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. 2014 Jan-Mar;15(1):2–14.

Table 2.

Methods of evaluation of sperm DNA fragmentation and sperm chromatin integrity

Sperm DNA fragmentation
Method Advantages Disadvantages
SCD

  • Technically simple

  • Precise

  • Highly reproducible

  • Inexpensive

  • Not requiring special equipment

  • Test results correlate with SCSA

  • Time-consuming

  • Labor intensive (microscopic evaluation of at least 500 spermatozoa)

  • Training required to avoid technician subjectivity
SCSA

  • 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

  • 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
TUNEL

  • 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

  • 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
COMET

  • 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

  • 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
Alkaline method:

  • 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
Neutral method:

  • low sensitivity

  • no reference limits correlating test results and prognosis in fertility potential
DNA ladder

  • Detects apoptotic spermatozoa in relation to low molecular weight DNA molecules present

  • Low molecular weight DNA-bearing spermatozoa correlate with TUNEL positive spermatozoa

  • Radioactive stains are required to observe the characteristic ‘ladder’ forms
DNA-break detection FISH

  • DNA fragmentation assessed directly in spermatozoa using genomic probes

  • New method

  • Test results not adequately validated yet
Sperm chromatin integrity
Aniline/Toluidine blue staining

  • 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

  • Precision dependent on staining efficiency

  • Inter-laboratory variability not tested
Chromomycin A3

  • Negative correlation with fertilization rates in IVF

  • Technically demanding

  • Current application only in research protocols

  • Inter-laboratory variability not tested