Evaluation of nuclear DNA damage in human spermatozoa in men opting for assisted reproduction

Transcription

1 Review Article Indian J Med Res 127, February 2008, pp Evaluation of nuclear DNA damage in human spermatozoa in men opting for assisted reproduction M.B. Shamsi, R. Kumar & R. Dada Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India Received April 20, 2007 Diagnosis of sperm DNA integrity of semen sample is important for consistently high reproductive efficiency. The conventional parameters of semen analysis take into account morphology, motility, and concentration of spermatozoa in the sample, which are insufficient for evaluation of reproductive potential. Current studies have implicated abnormal organization of genomic material in sperms as a probable cause in 20 per cent cases of male infertility. This is especially important in the era of assisted reproduction technique (ART) when a majority of infertile couples opt for assisted reproduction and in where cases DNA integrity is a better diagnostic and prognostic marker as compared to routine semen analysis. This article reviews and discusses some of the current techniques employed for evaluating chromatin structure or DNA damage in spermatozoa. These different techniques include single cell gel electrophoresis (COMET assay), Terminal tranferase dutp Nick End Labelling (TUNEL), sperm chromatin structure assay (SCSA), In situ nick translation (ISNT) and acridine orange test. These techniques are independent measure of sperm quality and assist in semen quality assessment by detecting defects in DNA integrity or chromatin structure. The discussed techniques vary in their level of accuracy, cost input, sophistication of analysis and their application depends upon the sensitivity required for analysis. The article also briefly outlines the DNA packaging and the causes of DNA damage in spermatozoa. During chromatin packing 85 per cent of the histones are replaced by protamine while the residual histones act as marker of genes which are expressed in early embryonic development. Among the different aetiological factors observed to be responsible for DNA damage in human spermatozoa increased reactive oxygen species (ROS), oxidative stress is highly correlated with greater DNA fragmentation index (DFI). Oxidative stress leads to single and double strand breaks in sperm DNA. Apoptosis and abnormal chromatin packing also contribute to DNA damage. The significance of chromatin structure studies is more stressed owing to the greater awareness to transmission of genetic diseases because of higher incidence of gene imprinting defects, increased cancer frequency and other congenital and non-congenital defects in children conceived through assisted reproduction techniques. Key words Assisted reproduction techniques - DNA fragmentation - DNA integrity - oxidative stress - sperm chromatin structure assay 115

2 116 INDIAN J MED RES, FEBRUARY 2008 Infertility is a major clinical problem with male factor responsible for 40 per cent cases of infertility. Current estimates indicate that nearly 20 per cent cases of idiopathic infertility have high level of sperm DNA fragmentation, a negative factor for infertility 1. Full sperm DNA integrity usually is defined as the absence of DNA nicks or single stranded (ss) breaks, double stranded (ds) breaks, and chemical modifications of the DNA. Of these, ds DNA breaks are the most mutagenic, because in the pronucleus stage zygote, the two genomes are separated, hence DNA template information for error free repair of the ds breaks is absent. Also in the G1 stage zygote, non homologous end joining (NHEJ) which is essentially error prone, is likely to prevail in the repair of ds DNA breaks 2. Much higher degree of negative association between the degree of DNA damage with various indices of fertility such as fertilization rate, embryo cleavage rate, implantation rate, pregnancy and live birth rate, have been observed. The spontaneous abortion rate is about 1.7-fold higher when more than 30 per cent of sperms contain fragmented DNA. The chances of live birth and conception decrease drastically when DNA fragmentation index (DFI) is > 30 per cent 3. Sperm DNA is increasingly being recognized as an important measure of fertilizing efficiency that has better diagnostic and prognostic capabilities than standard sperm parameters like sperm morphology, concentration and motility. Routine semen parameter does not always reflect the quality of sperm DNA. Men with normal spermiograms may still be infertile; the cause could be related to abnormal sperm DNA 4. Sperm DNA integrity may be evaluated in addition to routine sperm parameters (morphology, motility and concentration) to indicate the quality of spermatozoa. The assessment of quality of sperm DNA has become essential with the increase in use of assisted reproductive techniques (ART). In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) techniques bypass several natural selection barriers that are present throughout the male and female reproductive tracts and until the sperm enters the oocyte 4. Despite professional expertise and availability