoxyguanosine