Role of sperm DNA fragmentation in male factor infertility: A systematic review

Peer review under responsibility of Arab Association of Urology.

Figures & data

Fig. 1 Flow diagram illustrating study selection.

Fig. 2 Causes of SDF. Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography ©2017. All Rights Reserved.

Table 1 SDF testing methods.

Test	Principle	Method	Result	Advantage	Disadvantage
Acridine orange (AO) test	Metachromatic shift in fluorescence of AO when bound to DNA breaks	Acid denaturation, followed by staining by AO Uses fluorescent microscopy	Normal DNA fluoresces green Denatured DNA fluoresces orange-red	Rapid and simple Inexpensive	Inter-laboratory variations Lack of reproducibility
Aniline blue (AB) staining	Greater affinity for lysine-rich histones in immature sperm	Staining by AB Uses optical microscopy	Nuclei of immature sperm stain blue Protamine-rich nuclei of mature sperm remain unstained	Rapid and simple Inexpensive	Inter-laboratory variations Lack of reproducibility and precision dependent on staining efficiency
Toluidine blue (TB) staining	High affinity for sperm DNA phosphate residues associated with damaged chromatin	Staining by TB Uses optical microscopy	Producing an intense violet-blue colouration after incorporation into damaged chromatin	Rapid and simple Inexpensive	Inter-observer variability Precision dependent on staining efficiency
Chromomycin A3 (CMA3) staining	Compete with protamine for the same binding site in DNA	Staining by CMA3	Highly positive test reflects a low DNA protamination state associated with poorly packaged sperm chromatin	Strong correlation has been demonstrated with other SDF assays	Inter-observer variability Inter-laboratory variability not tested Technically demanding
TUNEL	Quantifies the enzymatic incorporation of dUTP into DNA breaks as percentage of fluorescent sperm	Labelled nucleotides are added to site of DNA fragmentation Fluorescence is measured by flow cytometry or fluorescence microscopy	Sperm with fragmented DNA showed fluorescence Result presented as percentage of fluorescent sperm	Direct assay Can be performed in fresh or frozen samples Can performed on few sperm Detects both single- and double-strand DNA breaks Commercial assay available Reference sample is not required	Requires standardization among laboratories Time-consuming Immature spermatozoa are not evaluated Variable clinical thresholds reported in the literature
SCSA	Measures the susceptibility of sperm DNA to denaturation	Acid denaturation, followed by staining by AO Measurement by flow cytometry Uses fluorescent microscopy	Normal DNA fluoresces green Denatured DNA fluoresces orange-red Result presented as DNA fragmentation index (%DFI) and high DNA stain ability (%HDS)	Standardised protocol available Rapid evaluation of large number of spermatozoa Correlations with results of other SDF assays Established clinical thresholds Can be performed on fresh or frozen samples	Indirect assay involving acid denaturation Proprietary protocol with no commercial assay Requires expensive instrument and highly skilled technicians
SCD/Halo test	Assess dispersion of DNA fragments after denaturation	Agarose-embedded sperm are subjected to a denaturing solution to remove nuclear proteins Uses fluorescent microscopy to observe chromatin dispersion after staining	Sperm with fragmented DNA do not produce halo Characteristic halo of dispersed DNA loops are observed in sperm with non-fragmented DNA Result presented as percentage of sperm with non-dispersed chromatin	Relatively simple test with commercial kit available	Indirect assay involving acid denaturation Inter-observer variability Time-consuming and labour intensive if using microscopic evaluation
SCGE/Comet assay	Electrophoretic assessment of DNA fragments of lysed DNA	Gel electrophoresis performed in alkaline or neutral conditions	Size of comet tail represents the amount of DNA fragments that stream out of the sperm head Result presented as mean amount of DNA damage per spermatozoon	Direct assay Can be performed on few sperm Detect multiple types of DNA damage of individual spermatozoon Result correlates well with other SDF assays	Requires fresh sample Inter-observer variability Time consuming Requires experienced observer

AB, aniline blue; AO, acridine orange; CMA3, chromomycin A3; TB, toluidine blue; SCD, sperm chromatin dispersion; SCGE, single cell gel electrophoresis.

Table 2 Summary of the effect on SDF using different treatment strategies.