of state of art technology in ART, the carry home live birth rate after several ART cycles is just 27 to 32 per cent. This is mainly due to genetic aberrations in germ cells which lead to embryonic lethality. The rate of carry home live birth is low especially when sperm with DNA damage is used in ART. Recurrent ART failure cause financial, emotional and psychological stress on the couples opting for such techniques. According to recent studies when sperm deformity index (SDI, i.e., the ratio of number of sperms with defects to the total number of sperms) is greater than 1.6, the chances of ART failure increases 5. DNA damage in sperm is promutagenic. It does not impair fertilization or cleavage as paternal genome is transcriptionally inactive till two days after fertilization 4. However, once the paternal genome is active it results in poor blastocyst development, unequal cleavage, implantation failure or early foetal loss. Small DNA damages in sperm are repaired by pre- and postreplication repair mechanisms, but large DNA damages cannot be repaired. Thus, though infertile men may have sperms with normal morphology, these germ cells may harbour damaged DNA. This results in pregnancy loss or birth of offsprings with major or minor congenital malformations, severe dysmormphogenesis or may lead to increased predisposition to certain cancers like retinoblastoma. Thus DNA integrity studies are of foremost importance in evaluation of all infertile males prior to assisted conception 4. This article reviews and discusses some of the current techniques employed for evaluating chromatin structure or DNA damage in spermatozoa. These techniques help to detect subtle defects in the chromatin structure or DNA integrity, and thereby assist in semen quality assessment. DNA integrity studies are a more reliable diagnostic and prognostic marker in ART studies as compared to routine semen analysis. DNA packaging in human spermatozoa The formation of mature spermatozoa involves a series of mitosis, meiosis, changes in cytoplasmic structure, topological rearrangements, alteration in transcription, replacement of histones with transition proteins and the final addition of protamines leading to highly condensed insoluble and stable chromatin. This highly organized, compact and insoluble nature of the sperm chromatin protects the genetic integrity during transport of the paternal genome through the male and female reproductive tracts 5. Human sperm DNA is organized into loops that are attached to the specific sites to the sperm nuclear matrix 6. There are three types of protamines : protamine 1 (P1),which forms the major component of sperm DNA binding protamines; protamine 2 (P2),which is deficient in cysteine residues and is expressed in spermatids prior to the widespread displacement of histones by transition

3 SHAMSI et al: TECHNIQUES FOR DNA DAMAGE EVALUATION IN HUMAN SPERMATOZOA 117 proteins. Transition proteins (TP) are intermediate in the process of histone replacement 7,8. Further in chromatin human sperm retention of 15 per cent histone nucleoproteins leads to less compact nucleosome structures 9. The histones in sperm chromatin are a subset of the histones in somatic chromatin and nucleosomes in the sperms are more closely packed than those found in somatic cells. In sperm histone H1 is absent, H2 takes the form of two minor variants, called H2A.X and H2A.Z, and the H3 and H4 are extensively acetylated 7,8. Absence of histone H1 and acetylation of histones are indicative of active chromatin. Histones in spermatozoa mark the sets of genes that are preferentially activated during early development. Thus histones in sperm influence the genes which are transcribed first after fertilization. During spermatogenesis and epididymal transit, nuclear histones are replaced by arginine/cysteine rich protamines 10. The thiol (-SH) groups of the cysteine residues are progressively oxidized to form disulphide bonds which provide stability and compactness to the sperm DNA 11. Protamines are also essential in successful egg-sperm fertilization process; any abnormality in the protamine level may thus lead to the damage in sperm DNA and may impair fertilization 12. Pathogenesis of DNA damage in human spermatozoa Various mechanisms can damage sperm DNA. Abnormal chromatin packing, reactive oxygen species (ROS) and apoptosis are the most important aetiological factors which disrupt DNA integrity. Apoptosis in testes controls the overproduction of male gametes and restricts the normal proliferation level during conditions unsuitable for sperm development 13. It also prevents clonal proliferation of germ cells with damaged DNA. Defects in the remodeling of cytoplasm during spermatogenesis may lead abortive apoptosis a phenomenon in which defective sperm cells escape programmed cell death and are present in the ejaculate. Presence of apoptotic markers as fas in such sperms support the theory of abortive apoptosis 14. During chromatin packaging endogenous nucleases (topoisomearse II) are activated to create and ligate nicks that facilitate replacement of histones with protamines during spermatogenesis by