TUNEL Assay for the Assessment of Sperm Chromatin Damage 467

TUNEL Assay for the Assessment of Sperm 48 Chromatin Damage

Rakesh K Sharma, Ashok Agarwal

Chapter Outline . Abstract . Reference Range of Sperm DNA Damage . Introduction . Factors Affecting the Assay Results . Etiology of DNA Damage . Association of Sperm DNA Damage with Semen • Causes of Sperm DNA Damage Parameters and ART Outcome . Mechanisms of Sperm DNA Damage • Association of DNA Damage with Sperm Parameters . Measuring Sperm DNA Damage • Lack of Association between the TUNEL Assay and the • Measurement of DNA Damage in Spermatozoa by TUNEL Assay Following Variables • Measurement of Sperm DNA Damage by Apoptosis • Association of TUNEL Test with ART Variables Detection Kit • Lack of Association of TUNEL Assay with ART Variables . Equipment and Reagents • Discriminating Power of the TUNEL Assay Natural or Intrauterine Insemination • Sample Preparation • IVF and/or ICSI • Staining Protocol • Staining Solution (Single Assay) . Conclusion • Flow Cytometry • Fluorescence Microscopy

ABSTRACT seeking infertility management (Lamb and Lipschultz, 2000; Oehninger, 2000; Sharma et al. 2004). DNA integrity is vital to the success of natural and assisted Sperm DNA integrity is essential for the accurate fertilization. Various assays have been developed to assess transmission of genetic information (Aitken and Sawyer, sperm chromatin integrity. However, the effect altered sperm 2003; Aitken and De Illuis, 2010). Sperm DNA is being chromatin integrity has on postembryonic development is recognized as an independent measure of sperm quality still unclear. Nonetheless, the assays are commonly used in that may have better diagnostic and prognostic identifying infertile couples as a way of diagnosing male capabilities than standard sperm parameters for both in infertility. In this chapter, we focus on one of the commonly vivo and in vitro fertility. Infertile patients tend to have used assays—TUNEL (terminal deoxynucleotidyl more DNA strand breaks than fertile men (Sakkas et al. transferase dUTP Nick End-Labeling). The chapter begins 1996; Kodama et al. 1997; Zini et al. 2001a,b; Zini et al. with a brief overview of the causes of DNA damage and 2009a,b). This is relevant in assisted reproductive then describes current assay protocols. We also report the techniques (ART), especially in cases where results of studies that have used the TUNEL assay to predict intracytoplasmic sperm injection (ICSI) is used, because the outcomes of assisted reproduction and discuss the the process bypasses natural selection barriers (Zini and limitations of this test. Sigman, 2009b). Reports suggest that markers of sperm DNA integrity may help differentiate fertile from infertile INTRODUCTION men. Therefore, assessing sperm DNA damage prior to Defective sperm function is one of the most common their use in ART is important (Zini and Libman, 2006). causes of male infertility, affecting 1 in 20 men in A number of tests that measure sperm DNA damage developed countries. It is associated with reduced have been developed in the past two decades (Evenson fertility, increased miscarriage rates and increased risk et al. 1999; Donnelly et al. 2000; Sakkas et al. 2000; Sharma of disease in the offspring. (Aitken RJ, 2006). Male factors et al. 2004; Chohan et al. 2006; Erenpreiss et al. 2006; are responsible for infertility in 30–50% of all couples Younglai et al. 2001; Zini and Sigman, 2009b). Each of 466 Manual of Assisted Reproductive Technologies and Clinical Embryology

