Procedure Of Measuring Sperm Dna Damage Biology Essay

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The principles and procedure of measuring sperm DNA damage by flow cytometry SCSA are described in details elsewhere Evenson et al., 1999; Spano et al., 2000; Bungum et al., 2004. SCSA is based on staining of sperm nuclei with acridine orange, to evaluate the ratio of single and double stranded DNA (following acid exposure which causes denaturation of double stranded DNA in sperm with an impairment of their chromatin structure). Sperm chromatin damage was quantified by the FCM measurements of the metachromatic shift from green (native, double-stranded DNA) to red (denatured, single-stranded DNA) fluorescence and displayed as red versus green fluorescence intensity cytogram patterns. The extent of DNA denaturation is expressed as the DFI, which is the ratio of red to total fluorescence intensity, i.e. the level of denatured DNA over the total DNA. Additionally, we have considered the fraction of cells with high DNA stainable (HDS) cells, which are thought to represent immature spermatozoa with incomplete chromatin condensation. The intra-laboratory coefficient of variation was found to be 4.5% for DFI and 10% for HDS, respectively.

Bungum M, Humaidan P, Spano M, et al. The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod. 2004; 19, 1401-1408.

Several methods have been developed to evaluate sperm DNA or chromatin integrity (67,68). A number of methods only detect breakage in single or double strands of sperm DNA. In contrast other methods are based on the fact that defects in the sperm chromatin structure have been associated with increase DNA instability and sensitivity to denaturing stress. Therefore these methods provide denaturation condition and the subsequent assessment of the sperm chromatin ability to maintain its integrity. Most of these methods are based on the staining of pre-treated spermatozoa by fluorescent or non-fluorescent dye staining. Detection of stained or unstained spermatozoa may be done by light or fluorescent microscopy. Since observational evaluations by individuals have too high interassay and intra-assay coefficient variation (69,70), recently most of the evaluations particularly for fluorescent dyes, are based on automated instruments such as flowcytometry. Acidic aniline blue staining

The Acidic Aniline Blue (AAB) stain discriminates between lysine-rich histones and arginine/ cysteine-rich protamines. This stain specifically reacts with lysine residues in nuclear histones and reveals differences in the basic nuclear protein composition of the sperm. Histone-rich nuclei of immature sperm are rich in lysine and will consequently take up the blue stain. On the other hand, protamine rich nuclei of mature spermatozoa are rich in arginine and cysteine and contain relatively low levels of lysine, which means they will not be stained by Aniline blue (71). Results of AAB have a negative correlation with sperm chromatin integrity and male fertility potential (72). However, there is a controversy on the correlation between the percentage of Aniline blue-stained spermatozoa and other sperm parameters that need to be further evaluated.

Toluidine blue staining

Toluidine Blue (TB) is a basic nuclear dye used for metachromatic and orthochromatic staining of chromatin. This stain is a sensitive structural probe for DNA. Due to the cooperative nature of metachromatic stain which is indicative only in severe DNA damaged conditions, it is revealed in only poor sperm DNA integrity. Therefore, TB staining should be used in combination with other more reliable staining methods for the assessment of sperm chromatin integrity (73). In general, the AAB and TB are two simple and cheap methods that have the advantage of providing suitable slides for use on a light microscope (74). The smears stained with the TB method can also be used for assessment of sperm morphology. However, these methods have the inherent limits of repeatability due to dye balance differences and a low number of sperm which can be reasonably counted (74).

Chromomycin A3 staining

Chromomycin A3 (CMA3) is a guaninecytosine- specific fluorochrome and indicative of poor chromatin packaged in human sperm via indirect counting of protamine-deficient sperm. Chromomycin A3 and protamines compete for the same binding sites in the DNA. Therefore, high CMA3 stained spermatozoa is a strong indicator of the defects in protamination (75). As a discriminator of ART success rate, CMA3 method has a sensitivity of 73% and specificity of 75%. Therefore, it may provide a prognosis on the success of ART (76).