relieving torsional stress 15. Persistence of endogenous nicks in spermatozoa indicate anomalies in spermatogenesis and an incomplete maturation process 16. The generation of ROS occurs physiologically during normal cell metabolism. Mitochondrial respiration is the main biological source of superoxide anion radicals under physiological conditions. High levels of ROS are potentially toxic to sperm quality and function 17. Semen of about per cent of infertile male patients has high ROS level and oxidative stress 18. High susceptibility of sperms to ROS-induced damages has been postulated due to presence of large quantities of polyunsaturated fatty acids in their plasma and low concentrations of scavenging enzymes (as most of the antioxidant enzymes are lost in spermiogenesis) in the cytoplasm, although tight packing of sperm chromatin offers protection against oxidative stress 17. ROS attacks nuclear and mitochondrial DNA, inducing fragmentation, aberrant recombination, and/or defective packaging. An increase in ROS changes the tertiary structure and expression of proteins, protein membrane receptors, and membrane transport proteins, which in turn result in the disturbance of ionic balance, though low concentration of ROS is essential and has a biopositive effect on normal functioning of the sperm cell. Low amount of free radicals in human semen enhances spermatozoa s ability to bind zona pellucida. Optimum concentration of ROS is also found to stimulate sperm capacitation, hyperactivation, acrosome reaction and oocyte fusion. While low ROS levels are associated with regulation of gene expression, notably upregulation of anti-oxidant proteins in oxidative stress 18. The leucocytes play an important role in immunosurveillence and phagocytic clearance of abnormal sperms 19. Increased ROS induced damage is also caused due to activated leucocytes 20,21. This mechanism operates when there is tissue inflammation. Also in smokers testis show inflammatory changes and there is increased production of free radicals by activated leucocytes 19. Cytokines as interleukin (IL)-6 and IL-8 recruit and activate leucocytes 3. Activated leucocytes produce hundred times more ROS than inactive leucocytes. These activated leucocytes overwhelm the antioxidant strategies and result in oxidative stress (OS). The formation of 8-hydroxy 2- deoxyguanosine (8-OhdG) is a key biomarker for oxidative DNA damage 22,23. Oxidative stress affects the integrity of sperm chromatin causing high frequencies of single and double strand breaks, base modifications, production of base free sites, deletions, frameshifts, cross-links and chromosomal rearrangements 24. Techniques for DNA damage measurement Finding the most appropriate technique to quantify the abnormal DNA and relate it to fertility has always

4 118 INDIAN J MED RES, FEBRUARY 2008 been a challenging task for researchers. Several techniques have been devised for studying DNA defects in human spermatozoa. Staining with aniline blue, toludine blue, and chromomycin A3 is used to identify sperm chromatin packaging defects. The integrity of sperm DNA can be evaluated with the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL), COMET, acridine orange staining, in situ nick translation, or sperm chromatin structure assay (SCSA) techniques. Sperm chromosomal aneuploidies are usually detected using fluorescence in situ hybridization (FISH) technique. Other genetic abnormalities as Yq microdeletions in sperm can be detected by PCR microdeletion analysis in infertile men with severe oligospermia and non obstructive azoospermia 25,26. In azoospermic men, germ cells can be retrieved from testis, matured externally and used for ART. Prior knowledge of germ cell Yq microdeletion status aids in providing comprehensive counselling and preventing iatrogenic transmission of this anamoly. Comet assay The comet assay is a very sensitive technique for detecting DNA damage. Comet assay measures the response of individual cells which enables the study of heterogeneity within a cell population, which is not possible with other techniques 27 like pulsed-field gel electrophoresis, filter elution or sucrose gradient sedimentation which only measure the average response of a large number of cells. The assay is based on the principle of faster rate of migration of smaller fragmented DNA towards anode in an electrophoretic field as compared to larger non fragmented DNA. The comet assay can be conducted in neutral (ph 9) or alkaline (ph >10) conditions depending upon the type of DNA damage to be investigated. The neutral comet assay detects ds DNA and is much more sensitive to identify DNA damage related to infertility. Alkaline comet assays identifies ss DNA breaks and alkali labile sites. These alkali labile fragments are abundant in spermatozoa and decrease the sensitivity of the assay to DNA fragments resulting from ds DNA breaks that are indicative of DNA damage and infertility 28,29. These alkali labile sites may lead to overestimation of true DNA breaks in a spermatozoa. Comet assay can detect 200 DNA strand breaks per cell, however with few modifications it may detect up to 50 