these tests has its own advantages and disadvantages. DNA damage can occur at a number of different stages This chapter focuses on one of these tests—the terminal of sperm development (e.g. testicular or post-testicular) deoxynucleotidyl transferase dUTP Nick End-Labeling and maybe a result of abortive apoptosis (Sakkas et al. (TUNEL) assay—particularly the role it plays as a test of 1999). Differentiated germ cells cannot be removed by measuring sperm DNA damage, its limitations and the apoptosis. It could also be a result of incomplete joining of results of ART outcome as measured by the TUNEL test the nicks within the DNA at the time of sperm chromatin either by microscopic evaluation or by flow cytometry remodeling. (Domínguez-Fandos et al. 2007). When sperm are transported from the epididymis, protamines are cross-linked by the formation of the ETIOLOGY OF DNA DAMAGE disulfide bridges, resulting in further compaction of the sperm chromatin. This essential mechanism is designed The structure and composition of sperm chromatin is to protect the male genome from insult (Saowaros and different from that of the somatic cells in that the sperm Panyim, 1979). Damage can occur during sperm chromatin is tightly packaged. This enables sperm to be migration through the epididymis as a result of reactive transported expeditiously. This also protects them from oxygen species (ROS)-producing immature sperm, which oxidative stress and other insults (Aitken and De Iulllis, co-migrate through the epididymis or by ROS-producing 2007). Sperm DNA is organized in a specific manner that epithelial epididymal cells during oxidation of thiol keeps the nuclear chromatin compact and stable. This groups and cross-linking of sperm chromatin (Moore, DNA organization not only permits the very tightly 2 1996). n

o packaged genetic information to be transferred to the egg, i

t but also ensures that the DNA is delivered in a physical c

e Extrinsic Factors S and chemical form that allows the developing embryo to easily access the genetic information. Fertile sperms Extrinsic or extratesticular factors that may cause sperm have stable DNA, which is capable of undergoing DNA damage include the following: decondensation at the appropriate time in the fertilization • Drugs, chemotherapy, radiation (Fossa et al. 1997; process and transmitting the DNA without defects. O’Flaherty et al. 2008) Sperm with high levels of DNA strand breaks are more • Cigarette smoking (Potts et al. 1999) often found in infertile men. These sperms fail to • Exposure to pesticides and air pollution (Sanchez- decondense after ICSI, and fertilization is often Pena et al. 2004; Rubes et al. 2005) unsuccessful (Sakkas et al. 1996; Lopes et al. 1998; Host • Genital tract infection and inflammation (Erenpreiss et al. 2000). et al. 2002) • Testicular hyperthermia (Sailer et al. 1997; Banks et Causes of Sperm DNA Damage al. 2005) • Hormonal factors (Xing et al. 2003; Meeker et al. 2008) A number of factors can cause sperm damage including • Varicocele (Zini et al. 2000; Saleh et al. 2003; Fischer intratesticular and extratesticular factors. Intrinsic factors et al. 2003). include defective spermatogenesis and spermiogenesis, and abortive apoptosis. During spermiogenesis, the majority of histones are initially replaced by transition MECHANISMS OF SPERM DNA DAMAGE proteins and then by protamines. These are small and Several theories have been proposed to explain why highly basic proteins that are bound to the sperm DNA DNA damage occurs in human spermatozoa. DNA (Olivia and Dixon, 1991; Ward and Coffey, 1991; Olivia damage is a major contributor to infertility, miscarriage R, 2006; Balhorn R, 2007). This makes the chromatin and birth defects in offspring (Aitken and De Iullis, 2010). highly condensed. Sperm DNA damage has been Studies have suggested that defective chromatin associated with protamine deficiency (Cho et al. 2003; packaging and DNA fragmentation may be induced by: Aoki et al. 2006). Infertile men have an increased sperm • Endogenous endonucleases (topoisomerases) as a histone-to-protamine ratio compared with fertile result of protamine deficiency (Sakkas et al. 1999; controls. Protamine deficiency can result in defective Marcon and Bioissoneault, 2004; Aoki et al. 2006) chromatin compaction (Aravindan et al. 1997) and • Oxidative stress caused by high levels of ROS and increased susceptibility to DNA damage (Aoki et al. low levels of antioxidant protection (Ollero et al. 2001; 2006), suggesting that DNA damage is caused by, at least Aitken and Sawyer, 2003; Saleh et al. 2003; De Iuliis in part, defects in spermiogenesis. et al. 2009) CHAPTER 48  TUNEL Assay for the Assessment of Sperm Chromatin Damage 467