Method	SDF relative reduction,%	SDF assay	Reference
Short abstinence	25	SCD	Gosálvez et al. [48]
	22	TUNEL	Agarwal et al. [49]
Oral antioxidant therapy	66	TUNEL	Greco et al. [50]
	18	TUNEL	Tunc et al. [51]
	19	SCSA	Ménézo et al. [52]
	58	TUNEL	Martínez-Soto et al. [53]
	29	SCD	Abad et al. [54]
Varicocelectomy	14	SCSA	Smit et al. [58]
	27	SCD	Zini et al. [59]
MACS	27	TUNEL	Lee et al. [60]
	0	TUNEL	Nadalini et al. [61]
IMSI	78	TUNEL	Hammoud et al. [62]
	0	SCD	Maettner et al. [63]
PICSI	68	SCD	Parmegiani et al. [64]
	0	SCSA	Rashiki Ghaleno et al. [65]
Density gradient centrifugation	22–44 ^*	SCD	Gosálvez et al. [48]
	57	SCD	Xue et al. [68]
Swim-up	33	SCD	Parmegiani et al. [64]
	38	SCD	Xue et al. [68]
Testicular sperm	80	SCD	Esteves et al. [10]
	80	TUNEL	Greco et al. [70]
	67	TUNEL	Moskovtsev et al. [71]

* Combined with short ejaculatory abstinence.

Table 3 Clinical indications for SDF testing.

Clinical varicocele

SDF testing is recommended in patients with Grade 2/3 varicocele with normal conventional semen parameters

SDF testing is recommended in patients with Grade 1 varicocele with borderline/abnormal conventional semen parameter results

Unexplained infertility/IUI failure/RPL

SDF testing should be offered to infertile couples with RPL or prior to initiating IUI

Early IVF or ICSI may be an alternative to infertile couple with RPL or failed IUI

IVF and/or ICSI failure

SDF testing is indicated in patients with recurrent failure of assisted reproduction

The use of testicular sperm rather than ejaculated sperm may be beneficial in men with oligozoospermia, high SDF and recurrent IVF failure

Borderline abnormal (or normal) semen parameters with risk factor

SDF testing should be offered to patients who have a modifiable lifestyle risk factor of male infertility

RPL, recurrent pregnancy loss.

Table 4 Indications, rationale and evidence for SDF testing.

Indications and rationale	References
Varicocele
Significant association between SDF and varicocele has been detected	Zini and Dohle [56]; Esteves et al. [76]
Varicocelectomy improves percentage of SDF resulting in improved pregnancy rates	Zini and Dohle [56]; Smit et al. [58]; Ni et al. [77]
Little is known about the effect of low-grade varicocele on SDF. High SDF has been reported in clinical varicocele, particularly Grade 2 and 3; improvement of SDF in all grades of varicocele have been reported after varicocelectomy	Ni et al. [77]; Sadek et al. [78]; Krishna Reddy et al. [79]
Unexplained infertility
High SDF is found in men with normal semen analysis	Saleh et al. [80]; Oleszczuk et al. [81]
SDF is an independent predictor of male fertility status	Bungum et al. [27]; Oleszczuk et al. [81]
SDF levels can predict the likelihood of natural pregnancy	Buck Louis et al. [25]; Zini [26]
Recurrent IUI failure
High SDF is associated with lower IUI pregnancy rates	Bungum et al. [27]; Duran et al. [28]
Recurrent pregnancy loss
High SDF is associated with greater incidence of abortion	Khadem et al. [39]; Ford et al. [82]
IVF and ICSI failures
SDF modestly affect IVF pregnancy rates	Zini and Sigman [30]; Osman et al. [35]; Jin et al. [41]
SDF does not affect ICSI pregnancy rates	Zini and Sigman [30]; Zhao et al. [32]
High SDF is associated with greater incidence of abortion in both IVF and ICSI	Zini [26]; Zini and Sigman [30]; Simon et al. [33]; Robinson et al. [38]
Testicular sperm have lower SDF than ejaculated sperm	Esteves et al. [10]; Greco et al. [70]; Moskovtsev et al. [71]
High IVF/ICSI success rates with testicular sperm	Esteves et al. [10]; Greco et al. [70]; Mehta et al. [72]; Pabuccu et al. [73]; Arafa et al. [74]
Lifestyle risk factor
Smoking and environmental/occupational exposures have detrimental effects on SDF	Yang et al. [83]; Wijesekara et al. [84]