DNA Breakage Detection-Fluorescent In Situ Hybridization (DBD-FISH)

In this assay, sperm was placed within an agarose gel on a slide that was exposed to an alkaline condition, which converts DNAstrand breakages into single-stranded DNA (ssDNA) motifs. After neutralizing and proteins extruding, ssDNA is available for hybridization with specific DNA probes. Theprobe indicates the chromatin area to be analyzed. As DNA breakages increase, more ssDNA is created by the alkaline condition and more probe hybridizes, which leads to an increase in the fluorescence strength and surface area of the fluorescent in situ hybridization (FISH) signal. Defects in sperm chromatin packaging significantly increase the availability of DNA ligands and the sensitivity of DNA to denaturation by alkaline condition. Therefore, DBDFISH used for in situ evaluation and quantification of DNA breakages, brings to light the structural aspects of the sperm chromatin (77, 78). Although this method shows structural aspects of sperm chromatin, it is expensive and time-consuming. In addition this assay has less clinical value and its results are not superior to the other methods (78).

In situ nick translation

The Nick Translation (NT) method measures the insertion of biotinylated deoxyuridine triphosphate (dUTP) at single strand DNA breakages that is catalyzed by DNA polymerase I. It particularly stains spermatozoa with considerable quantity of endogenous DNA breakage. The NT method shows abnormalities that have risen during remodeling of the sperm chromatin. As a result most of these anomalies have not been shown by standard semen analysis such as sperm morphology (79). Application of NT assay shows an association between sperm chromatin integrity and sperm motility and morphology and to a lesser extent, sperm concentration. The NT method is used for detection of sperm DNA damage arising from causes such as heat exposures or the production of ROS following leukocytospermia and contact of sperm with leukocytes within the urogenital tract of men (79). The benefit of the NT method is direct labeling of the DNA breakage sites, and consequently the breakage sites are detectable at the molecular level (80).

Acridine Orange staining

The Acridine Orange (AO) staining as a fluorochrome measures the susceptibility of sperm DNA to acid-induced denaturation and subsequently shifts of AO fluorescence from green (double strand) to red (single strand). AO interacts with double-stranded DNA as a monomer; however it binds to single-stranded DNA as an aggregate. The monomeric AO binding to native double strand DNA emits green fluoresces, while the denatured DNA binds to aggregated AO and produces red fluoresces (81). The AO assay, also named as Sperm Chromatin Structure Assay (SCSA), is a functional assay that measures sperm quality. The variation of its results between different individuals (inter-assay) and between several assays (intra-assay) for the same sample is very high. If the inter-assay coefficient variations of AO staining method were less than 5%, it is rendered as a highly reproducible technique. To increase the accuracy and precision of AO staining results for sperm chromatin, there is a need for more expensive instrumentation such as flowcytometer to differentiate different colors and interpret the results. Also, individual subjectivity may hinder the results if fluorescent microscopy is used (82). Since the SCSA is highly constant over a long period of time as compared to the standard parameters of semen analysis, it may be applied successfully in the epidemiological studies in the field of andrology (83).

Sperm chromatin dispersion

The Sperm Chromatin Dispersion (SCD) test is based on this fact that when sperm are exposed to an acid solution prior to lysis following the removal of nuclear proteins, the DNA dispersion halos will be observed. It presents minimally in sperm chromatin without DNA fragmentation or not produced at all in sperm chromatin with fragmented DNA (84). The major advantage of the SCD test besides the above mentioned methods is that it does not need to detect the color or fluorescence intensity. Furthermore, the test is easy, fast, and reproducible and its results are as good as to those of the SCSA (85).

Comet assay

The comet assay is a single-cell gel electrophoresis for detection of DNA fragmentation in a single cell (86). In this assay, sperms are stained with a Fluorochrome that binds to DNA. During electrophoresis, the movement of fragmented double-stranded DNA from a damaged sperm chromatin becomes visible as a comet with a tail (86). Singh et al modified the comet assay in 1988 (87) by performing electrophoresis under alkali buffers to expose alkali-labile sites on the DNA. This alteration changed the sensitivity of the assay for detection of both single and double-stranded DNA breakages (88). Recently using particular software the amount of fragmentations is quantified by measuring the displacement between the nucleus "comet head" and the resulting tail. The tail lengths are used as an index for the intensity of DNA fragmentation. However, determination of both intensity and length of the tail defines it more precisely (89). This method is fruitfully used in the evaluation of DNA fragmentation after cryopreservation (90). It may also prognoses the success of IVF and ICSI, and embryo quality on the base of sperm chromatin integrity particularly in couples with idiopathic infertility (91, 92). The comet is a well-standardized assay that correlates significantly with TUNEL and SCSA methods (93). It is simple to perform, has a low intra-assay coefficient of variation, and not expensive (68). It is based on fluorescent microscopy, therefore, it requires a well experienced individual to examine the slides and interpret the results.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine Triphosphate-Nick End Labeling (TUNEL) assay