• Abortive apoptosis anti-Br-dUTP-FITC system; FITC labeled • Alterations in topoisomerase II activity (Weng et al. dUTP system (in situ Cell Detection kit, Catalog No. 11 2002). 684 795 910, Roche Diagnostics GmBH, Mannheim, Recent studies show that oxidative stress is a major Germany or Roche Diagnostics, Indianapolis, IN), and contributor to sperm DNA damage (Aitken De Iuliis, Apoptosis detection kit (Apo-Direct kit; Catalog No. 2010). DNA damage may manifest as condensation or 556381; BD Pharmingen, San Diego, CA). nuclear maturity defects, DNA breaks or DNA integrity defects and sperm chromosomal aneuploidy. Measurement of Sperm DNA Damage by Apoptosis Detection Kit MEASURING SPERM DNA DAMAGE We describe the measurement of sperm DNA damage Over the years, several tests have been described that using the apoptosis detection kit which can be run using measure different aspects of DNA damage (Table 48.1). either flow cytometry or fluorescence microscopy These tests are used to diagnose male infertility and predict (Flow chart 48.1). the chances for pregnancy in the context of assisted reproduction. All of these tests have advantages and limitations. One of the most commonly used assays is the Flow chart 48.1: Flow diagram showing the steps involved in the TUNEL assay. The assay causes a reaction based on the measurement of sperm DNA damage by the TUNEL assay S terminal deoxynucleotidyl transferase (TdT), which is e c t specific for apoptotic cells and allows DNA strand breaks i o n

in apoptotic cells to be distinguished from those of necrotic 2 cells (Gorczyca et al. 1993). This single step staining method labels DNA breaks with fluorescein isothiocyanate- deoxyuridine triphosphate (FITC-dUTP) followed by flow cytometric analysis. TdT catalyzes a template-independent addition of bromolated deoxyuridine triphosphatase to the 3’-hydroxyl (OH) termini of double and single stranded DNA. After incorporation, these sites are identified by flow cytometric means by staining the sperm. TUNEL identifies what is termed as “real” or actual DNA damage that is, damage that has already occurred as opposed to “potential” damage caused by exposing sperm to denaturing conditions. Some of the tests in Table 48.1 generally correlate well with one another. For example, the TUNEL assay correlates well with sperm chromatin structure assay (SCSA) (Sailer et al. 1995; Chohan et al. 2006), toluidine blue staining (Erenpreiss et al. 2004), the acridine orange test (Zini et al. 2001a,b) and comet assay (Aravindan et al. 1997; Donnelly et al. 2000). While the SCSA and TUNEL assay predict similar levels of DNA fragmentation, the acridine orange test consistently demonstrates higher levels of DNA fragmentation (Chohan et al. 2006). TUNEL DNA damage is correlated with the DNA damage associated the 8-OHdG type (Agbaje et al. 2008).

Measurement of DNA Damage in Spermatozoa by TUNEL Assay DNA damage can be measured via the TUNEL assay using various protocols, such as: Biotin-dUTP/avidin; BrdUTP/ 468 Manual of Assisted Reproductive Technologies and Clinical Embryology Contd... software s nduce breaks at fragmentation “alkaline-labile” sites 5. Requires imaging Identifies all 7. Alkaline: May identify May identify more lly relevant breaks 8. May i Alkaline: Can be performed on 1. Thresholds not not required 3.parameters Not specific to oxidative commercial kits cells required 4. protocols Lack of standard Neutral: clinica 3. Simple and fast4. High sensitivity5. Indicative of apoptosis6. Correlated with semen required (flow cytometer) damage 7.equipment 4. Special Associated with fertility 8. in Available 4. Small number of5. Can be performed on fertility 2

