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EVALUATION OF CHROMOSOME BREAKAGE AND DNA INTEGRITY IN SPERM: AN INVESTIGATION OF REMOTE SEMEN COLLECTION CONDITIONS
Citation:
Young, K. E., W. A. Robbins, L. Xun, D. Elashoff, S. A. Rothmann, AND S D. PERREAULT. EVALUATION OF CHROMOSOME BREAKAGE AND DNA INTEGRITY IN SPERM: AN INVESTIGATION OF REMOTE SEMEN COLLECTION CONDITIONS. JOURNAL OF ANDROLOGY 24(6):853-861, (2003).
Description:
Home collection of ejaculated semen would facilitate participation rates and geographic diversity in reproductive epidemiology studies. Our study addressed concerns that home collection and overnight mail return might induce chromosome/DNA damage. We collected semen from 10 healthy men. Part of each sample was snap-frozen and the rest held at 22 ? 1?C for 24 hours (simulating ambient temperature during overnight return) then snap-frozen. Chromosome breakage was measured using tandem-label sperm-FISH. DNA fragmentation was measured using TUNEL and neutral comet assay. Tandem-label sperm-FISH and TUNEL detected no statistically significant difference between sperm fresh frozen (FF) and sperm frozen after 24 hours (F24). Mean frequency of chromosome breakage per 10,000 cells scored in FF and F24 was 10.5 ? 1.3 and 11.2 ? 1.1, respectively (pStudent?s t-test = 0.69). Mean percentage of TUNEL positive cells per 2,000 cells scored in FF and F24 was 136 ? 29 and 213 ? 28, respectively (pStudent?s t-test = 0.07). The neutral comet assay detected a statistically significant difference in DNA strand breakage between the two groups (percentage of DNA in the tail pStudent?s t-test = 0.037; tail moment pStudent?s t-test = 0.006; and tail length pStudent?s t-test = 0.033). Mean frequency of damage (denoted by tail length) per 100 cells scored in FF and F24 was 175.0 ? 15.5 and 152.2 ? 17.6, respectively. Tandem-label sperm-FISH, TUNEL, and neutral comet assay are useful analytical techniques for laboratory-based studies of human sperm genomic integrity; however, for field studies incorporating 24 hours non-refrigerated return of semen, only measurement of chromosome breakage using sperm-FISH was clearly unaffected. TUNEL and neutral comet assay need further study before incorporation into home collection.
Key words: snap-freezing, sperm-FISH, TUNEL, neutral comet assay