SCD |
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SCSA
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Rapid evaluation of a large number of spermatozoa (∼5,000)
Rapid assessment of many samples
Flexibility in routine laboratory practice (also used in frozen samples)
Highly reproducible
Correlations with the results of other methods evaluating different types of DNA damage (TUNEL, COMET)
Application in environmental studies
Sensitive
Statistically robust
DFI: unique reference limits associated with fertility prognosis
HDS: provides information on chromatin condensation, associated with sperm cell immaturity
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High cost equipment is required
Precision is based on the evaluation of a large number of spermatozoa
Reference sample is required for flow cytometer calibration
The evaluation of partially stained spermatozoa reduces the objectivity
Does not reflect a distinct physiological process
Indirect evaluation of the actual fragmentation of the DNA
Result interpretation can be difficult
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TUNEL
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Assessment of a small number of spermatozoa (∼200)
The use of bright field microscopy may reduce the cost
Effective even in low concentration samples (eg. testicular biopsy)
Reference sample is not required
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Time consuming (∼3 hours of laboratory time per assay)
Not clear correlation between suggested reference limits and prognosis in ART
Immature spermatozoa are not evaluated (eg. high HDS cells of SCSA)
High intra-assay and inter-laboratory variability
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COMET
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Quantifies the actual DNA damage of each examined spermatozoon (strand breaks)
More sensitive in alkaline conditions (identifies both single and double DNA strand breaks)
Correlates well with TUNEL and SCSA
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software required
Experience in data collection and interpretation required
Special equipment required (electrophoresis unit connected to fluorescence microscope)
Difficult to standardize (high intra- assay and inter-laboratory protocol variability)
Time consuming
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Alkaline method: |
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Possible overestimation of DNA breaks due to induced conversion of alkali-labile sites into breaks
Does not provide clear distinction between fertile normospermic and infertile normospermic/asthenozoospermic men
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Neutral method: |
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DNA ladder
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DNA-break detection FISH
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Sperm chromatin integrity |
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Aniline/Toluidine blue staining
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DNA-protein interaction better evaluated in comparison to SCSA
Assessment of a small number of spermatozoa
Inexpensive
Applied with bright field microscopy
Test results correlate with TUNEL, SCSA, COMET
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Chromomycin A3
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