n o i t c e S cytometry/fluorescence 1. Measured Parameter Detection Method Advantages Disadvantages denatures all DNA Labels SS and DS breaks single sperm cells (tail long tails SS breaks 2. Simple and inexpensive 2. Not specific to oxidative may be some SS breaks DS breaks bind to SS DNAbreaks DNA of Assay Principle 1. Alkaline conditions, few sperm 6. assay protocols Variable 1. Electrophoresis of of sperm with Percentage microscopy Fluorescence 1.sensitivity High Alkaline COMET 1.2.intensive Labor Identifies both DS and breaksunimportant clinically Neutral COMET 1. Does not denature DNA 2. Identifies DS breaks, 1.biotinylated Incorporates of cells with Percentage microscopy Fluorescence 2. Template-dependent Simple3. Labels SS breaks, not 1.DNAnicked Denatures 2. Whole genome probes Amount of fluorescence proportional to numbermicroscopy Fluorescence 1.thresholds Unclear Can perform on few spermanalyzer image and Limited clinical data Table 48.1: Common sperm DNA integrity assays describing their principle, end measurement and advantages disadvantage (Haines et al., 1998; Klaude et al., 1996; Hughes 1996; Singh NP and 2. form tail fragments DNA 1998; Morris Stephens, in tail) length, % of DNA 3. in head stays DNA Intact 3. Correlates with damage seminal parameters 3. No evident correlation in 1993; Sailer et al., 1995;Agarwal and Said, 2004;Henkel et al., 2004; GrecoendsDNA free to 2.independent Template et al., 2005; Sergerie al. 3. 2005a,b; Sharma et al., 2010) DNA labeled microscopyCOMET et al., 2002) few sperms standardized 2.equipment Expensive 2.protocols assay Variable DIRECT ASSAYS Type of Assays TUNEL (Gorczyca W et al., 1. Adds labeled nucleotides of Percentage cells with Flow In situ Nick Translation J et al., 1998; Irvine(Twigg et al., 2000)breaksDNA SS at dUTP dUTP incorporated INDIRECT ASSAYS and image analyzerDNA Break Detection FISH with DNA polymerase I (fluorescent cells) 2. Less sensitive CHAPTER 48  TUNEL Assay for the Assessment of Sperm Chromatin Damage 469 affect results affect heterogeneous staining 4. equipment required Special 5. protocols Lack of standard TUNEL = terminal deoxynucleotidyl TUNEL ormance liquid chromatography; SCD = sperm can use bright-fielddata clinical Limited Correlates with sperm 2. Large amount of sample 5. Associated with fertility 3. Introduction of artifacts S e c t i o n

