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Objective: To evaluate sperm nuclear DNA fragmentation and chromatin structure after 18 hours’incubation at room temperature. Methods: Twenty-eight male partners who participating IVF treatment were prospectively included in this study. Ejaculated sperm count and motility were assessed. The sperm was then immediately processed by the conventional swim-up method. After utilization of some of the sample for routine clinical use, the remainder of each of the samples was divided into two aliquots. One aliquot was immediately assessed for sperm nuclear DNA fragmentation (TUNEL assay) and chromatin structure (toluidine blue [TB] staining). The other aliquot was incubated at room temperature for 18 hours and then assessed by two methods. Only dark-TB sperms were considered as having abnormal chromatin structure. Data before and after extended incubation were compared using a paired Student’s t-test. Results: Before and after extended culture, nuclear DNA fragmentation assessed by TUNEL was 4.9±4.7% and 7.0±6.4%, respectively (p=0.008). The proportion of abnormal chromatin structure (dark-TB sperm) was 8.2±5.6% and 10.3±6.5% (p<0.001), before and after incubation, respectively. Conclusion: After 18 hours’ incubation at room temperature, sperm nuclear DNA and chromatin structure were significantly affected. The IVF practitioner should bear this information in mind when performing delayed insemination, especially for in vitro maturation cycles.