Terminal deoxynucleotidyl transferase mediated deoxyuridine Triphosphate-Nick End Labeling (TUNEL) assay quantifies the integration of the flurochrome or biotin labeled dUTP at single and double-strand DNA breakages in a reaction catalyzed by the Terminal deoxynucleotidyl Transferase (TdT) enzyme that is not dependent on the template. This enzyme inserts biotinlyated dUTP or FITC-dUTP at 3'-OH end of DNA breaks to prodsuce a signal. Intensity of signals depends on the number of DNA breaks at the head of spermatozoa. Therefore, sperm with normal chromatin integrity have only background fluorescence, while sperm with fragmented DNA (multiplechromatin 3'-OH ends) emit highly fluorescence light (94). The TUNEL assay has been usually used in andrology research related to sperm chromatin integrity and it abnormalities. It gives valuable data in numerous cases of infertile and subfertile men (95,96). The flowcytometric quantification of labeled DNA 3'-OH ends in sperm head is generally more precise and reliable; but it is much more expensive (96).

High-performance liquid chromatography

This method determines the concentration of 8-hydroxy-2-deoxyguanosine (8 OHdG), which is a byproduct of oxidative damage of DNA in the sperm chromatin. It is the regularly studied biomarker for oxidative damage of sperm chromatin. Along with different oxidative adducts to DNA, 8-OHdG has been selected as a representative of oxidative damage of DNA due to its high specificity, strong mutagenicity, and relative abundance in DNA (97). This method presents the most direct evidence suggesting the contribution of oxidative damage of DNA sperm in male infertility, based on the result that levels of 8-OHdG in sperm are significantly higher in infertile men than in fertile controls and have an opposite relation with sperm concentration (98). 8-OHdG in sperm DNA has been shown to increase in smokers, and they inversely correlate with the intake and the seminal plasma concentration of vitamin C. If 8-OHdG modifications in DNA were not repaired, it will be mutagenic and may lead to early abortion, malformations, or malignancy in children. Furthermore, this modification could be a marker of OS in sperm, which may have negative effects on sperm function (99).

Significance of sperm chromatin \ integrity on male fertility

It is believed that sperm chromatin integrity is correlated with male fertility (47), thus it has been shown that unexplained infertile men with normal routine semen parameters have a higher DNA Fragmentation Index (% DFI) (100). Evenson et al (101) have shown that the DFI is the most excellent predictor of couple fertility and their ability to get conceive. There are many studies evaluating the effect of DFI on ART outcomes, especially in cases with recurrent ART failure. The relationship between sperm chromatin integrity and IUI outcome has been shown in several studies (102). Host et al found no correlation between sperm DNA breakages and the fertilization rate following ICSI (103). In contrast several other studies have found a significant negative correlation between sperm DNA fragmentation and the ICSI results (r=-0.23, p=0.017) (104). Sun et al (105) found a negative correlation between sperm semen analysis parameters and sperm DIF. In a new study, the proportion of sperm with fragmented DNA correlated with numbers and embryo quality, embryo development and the rate of the ongoing pregnancies. DNA fragmentation may not influence the fertilization rate following IVF or ICSI (106). However, when the patients were divided into two category according to cut-off value of10%, the fertilization rate was significantly higher for DNA fragmentation lower than 10% (84.1 vs. 70.7%, p<0.05). In a prospective study (100), Saleh et al examined the relationship between sperm DNA damage and ART outcomes in 33 couples with approved male factor infertility. They found that the sperm DFI was negatively correlated with sperm concentration (r=-0.31, p=0.001), p motility (r=-0.47; p<0.001) and normal sperm morphology (r=-0.40; p<0.0001) (100). The current data suggest that fertilization and pregnancy rates following ICSI, are not related to the severity of the sperm defects (106). This finding has caused debate on the

Table (1): Methods to assess sperm chromatin integrity. COMET, single-cell gel electrophoresis assay; DSB, double strand break; SCSA, sperm chromatin structural assay; SSB, single strand break; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (Erenpreiss et al., 2006- modified).

Erenpreiss J, Spano M, Erenpreisa J, Bungum M, et al. Sperm chromatin structure and male fertility: biological and clinical aspects. Asian. J. Androl. 2006; 8 (1): 11-29.