2 Fluorescence microscopy Easy, Percentage of sperm with sperm DNA in nuclei with chromatin loosely packed and/or defective DNA are more liable to binding with basic dyes. produce halo withDNA denatures with a ratio of red tofluoresces green fluoresces red thousands of cells examined required 1. Basic dye used to Normal sperm not stained.microscopy Optical Easy to measure with sperm chromatinDetects Mature sperm arerobust not method, Subjective content 1. DNA adduct formationmicroscopy Optical adducts are DNA measured HPLC with electrochemical 1. High specificity Easy to measure withrobust not method, Subjective 1. Difficulty with indistinct 3. Normal sperm 1. Mild acid treatmentspermof DFI: Percentage cytometry Flow 3. DS DNA (nondenatured) 4. SS DNA (denatured) 1.rapidlycells Many 5. Flow cytometry counts Same as above, 1.equipment Expensive of cells with Percentage microscopy Fluorescence Simpleindistinct with Difficulty 1. Individual cells immersed (Mello ML, 1982;Erenpreis J, 2004) 2. Phosphate residues of integrity. DNA evaluate blue Damaged sperm stain Aniline Blue (Auger et al., 1990;Dadoune et al., 1988)8-OHdG Analysis of sperm nuclei defects related to the nucleoprotein blue sperm are stained (Fraga et al., 1991; LoftAgbaje et al.,et al., 2003; and immature unstained 2008; Kodama et al., 1997; 2. 8-OHdG is the major due to oxidative stress bright field microscope adduct formed when DNA bright field microscope detection 2. Quantitative 3. 4. sensitivity High colors, rapid fading, heterogeneous staining Toulidine Blue Toulidine De Iuliis et al., 2009) is subjected to attack. function required Acridine Orange Flow Cytometric Assays al.,et (Darzynkiewicz 1975; Evenson et al.,SDFA)1980)(SCSA, SS or DS breaks 2. Acridine orange binds to DNA (red + green) fluorescence greater than the mainAcridine Orange Test Manual cell population (Chohan et al., 2006; et al., 1984)Tejada hand-counting of green and red cells red fluorescence 2. Most published studies 2. Small variations in reproducibleconditions laboratory colors, rapid fading, (Muriel et al., 2006; Fischeret al., 2003; Fernadez in agaroseet al., 2005) 2. Denatured with acid small or absent halolysed then microscopy chromatin dispersion test; SCSA = sperm chromatin structure assay; SDFA = sperm DNA fragmentation assay; SS = single-stranded; fragmentation = sperm DNA = sperm chromatin structure assay; SDFA chromatin dispersion test; SCSA transferase-mediated dUTP nick end-labeling Contd... SCD index; DS = double-stranded; FISH fluorescence in situ hybridization; HPLC High-perf fragmentation Abbreviations: DFI = DNA 470 Manual of Assisted Reproductive Technologies and Clinical Embryology EQUIPMENT AND REAGENTS • Place the cell suspension on ice for 30–60 minutes/ overnight 1. APO-Direct Kit (BD Pharmingen, Catalog # 556381) • Centrifuge to pellet the cells at 300 g for 7 minutes. The assay kit consists of two parts: part A (Component o Discard the supernatant and suspend the pellet in 1 No. 6536AK, which must be stored at 4 C, and part B ml of ice-cold 70% (v/v) ethanol at – 20°C until use. (Component No. 6536BK), which must be stored at Cells can be stored at – 20°C several days before use. –20oC. Component No. 6536AK comprises the following reagents: Staining Protocol • PI (Propidium Iodide)/RNase Staining Buffer • Resuspend the positive (6552LZ) and negative (5 g/ml PI, 200 g/ml RNase) (51-6551AZ) (6553LZ) control cells by swirling the vials. Remove • Reaction buffer (contains cacodylic acid 2 ml aliquots of the control cell suspensions (dimethylarsenic) (51-6549AZ) (approximately 1 x 106 cells/ml) and place in 12 x 75 • Rinsing buffer (contains 0.05% sodium azide) (51- mm centrifuge tubes. Centrifuge the control cell 6550AZ) suspensions for 5 minute at 300 x g and remove the • Wash buffer (contains 0.05% sodium azide) (51- 70% (v/v) ethanol by aspiration, being careful to not 6548AZ) disturb the cell pellet. Component No. 6536BK comprises the following reagents: Note: Use of polystyrene, and not polypropylene tubes, (12 x 75 mm) is recommended to avoid cell build up, 2

