Revalidation of DNA Fragmentation Analyses for Human Sperm-Measurement Principles, Comparative Standards, Calibration Curve, Required Sensitivity, and Eligibility Criteria for Test Sperm

(1) Background: Double-strand breaks (DSBs) in a single nucleus are usually measured using the sperm chromatin structure assay (SCSA), sperm chromatin dispersion (SCD) test, and comet assay (CA). Mono-dimensional single-cell pulsed-field gel electrophoresis (1D-SCPFGE) and angle-modulated two- dimensional single-cell pulsed-field gel electrophoresis (2D-SCPFGE) were developed to observe DNA fragmentation in separated motile sperm. (2) Methods: Comparative standards, calibration curves, required sensitivity levels, and eligibility criteria for test sperm were set up to validate the measurement principles of these tests. (3) Results: The conventional methods overlooked the interference of nucleoproteins in their measurements. In-gel proteolysis improves the measurement accuracies of 1D- and 2D-SCPFGE. Naked DNA is suitable for comparative standards and test specimens. Moreover, several dysfunctions that might induce DNA damage are observed in the separated motile sperm. Overall, the discussion highlights the need to revisit the conventional univariable analyses based on the SCSA, SCD test, and CA. (4) Conclusions: Human infertility is a complex syndrome, and the aim of quality control in intracytoplasmic sperm injection is to identify the underlying dysfunctions remaining in the separated motile sperm that render them ineligible for injection. Multivariable analyses with special consideration to confounding factors are necessary in future cohort studies.