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DNA integrity tests
Fernandez et al. (2003) J Androl. 24(1):59‐66.
Tests
Mechanism
Sperm chromatin structure assay (SCSA)
Acridine orange staining in acid condition (acid induced denaturation preferentially at DNA breaks) • green fluorescence - intact dsDNA
• red fluorescence – fragmentation (DFI>30%)
TUNEL
Enzymatic incorporation of marked nucleotides at free DNA ends (dsDNA +ssDNA)
COMET
Single-cell electrophoresis after lysis of nuclear proteins binding DNA (1) dsDNA (neutral) (2) dsDNA+ssDNA(alkaline)
Sperm chromatin dispersion test
Fragmented DNA fail to produce a halo of dispersed DNA loops after acid denaturation and removal of nuclear protein
Chromatin packaging
Chromomycin A3 competes with protamines for DNA. CMA3 fluorescence (bright yellow-green fluorescence) implies poor protamination.
Overview
• Sperm chromatin structure
• Causes of sperm DNA damage
• Tests for DNA fragmentation
Effects on IVF outcomes
• Effects on reproductive o
• What can be done?
• Conclusions
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