• FITC-dUTP (0.25 nmole/reaction; contains 0.05%

n improve staining and/or loss of cells. o

i sodium azide) (51-6555EZ) t

c • Negative control cells (contains 70% vol./vol. • Resuspend each tube of control and sample tubes with e S ethanol) (51-6553LZ) 1.0 ml of Wash Buffer (6548AZ) (blue cap) for each • Positive control (contains 70% vol./vol. ethanol) tube. Centrifuge as before and remove the supernatant by aspiration (51-652LZ) • Repeat the Wash Buffer treatment. • TdT enzyme (10,000 U/mg) 20 g/ml in 50% vol./ vol. glycerol solution) (51-6554EZ) Note: Washing should be carried out by gentle mixing 2. Pipettes and Pipette Tips (1000 l, 100 l and 50 l) and not by pipetting to avoid cell loss. 3. Serological Pipettes (2 ml and 5 ml) • Resuspend each tube of the control cell pellets in 50 l 4. Microcell Counting Chamber (Conception Technolo- of the staining solution (prepared as described below). gies, San Diego, CA) 5. Paraformaldehyde (3.7%) in Phosphate Buffered Staining Solution (Single Assay) Saline (PBS), pH 7.4 • The staining solution consists of the following: reaction 6. Microfuge Eppendorf Tubes buffer (green cap); TdT enzyme (yellow cap); FITC- 7. Ethanol (70%) dUTP (orange cap). The staining solution for a single 8. Flow Cytometer assay is prepared by mixing the staining reagents as follows: Reaction buffer 10 l + TdT enzyme 0.75 l + Sample Preparation FITC-dUTP 8.00 l and distilled water 32.25 l to give a total staining solution volume of 51.00 l. • Following liquefaction, evaluate semen specimens for volume, sperm concentration, total cell count, motility Note: The appropriate volume of staining solution to and morphology prepare for a variable number of assays is based upon multiples of the component volumes needed for one • Aliquot and load a 5 μl aliquot of the sample on a assay. Mix only enough staining solution to complete the Microcell slide chamber for manual evaluation of number of assays prepared per session. The staining concentration and motility. Check the concentration solution is active for approximately 24 hours at 4°C. of sperm in the sample. Adjust it to 2–3 × 106/ml (Flow • Incubate the sperm in the staining solution for chart 48.1) 60 minutes at 37°C • Suspend the cells in 3.7% (w/v) paraformaldehyde • At the end of the incubation time, add 1.0 ml of Rinse prepared in PBS (pH 7.4) Buffer (6550AZ) (red cap) to each tube and centrifuge Note: Fixing in paraformaldehyde is important to avoid each tube at 300 x g for 5 minutes. Remove the loss of smaller fragments of DNA that are not chemically supernatant by aspiration fixed prior to washing the samples. • Repeat the cell rinsing with 1.0 ml of Rinse Buffer, CHAPTER 48  TUNEL Assay for the Assessment of Sperm Chromatin Damage 471

centrifuge and remove the supernatant by aspiration Dickinson, San Jose, CA). The excitation wavelength is • Resuspend the cell pellet in 0.5 mL of the PI/RNase 488 nm supplied by an argon laser at 15 mW. Green Staining Buffer (6551AZ) fluorescence (480–530 nm) is measured in the FL-1 Note: If the cell density is low, decrease the amount of channel and red fluorescence (580–630 nm) in the FL-2 PI/RNase Staining Buffer to 0.3 ml. channel. Spermatozoa obtained in the plots are gated using a forward-angle light scatter and side-angle light • Incubate the cells in the dark for 30 minutes at room scatter dot plot to gate out debris, aggregates and other temperature cells different from spermatozoa. TUNEL positive • Analyze the cells in PI/RNase solution by flow spermatozoa in the population are measured after cytometry converting to a histogram (Figs 48.1A and B). The Note: The cells must be analyzed within 3 hours of percentage of positive cells (TUNEL-positive) are staining. Cells may begin to deteriorate if left overnight calculated on a 1,023 channel scale using the appropriate before analysis. flow cytometer software FlowJo Mac version 8.2.4 In addition to the negative and positive controls (FlowJo, LLC, Ashland, OR) as described by authors provided with the kit, it is also important to include the earlier (Sharma et al. 2010). negative and positive sperm control samples. Fluorescence Microscopy

Negative Control S If the flow cytometry is not accessible, the same stained e c t

