Measurement of DNA fragmentation in human sperm and their exclusion by means of in vitro processing

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The assurance of the integrity in DNA structure is minimum premise for the clinical application of ICSI. The establishment of the quality certification system is certainly the most urgent task. We newly developed single cell pulse field gel electrophoresis(SCPFGE) to observe double strand breaks in DNA. The specimen (2 X10^5 sperm/ml) was suspended into 2.0% melted agarose, and thin film of agarose was made on a glass slide with hyddrophylic surface. Our preliminary experiments by SCPFGE have revealed that the mode of DNA fragmentation in human sperm nuclei have been extremely variable. They were classified as follows ; 1.intact (continuous DNA fibers were elongated form the origin), 2.light fragmentation (a few long fragments were found beyond the continuous fibers), 3.moderate fragmentation (two or three mass of short fragments were extended from the origin) and 4.severe fragmentation (origin was almost disappeared and the mass of short fragment was found).Human sperm with DNA fragmentation was excluded by means of differential velocity sedimentation in Percoll density gradient, equilibrium sedimentation in Optidenz and swim up procedure. The sediment of Percoll density gradient was farther separated by equilibrium sedimentation, the sperm were distributed into two peaks. The profiles of SCPFGE showed that almost the sperm in lower peak showed DNA fragmentation, whereas those in the upper peak gave elongated continuous DNA fibers. The motile sperm was further separated by means of the swim up method.In conclusion, DNA denature in human sperm accompanied with the alteration of apparent density and the sperm with double strand breaks in DNA structure could be excluded by the in vitro processings.