Журнал медицинских методов диагностики

Журнал медицинских методов диагностики
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ISSN: 2168-9784


Revalidation of the Sperm Chromatin Dispersion Test and the Comet Assay Using Intercomparative Studies Between Purified Human Sperm without and with End-Stage DNA Fragmentation

Satoru Kaneko*, Kiyoshi Takamatsu

Objectives: To perform intercomparative studies to revalidate the principles and quantitative performance of the Sperm Chromatin Dispersion Test (SCD) and Comet Assay (CA).

Methods: Human sperm without and with granular fragments in end-stage fragmentation were purified from normozoospermic semen and used as naturally occurring negative and positive controls (NC and PC, respectively). Both SCD and CA extracted nucleoproteins with 2.0 mol/L NaCl, 1.0 mmol/L DTT. SCD determined that DNA damage as inversely proportional to the area of the violet halo. The CA estimated the level of DNA damage electrophoretically from the number of granular fragments; the so-called comet tail, their electrophoretic features were compared with the Single-Cell Pulsed-Field Gel Electrophoresis (SCPFGE).

Results: The violet halo in SCD was determined as consisting of Crystal Violet (CV)-stainable nucleoproteins that adhered to the radiated DNA fibers. SCD could not distinguish between NC and PC. The residual nucleoproteins blocked the migration of DNA in the natural CA; in contrast, SCPFGE with in-gel tryptic digestion discharged the fibrous and granular fragments beyond the elongated DNA fibers. The alkaline CA was run DNA in 0.3 mol/L NaOH. Although DNA was shredded to granular fragments, the residual nucleoproteins retained their binding capacity to DNA and still fixed the newly generated fragments.

Conclusion: DNA fragmentation analyses require measuring an early stage of fragmentation in separated motile sperm, a lack of proteolysis diminishes the quantitative performances of neutral and alkaline CA. Overall, the results suggested that SCD and CA were insufficiently sensitive as tools to harvest data for clinical statistics.