breaks per cell. Comet image analysis: The head region of a comet represents unfragmented DNA that does not migrate outside the region of the nucleus while tail represents DNA which migrates due to fragmentation and loss of structure. In two dimensional planar imaging the fluorescent intensity at a location is directly proportional to the amount of DNA at that location 28. There are different approaches to analyze and calculate the amount of DNA damage depending upon the sensitivity and accuracy of analysis (i) Emperical scoring based on the presence or size of the tail, by taking the ratio of flourescent intensity at a fixed distance from the head to the intensity at the centre of the head. If this ratio is 1, then it indicates no DNA damage. (ii) By measuring the tail length from negative photomicrographs. Greater the tail length more is the degree of DNA damage. (iii) Use of accurate and sensitive digital image acquisition systems and analysis software. Tail moment a factor which combines tail length and distribution of DNA in the tail is the best way to quantify the amount of DNA damage using the digital comet image. Only tail length is not a sufficient indicator of DNA damage because with increase in damage the tail length increases steadily, however, it attains a plateau beyond a certain degree. At higher degree of damage the tail length becomes steady whereas the amount of DNA migrating into the tail still increases. The analysis software calculates the intensity integral at each column of pixel and then multiplies it with the horizontal distance from the centre of comet head. These individual moments are then summed for the entire comet tail and normalized to the total comet intensity to give the tail moment. The tail moment approach increases the sensitivity of analysis, in cases where two different comet images have same tail length but the degree of damage will be higher for the image which has greater tail moment due to higher intensity of fluorescence produced by the larger number of fragmented DNA present 30,12. Limitations of comet assay: DNA damage can be overestimated in comet assay due to the presence of residual RNA which creates background during analysis, or can be underestimated because of proteins which hamper the movement of fragments during electrophoresis. Other factors responsible for underestimation are masking, overlapping and entangling of migrating filaments. Incomplete chromatin decondensation may not allow breaks to be revealed. During processing a few small fragments are lost and moreover a few fragments are too small to be visualized.

5 SHAMSI et al: TECHNIQUES FOR DNA DAMAGE EVALUATION IN HUMAN SPERMATOZOA 119 Overlapping comet tails decrease the credibility and accuracy of the assay. Due to high inter-laboratory variation comet assay is not suitable for clinical use 31,32. Advantages of comet assay: In comet assay only a few number of cells are required, data can be collected at the level of individual cells, wide range of eukaryotic cells can be used, the assay is sensitive, simple and can detect non uniform responses within a mixed population. TUNEL TUNEL incorporates biotinylated deoxyuridine (dutp) to 3 OH at single and double strand breaks to create a signal, which increases with the number of DNA breaks. The principle of TUNEL involves labelling the 3 ends of fragmented DNA with biotinylated dutp using recombinant terminal deoxynucleotidyl transferase (TdT) enzyme in a template independent manner. These incorporated labelled nucleotides can be detected in spermatozoa by flowcytometry, flourecence microscope or light microscope. In detection by fluorescence microscopy spermatozoa with normal DNA having capped telomeres at the 3 OH end do not fluoresce while those with fragmented DNA (multiple 3 OH ends) have bright fluorescence 33,12. In enzymatic detection of fragmented DNA by TUNEL horseradish peroxidase-labelled peroxidase streptavidin (Streptavidin HRP) is bound to the biotinylated nucleotides, which are detected using the peroxidase substrate hydrogen peroxide, and the stable chromogen, diaminobenzidine (DAB) which produces brown stain 34. Limitations of TUNEL assay: By TUNEL the degree of DNA damage within a cell cannot be quantified, it only reveals the number of cells within a population with DNA damage. Since the analysis involves the visual observation by light microscopy of stained cells (with damaged DNA) and non stained cells (with non damaged DNA) the background staining decreases the efficiency of the assay. It is worth mentioning that such cells can also be identified using flowcytometry or fluorescence microscopy. Due to lack of useful thresholds and the non validated standardized forms of TUNEL, the assay cannot be used for routine clinical tests with prior training in order to obtain reproducible data. Moreover, only a limited number of cells are labeled due to unique DNA packaging in spermatozoa 12. Advantages of TUNEL assay: TUNEL can simultaneously detect single and double strand breaks unlike comet assay which requires different protocols for studying both type of strand breakages 12. In situ nick translation