Acridine orange/SCSA

To assess the susceptibility of sperm DNA to acid-induced denaturation, the SCSAw was done using a method described previously (1). corresponding to the percentage of cells outside the main population) and as percentage of spermatozoa with high green fluorescence or high DNA stainability (%HDS), as an indication of sperm DNA compaction (2)

TUNEL assay

DNA strand breaks were analyzed An aliquot consisting of cells stained with the staining solution lacking the terminal deoxynucleotidyl transferase enzyme was included as a negative control for each sample (3).

Comet assay

DNA strand breaks present in spermatozoa were determined by the comet assay (4)

mBBr thiol labeling assay Thiol (SH)

Labeling was done according to the method described by ().

CMA3 assay

1. Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 2002; 23: 25-43.

2. Evenson DP, Wixon R. Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA®). Toxicol Appl Pharmacol 2005;207: 532-537.

3. Zubkova EV, Robaire B. Effects of ageing on spermatozoal chromatin and its sensitivity to in vivo and in vitro oxidative challenge in the Brown Norway rat. Hum Reprod 2006;21: 2901-2910.

4. Codrington AM, Hales BF, Robaire B. Spermiogenic germ cell phase-specific DNA damage following cyclophosphamide exposure. J Androl 2004; 25: 354-362.

5. Zubkova EV, Wade M, Robaire B. Changes in spermatozoal chromatin packaging and susceptibility


Sperm Chromatin Structure Assay (SCSA)

The SCSA was first described over 25 years ago (1). This assay is based on the premise that DNA in sperm with abnormal chromatin structure is more prone to acid or heat denaturation (2). Using the metachromatic properties of acridine orange (AO), SCSA measures susceptibility of sperm DNA to acid-induced denaturation in situ. By quantifying this metachromatic shift of AO from green to red after acid treatment using flow cytometry, the extent of DNA denaturation is determined (1). The parameter obtained by SCSA most commonly referred to in the literature is DNA fragmentation index (DFI), a measure of DNA denaturation.

Acridine orange test

The acridine orange test (AOT) is based on similar principles as the SCSA in which the metachromatic shift of AO from green to red is used to determine extent of DNA denaturation. The AOT is simpler and less expensive than the SCSA since it can be done by visual interpretation under fluorescent microscopy without the need for flow cytometry or a SCSA trained technician (2). However, issues of indistinct colors, rapid fading, and the heterogeneous staining can cause difficulties during visual interpretation (3).

Toluidine blue

Toluidine blue (TB) is a basic dye used to evaluate sperm chromatin integrity. Phosphate residues of sperm DNA in nuclei with loosely packed chromatin and/or impaired DNA are more liable to binding with basic dyes such as TB (1).

Thus, using light microscopy, damaged sperm will be

stained blue while normal sperm will remain colorless.

Aniline blue

Aniline blue is an acidic dye which is used to evaluate sperm chromatin integrity. Sperm with impaired DNA often display the presence of residual histones.

These residual histones lead to looser chromatin packaging allowing increased accessibility of basic groups of the nucleoprotein and subsequently liable to bind acidic dyes such as aniline blue (5).

TUNEL The terminal deoxynucleotidyl transferase-mediated (TdT) deoxyuridine triphosphate (dUTP) nick end labeling assay (TUNEL)

is a direct quantification of sperm DNA breaks (6). dUTP is incorporated at single-stranded and double stranded DNA breaks in a reaction catalyzed by the enzyme TdT. The DNA breaks based on the incorporated dUTP are then labeled and can be measured using bright field or fluorescent microscopy as well as flow cytometry (6). Sperm are then classified as TUNEL positive or negative and expressed as a percentage of the total sperm in the population. Typical results of the TUNEL assay are shown in Fig. 1A. In situ nick translation assay The in situ nick translation (NT) assay is similar to the TUNEL assay in that it quantifies incorporation of Dutp into DNA breaks. However, in contrast to TUNEL which identifies both single-stranded and double-stranded DNA breaks, the in situ NT assay only identifies single-stranded DNA breaks in a reaction catalyzed by the templatedependent enzyme, DNA polymerase I. Although this can be a relatively simple test to perform, it lacks sensitivity when compared to other assays (2).


