Human Reproduction, Vol. 16, No. 8, 1703-1707,
August 2001
© 2001 European Society of Human Reproduction and Embryology
Low rates of DNA fragmentation in selected motile human spermatozoa assessed by the TUNEL assay
Fertility Laboratory, Department of Obstetrics and Gynaecology, University Medical Centre St. Radboud, Geert Grooteplein 8, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
BACKGROUND: In this study we present the physiological changes observed in ejaculated spermatozoa of normospermic men after exposure to hydrogen peroxide (H2O2) or 
irradiation. METHODS: Motility changes as well as membrane and DNA-damage were determined in spermatozoa after incubation with 25 µmol/l of H2O2 during increasing intervals of time (0–60 min and after 24 h) or after irradiation of cells using 
rays. Annexin V-binding in combination with propidium iodide was used for the assessment of membrane changes after each incubation time. TdT-mediated-dUTP nick-end labelling (TUNEL) was used to evaluate DNA damage. RESULTS: After 1 h incubation of the spermatozoa with H2O2, almost all cells were positive for Annexin-V, while no significantly increase in TUNEL positivity was observed. TUNEL results were significantly higher 24 h after incubation with H2O2 (10–16.3%, P = 0.03). In the control group (cumulus cells), an increase in the percentage of TUNEL positive cells was observed after 15 min of incubation with H2O2 and showed a five-fold increase after 24 h (from 8.1–72.1%, P < 0.001). TUNEL positive cells after irradiation increased with the doses and post-irradiation time (from 10.8–47.2%). Interestingly, when only motile spermatozoa from irradiated samples were analysed, only 0.5% were TUNEL positive. CONCLUSION: Motility may be a relevant physiological marker for DNA-intact sperm after exposure of spermatozoa to H2O2 and irradiation.
Key words: apoptosis/gamma radiation/ICSI/reactive oxygen species/TUNEL
1 To whom correspondence should be addressed. E-mail: l.ramos{at}obgyn.azn.nl
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