assay (ISNT) ISNT is a modified form of TUNEL, which utilizes incorporation of biotinylated-dutp at the ssdna in a reaction catalyzed by template dependent enzyme, DNA polymerase 1 (DNA Pol 1), unlike TUNEL which utilizes template independent TdT. Moreover ISNT can only be used for single strand breaks not for both ss and ds breaks as in TUNEL 29. Limitations of ISNT: ISNT has a very dynamic range and lacks sensitivity compared with other assays. According to Irvine et al 29 the clinical value of ISNT assay is severely limited because no correlation has been proven with fertilization in in vivo studies. These authors have reaffirmed that the neutral comet assay is more sensitive measurement of sperm DNA damage than ISNT assay. Advantages of ISNT: ISNT assay could identify spermatozoa that contain variable and appreciable levels of endogenous DNA damage. Sperm chromatin structure assay (SCSA) SCSA is a technique, in which 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) 35. The most important parameter revealed by SCSA is the DNA fragmentation index (% DFI). Limitations of SCSA: The SCSA does not give much information about the extent of DNA damage in spermatozoa, since it focuses on measuring the percentage of spermatozoa with dispersed or non dispersed nuclei 12. Advantages of SCSA: It is simple and less time consuming method for the analysis of human spermatozoa 12. Acridine orange test (AOT) AOT is a simplified microscopic method of SCSA which does not require expensive flowcytometry and relies on visual interpretation of fluorescing spermatozoa and debris that fall into a broad range of colours under microscopic examination. Pretreated sperm samples (citric acid solution ph 2.0) stained with acridine orange (0.2 mg/ml H 2 O) are washed and covered with coverslip for examination 36. Sperms with ds DNA fluoresce green while those with ssdna fluoresce red under a fluorescence microscope.

6 120 INDIAN J MED RES, FEBRUARY 2008 Limitations of AOT: Indistinct colour, rapidly fading fluorescence and heterogeneous slide staining exacerbate problems with interpretation 37. The AOT has less sensitivity to detect comet tails and its use in assessment of male infertility remains controversial. Correlation between DNA damage and assisted reproduction outcome ART has revolutionized the management of severe male factor infertility. ART has increased the chances of sperm with abnormal genome fertilizing a oocyte. Extensive data exist on the relationship between DNA damage and outcomes after ART. Most of the studies have shown a significant association or negative trends with fertility outcome parameters after ARTs. Recent studies have reported that cases harbouring cytogenetic abnormalities and Yq microdeletions experience recurrent ART failure. These studies report that germ cells with chromosomal aneuploidies and microdeletions result in poor blastocyst development and pre- and/or post-implantation failure 38,39. Some studies have found no correlation between levels of DNA damage and fertilization rates but rather an association between DNA damage and post fertilization development 4. ICSI studies have found a lack of correlation between fertilization rates and DNA damage, suggesting that ICSI bypasses the natural selection process and allows spermatozoa with DNA damage to fertilize oocytes. In ICSI studies an association between pregnancy rate and sperm DNA damage is found indicating that embryo employs certain mechanisms to prevent defective genomic material from passing on to the offspring 4. In a study conducted by Duran et al 39, degree of DNA fragmentation assessed by TUNEL and AOT was significantly lower in sperm samples that initiated pregnancy by intra uterine insemination (IUI). When the sperm sample has > 12 per cent fragmented DNA the reproductive outcome was zero 40. A good correlation was found between the percentage of sperm with normal DNA and IVF rates 40. It was found that sperm bound to zona pellucida had low amounts of DNA damage indicating that the human zona pellucida selectively binds sperm with a mature nuclei and normal DNA 41. However, all these steps are bypassed during ICSI. Morris et al studied the DNA damage in sperm assessed by COMET, IVF and ICSI. Per cent cleavage/ fertilization, embryo cell number and embryo grade were lower in patients who had severe DNA damage 42. When patients undergoing ICSI were analyzed separately, a significant correlation between DNA damage in sperm and impairment of post-fertilization embryo cleavage was noted 4. Discussion The need of DNA integrity studies of human spermatozoa has gained importance because of the advancements in in vitro fertilization techniques and ICSI. Children conceived through assisted reproductive technology account for 5 per cent of the total births in some countries 4. This focus on genomic integrity of male gamete has been further intensified by growing concern about the transmission of genetic diseases via ART. Normally in an infertile couple there is no propagation of genetic anomaly by virtue of they being infertile. However, by ART these abnormalities are transmitted and these manipulations