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Hum. Reprod. Advance Access published online on June 12, 2009
Human Reproduction, doi:10.1093/humrep/dep214
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© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis

L.K. Thomson1,4, S.D. Fleming2, R.J. Aitken3, G.N. De Iuliis3, J.-A. Zieschang1 and A.M. Clark1

1 Fertility First, PO Box 807, Hurstville, NSW 2220, Australia 2 Department of Obstetrics and Gynaecology, Westmead Hospital, University of Sydney, Westmead 2000, Australia 3 ARC Centre of Excellence in Biotechnology and Development and Discipline of Biological Sciences, University of Newcastle, Callaghan, Australia 2308

4 Correspondence address. E-mail: lthomson{at}fertilityfirst.com.au

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 µM 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.

Key words: Sperm DNA fragmentation/cryopreservation/8OHdG/genistein/caspase inhibitor

Submitted on February 12, 2009; resubmitted on April 15, 2009; accepted on May 20, 2009.


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