The single-cell gel electrophoresis (Comet) assay is another test for direct assessment of sperm DNA breaks (7). Decondensed sperm are suspended in an agarose gel, subjected to an electrophoretic gradient, stained with fluorescent DNA-binding dye, and then imaged with imaging software. Low-molecular weight DNA, short fragments of both single-stranded and double-stranded DNA, will migrate during electrophoresis giving the characteristic comet tail (2). High-molecular weight intact segments of DNA will not migrate and remain in the head of the "comet." Imaging software is then use to measure comet tail length and tail fluorescent intensity, which are increased in sperm with high levels of DNA strand breaks (8).

Sperm chromatin dispersion test

The sperm chromatin dispersion (SCD) test is based on induced condensation which is directly linked with sperm DNA fragmentation (9). Intact sperm are immersed in an agarose matrix on a slide, treated with an acid solution to denature, and then treated with a lysis buffer to remove sperm membranes and proteins giving rise to nucleoids with a central core and a peripheral halo of dispersed DNA loops. Sperm with non-fragmented DNA release their DNA loops forming large halos.

However, sperm which produce a very small halo or no halo at all contain DNA fragmentation (10). Sperm can be stained with Wright's stain for visualization under bright field microscopy or an appropriate fluorescent dye for visualization under fluorescent microscopy.


1. Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science. 1980; 210: 1131-3.

2. Schulte RT, Ohl DA, Sigman M. et al.Sperm DNA damage in male infertility: etiologies, assays, and outcomes. J Assist Reprod Genet. 2010; 27: 5-6.

3. Chohan KR, Griffin JT, Lafromboise M, et al. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J Androl. 2006; 27: 53-9.

4. Mello ML. Induced metachromasia in bull spermatozoa. Histochemistry. 1982;74:387-92.

5. Auger J, Mesbah M, Huber C, et al. Aniline blue staining as a marker of sperm chromatin defects associated with different semen characteristics discriminates between proven fertile and suspected infertile men. Int J Androl. 1990; 13: 452- 62.

6. Gorczyca W, Gong J, Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 1993;53: 1945-51.

7. Haines G, Marples B,Daniel P, et al DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the comet assay. Adv Exp Med Biol. 1998;444: 79-91. discussion 92-73.

8. Hughes CM, Lewis SE, McKelvey-Martin VJ, et al. A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay. Mol Hum Reprod. 1996; 2: 613- 9.

9. Muriel L, Garrido N, Fernandez JL, et al. Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril. 2006; 85: 371- 83

10. Fernandez JL, Muriel L, Goyanes V, et al. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil Steril. 2005; 84: 833- 42.


PDF21 Sperm chromatin structure

Chromatin of mammalian sperm has a unique structure that is highly organized, condensed, and compacted. This allows protection of the paternal genome during transport through the male and female reproductive tracts and its subsequent delivery to the ova in good condition. Mammalian sperm DNA is the most tightly compacted eukaryotic DNA (1). This feature is in contrast DNA structure in somatic cell nuclei. Somatic cell nuclear DNA is wrapped around an octamer of histones and packaged into nucleosomes and then further coiled into a solenoid [84]. This type of packaging adds histones, which increase chromatin volume. Sperm cell nuclei simply do not have the volume for this type of packaging and thus must undergo a different type of packaging [124]. During spermiogenesis, sperm chromati undergo a series of modifications in which histones are lost and replaced with transition proteins and subsequently with protamines [27, 68, 77]. Protamines are approximately half the size of histones [44]. The DNA strands are highly condensed by these protamines and form the basic packaging unit of sperm chromatin, a toroid. The toroids are further compacted by the intramolecular and intermolecular disulfide cross-links between cysteine residues present in protamines [72]. All of these levels of compaction and organization help to protect sperm chromatin during transport through the male and female reproductive tract and also ensures the paternal genome is delivered in a form that allows developing embryo to accurately express genetic information [103]. Although human sperm chromatin contains this highly organized and compact structure, it is less compact than in other mammals. Approximately 15% of histones are retained in human sperm chromatin subsequently making chromatin less tightly compacted [11, 47]. Infertile men have been reported to have a higher histone to protamine ratio in their sperm chromatin [94, 116]. Human sperm also contain two types of protamines, P1 and P2. P2 protamines contain fewer cysteine groups and thus contain less disulfide crosslinks [25]. This theoretically leaves the DNA more susceptible to damage. It has been reported that altered P2 expression is common in men with infertility [18]. Etiologies and mechanisms of sperm DNA damage There are several different levels of sperm chromatin abnormalities that are important to consider: 1) damage to the actual DNA physical integrity in the form of singlestranded or double-stranded DNA strand breaks, 2) nuclear protein defects that may interfere with histone to protamine conversion and subsequent DNA compaction, and 3) chromatin structural abnormalities causing altered tertiary chromatin configuration. Environmental stress, gene mutations, and chromosomal abnormalities can all disturb biochemical events that occur during spermatogenesis, which can ultimately lead to abnormal chromatin structure incompatible with fertility [41]. Ova are able to repair sperm DNA damage to a certain extent [48]. However, when sperm DNA damage is extensive, ovum may not have repair capacities to allow normal development.

