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The Acidic Aniline Blue 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).
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