Semiquantitative Analyses of Naturally and Chemically Induced DNA Fragmentation in Human Sperm by Means of Angle-Modulated Two-Dimensional Single-Cell Pulsed-Field Gel Electrophoresis – Technical Instructions to Exclude False-Positive and -Negative

Background To date, single-nuclear DNA damages in human sperm have been examined by means of the comet assay and the sperm chromatin structure assay. Revalidation using the comparative standards revealed their technical shortcomings. Methods We developed angle-modulated two dimensional single-cell pulsed-field gel electrophoresis (2D-SCPFGE) to detect the early symptoms of naturally occurring DNA fragmentation. Results The mass of naked DNA fibers was prepared by means of simultaneous in-gel swelling and proteolysis. They were fragile for reactive oxygen species (ROS), we developed the anti-ROS 2D-SCPFGE system to exclude false-positive. The first run discharged long-chain fibers were from the origin, and fibrous and granular segments were separated beyond the tips of the fibers. After 150° rotation, the second run unexpectedly extended long-chain fibers obliquely backwards from the origin, and long fibrous segments were drawn out from a bundle of long-chain fibers that extended during the first run. Two-directional current is essential to exclude false-negative. Ice crystals generate artifactual DNA fragmentation during cryopreservation. 2D-SCPFGE visualized the dose-dependent chemical and enzymatic DNA cleavage as the increment of fibrous segments and shortening their length. Conclusions Quantification of DNA fragmentation in human sperm is an unprecedented work; the present review illustrates the technical instructions to exclude false-positive and -negative.