lead to cloning of infertile population. These technologies are known to be associated with high rates of multiple births, low birth weight and a number of major and minor congenital abnormalities like hypospadias, neural tube defects and other non congenital abnormalities. These anomalies are being attributed to new methods of fertilization that use intracellular and biological manipulations as well as to bypassing the process of natural selection in sperms. IVF births are associated with increased risk of cardiac, urogenital, neurodegenerative and visual impairments. ART has also been implicated as an aetiological factor in increased incidences of certain malignancies as imprinting defects like retinoblastoma, Angelman syndrome and Beckwith-Wiedeman syndrome. Six-fold increase in retinoblastoma (Rb) had been observed in children born through ART 4. Patients seeking IVF have higher DNA breakages or abnormal sperm chromatin 4, thus as per the Knudsons two hit hypothesis, the chances for second hit for the inactivation of Rb gene on chromosome 13 is higher in such germ line cells with defective chromatin 43. Therefore further research is essential to establish the long term safety of microassisted fertilization and consideration should be given to incorporating these concerns into the counselling of couples going for such treatment. Establishing the aetiology of the sperm chromatin damage may serve as a basis to provide medical treatment to individuals with high base line damage prior to assisted conception.

7 SHAMSI et al: TECHNIQUES FOR DNA DAMAGE EVALUATION IN HUMAN SPERMATOZOA 121 DNA damage due to apoptosis occurs in testis during spermatogenesis predominantly at spermatogonia and dividing cells level. It also increases in spermatozoa of poor quality as measured with TUNEL or comet assay. The increased sensitivity to DNA damage in abnormal spermatozoa is probably due to failed chromatin condensation, which makes DNA more accessible to damage. Therefore the presence of DNA fragmentation in ejaculated spermatozoa might correlate with defects in spermatogenesis and thus impair fertility. Accumulating evidences for the presence of damaged DNA in morphologically normal spermatozoa and its effect in pregnancy outcomes had stressed the need to develop assays that can quantify sperm DNA damage with high sensitivity. In terms of sensitivity the comet assay has been shown to be one of the best for the detection of DNA damage 44,45. Sensitivity to the detection of single strand DNA breaks comes from the use of alkaline lysis buffer, which reverses DNA supercoiling and separates the DNA duplex into single strands. Further sensitivity to subtle changes in DNA is gained by incubation of the cells in the presence of Proteinase K, which removes protamines that otherwise impede DNA migration through the agarose. Minor variations in the conventional protocols as inclusion of antioxidants in electrophoresis buffer, alterations in electrophoresis time and differences in ph can all influence the sensitivity of the assay. The comet assay may even overestimate true DNA strand breakage in spermatozoa because of artificial damage induced at alkali labile sites within the DNA strand 45. ISNT condensed procedure effectively correlates the DNA damage with semen quality. The high correlation observed between the ISNT condensed assay and semen quality reflects that this assay measures more than just DNA damage, it also provides an assessment of the efficiency of DNA compaction. The ISNT-condensed assay is reliant on the access DNA polymerase 1 gains to the genome as a result of incomplete compaction of the sperm nucleus, the level of DNA nick end labelling being highly correlated with the levels of sperm nucleus protamination 46. Therefore this assay describes a relationship between semen quality and DNA damage because it incorporates a measure of chromatin condensation that in turn reflects the quality of processes controlling the differentiation and maturation of the spermatozoa. The major problem with this procedure is that it has a very dynamic range, given that less than 10 per cent of all spermatozoa are labelled. TUNEL assay unlike comet assay which requires much manual labour, is simple to handle and is much easy to work upon. It can be used in both tissue sections and individual or cultured cells to measure DNA fragmentation. The specificity of the streptavidin labelled enzyme HRP for the substrate hydrogen peroxide provides specifity and accuracy to the assay without producing background interference. However, considering factors such as simplicity, cost, and availability of resources, techniques as SCSA, AOT, etc., are an edge above other techniques and will remain integral to scientific research. Many technical and biological factors determine the accuracy to evaluate the DNA damage in sperm cells. In spite of the variety of different methods available to evaluate sperm chromatin maturity levels, the bulk of evidence points to the pivotal role of chromatin /DNA integrity during spermatogenesis and in the fertilization process. 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