PDF26 Evaluation of sperm nuclear DNA damage

Based on the critical importance of accurate transmission of genetic information to the offspring, several assays have been developed to evaluate sperm chromatin/DNA integrity. These assays have been also used in an attempt to establish a significant correlation with male infertility.

Comet assay

The comet assay measures DNA damage by quantifying the single- and double-stranded breaks associated with DNA damage (1). In this assay, spermatozoa are stained with a fluorescent DNA-binding dye. The resulting images, which resemble `comets', are measured after staining to determine the extent of DNA (2). The characteristics that have been used for analysis include the diameter of the nucleus and the comet length (3). One of the principles of the comet assay is that nicked double-stranded DNA tends to remain in the comet head, whereas short fragments of nicked double- and single-stranded DNA migrate into the tail area (4). Thus, spermatozoa with high levels of DNA strand breaks would show increased comet tail fluorescent intensity (5) and comet tail length (6). However, useful thresholds have not been established for the comet assay.

In-situ nick translation (NT) assay

The NT assay quantifies the incorporation of biotinylateddeoxyuridine triphosphate (dUTP) at single-stranded DNA breaks in a reaction that is catalysed by the template-dependent enzyme, DNA polymerase I. The NT assay identi®es spermatozoa that contain appreciable and variable levels of endogenous DNA damage (7). The clinical value of the NT assay is severely limited because no correlation has been proven with fertilization during in-vivo studies (8), and because of its lack of sensitivity compared with other assays (9).

TUNEL assay

The TUNEL assay quantifies the incorporation of deoxyuridine triphosphate (dUTP) at single- and double-stranded DNA breaks in a reaction catalysed by the template-independent enzyme, terminal deoxynucleotidyl transferase (TdT) (10). Incorporated dUTP is labelled such that breaks can be quanti®ed by ¯ow cytometry, ¯uorescent microscopy or light microscopy. The TUNEL assay cannot be employed for routine clinical use due to a lack of useful thresholds.

Sperm chromatin structure assay (SCSA)

The SCSA is a flow cytometric assay that relies on the fact abnormal sperm chromatin is highly susceptible to physical induction of partial DNA denaturation in situ (11). The extent of DNA denaturation following heat or acid treatment is determined by measuring the metachromatic shift from green fluorescence (acridine orange intercalated into double-stranded nucleic acid) to red fluorescence (acridine orange associated with single stranded DNA) (11). The most important parameter of the SCSA is the DNA fragmentation index (%DFI), which represents the population of cells with DNA damage (12).

Acridine orange test

The acridine orange test (AOT) was introduced as a simplified microscopic method of the SCSA that does not require expensive flow cytometry equipment and a SCSA-trained technician (11). It relies on visual interpretation of fluorescing spermatozoa and debris that fall into a broad range of colors under microscopic examination. Indistinct color, rapidly fading fluorescence and heterogeneous slide staining exacerbate problems with interpretation (13). Such conditions preclude using the AOT for critical clinical diagnosis and prognosis of a semen sample (14), since the AOT may introduce many sources of variation. Although some laboratories have used the AOT in an attempt to improve male fertility evaluations (15), the predictive value of the test for human fertility remains controversial. However, in relation to the clinical significance of this assay, a strong positive correlation exists between the AOT and TUNEL assays. In addition, the AOT correlates negatively with sperm motility (16).

Sperm chromatin dispersion (SCD) test

This assay has been recently described as a simple and inexpensive method for the analysis of sperm DNA fragmentation. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo when mixed with a aqueous agarose following acid denaturation and removal of nuclear proteins (17).

Other methods

Other methods may be used to detect DNA damage in human spermatozoa, such as electron microscopy (18), enzyme-linked immunosorbent assay (ELISA) (19), FISH (20), and highperformance liquid chromatography, which is used to measure the level of 8-OhdG (21)