Human Reproduction, Vol. 17, No. 5, 1274-1280,
May 2002
© 2002 European Society of Human Reproduction and Embryology
Reliability of the comet assay in cryopreserved human sperm
1 Environmental Health Department, Occupational Health Program and 2 Biostatistics Department, Harvard School of Public Health, Boston, MA, 3 Department of Bioengineering, University of Washington, Seattle, WA, 4 Department of Biostatistical Science, Dana-Farber Cancer Institute, Boston, MA, and 5 Vincent Obstetrics and Gynecology Service Andrology Laboratory and IVF unit, Massachusetts General Hospital, Boston, MA, USA
BACKGROUND: Although the comet assay has potential value for measuring DNA damage in large epidemiological human sperm studies, it is impractical to perform the assay daily on fresh semen samples. Therefore, before its use in epidemiological studies, the reliability of the comet assay in measuring DNA damage in cryopreserved sperm should be compared with that in fresh human sperm. METHODS: Semen samples from 16 men were cryopreserved in liquid nitrogen (LN) using four methods: flash freezing with and without cryopreservative, and programmable freezing with and without cryopreservative. Neutral microgel electrophoresis was performed and comets were stained with YOYO-1. Comet length was measured using an eyepiece micrometer at x400 magnification. RESULTS: The highest correlation was between comet assay results obtained from fresh human semen compared with semen flash frozen without cryopreservative (R = 0.88). However, the method of cryopreservation, as compared with other sources of variability, accounted for only 6% of the variability. Inter-individual variability accounted for 20%, and individual sperm-to-sperm variability within an ejaculate accounted for 65%. CONCLUSIONS: Flash-freezing in LN without cryopreservative most closely reproduced the results obtained using fresh human semen samples, and thereby represents the most appropriate cryopreservation method for human semen in epidemiological studies utilizing the neutral comet assay.
Key words: comet assay/cryopreservation/human sperm/intra-class correlation/reliability
6 To whom correspondence should be addressed at: Environmental Health Department, Occupational Health Program, Building 1 Room 1405, 665 Huntington Avenue, Boston, MA 02115-9957, USA. E-mail: rhauser{at}hohp.harvard.edu
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