In this, the TdT enzyme is omitted from the reaction mixture. cells can be scored with a fluorescence microscope. An i o n

aliquot of the stained sample is loaded on a slide and 2 Positive Control covered with a coverslip. A minimum of 500 spermatozoa per sample are scored under 40X objective of the DNA damage is induced by adding 100 l of DNase I (1 epifluorescence microscope (excitation between 460 nm mg/ml) for 1 hour at 37oC. and 490 nm and an emission > 515 nm). The number of spermatozoa per field stained with PI (red) is counted Flow Cytometry first followed by the number of cells emitting green For flow cytometry evaluation, a minimum of 10,000 events fluorescence (TUNEL positive); the numbers are are examined for each measurement at a flow expressed as a percentage of total count of the sample rate of about 200 events/sec on a flow cytometer (Domínguez-Fandos et al. 2007). (Fluorescence activated cell sorting caliber, Becton and REFERENCE RANGE OF SPERM DNA DAMAGE

Figs 48.1A and B: Representative images of sperm DNA damage as measured by the TUNEL assay. (A) Negative sample; (B) Positive sample showing DNA damage 472 Manual of Assisted Reproductive Technologies and Clinical Embryology

We have recently reported the intra-assay, interassay, pregnancy loss) is more important than reporting the odds interobserver and intraobserver values for the TUNEL ratio (Zini and Sigman, 2009b). assay and reported a cutoff value (19.2%), sensitivity Unfortunately, none of assays for DNA damage can (64.9%) and specificity (100%) for this test (Sharma et al. selectively differentiate clinically important DNA 2010). This cutoff value is similar (20%) to that reported fragmentation from clinically insignificant fragmentation. by Sergerie et al. (2005). The assays also cannot differentiate the DNA nicks that Establishing interobserver and intraobserver as well occur normally (physiological) from pathological nicking. as interassay and intra-assay variations is extremely These assays can only determine the amount of DNA important. We have recently shown that these variations fragmentation that occurs with the assumption that can be minimized to acceptable values (Sharma et al. higher levels of DNA fragmentation are pathological. 2010). It is also important to determine whether the test is to be utilized as a screening/diagnostic test or used to FACTORS AFFECTING THE ASSAY RESULTS predict an established endpoint. Sensitivity is important The methods used to assess DNA damage were originally and must be high for a test to be used in screening or developed and validated for investigation of DNA in diagnostic purposes. Specificity becomes critical if a test somatic cells. The TUNEL assay includes specific is offered as a predictive marker of a defined endpoint detection of free DNA ends (“nicks”) by enzymatic (Sharma et al. 2010). Both sensitivity and specificity are incorporation of marked nucleotides, and whether highly associated with intrinsic performance of the TUNEL assay compacted sperm DNA is accessible without inducing 2

n and are not dependent upon prevalence of the sample.

o DNA damage is unclear. Several factors are important i A cutoff value of 20% DNA fragmentation was able t to consider when performing this test. c

e to differentiate fertile men from infertile men (Sergerie S • Accessibility of the DNA et al. 2005a). At this cutoff, the sensitivity was 96% with • Sperm preparation specificity of 89.4%. A cutoff of 24% was obtained by • Presence of dead cells Lopes et al. (1998) and 25% in normozoospermic infertile • Number of cells examined men by Saleh et al. (2002). It is important that each • Interobserver and intraobserver as well as interassay program establishes its own thresholds by adopting strict and intra-assay variations. quality control standards and implementing observer and assay variations and appropriate assay controls. ASSOCIATION OF SPERM DNA DAMAGE WITH The negative predictive value (NPV) and positive predictive value (PPV) depend on the prevalence of SEMEN PARAMETERS AND ART OUTCOME infertility in the tested population. Thus, they will be A large number of studies have described an association different in populations where the percentage of fertile between DNA damage (as measured by various tests) subjects may be higher. The high PPV reported in our and semen parameters and ART outcome (Morris et al. study (Sharma et al. 2010) suggests that sperm DNA 2002; Henkel et al. 2003; Gandini et al. 2004; Zini et al. fragmentation measured by the TUNEL assay is a good 2005; Boe-Hanson et al. 2006; Borini et al. 2006; Benchaib predictive parameter to identify an infertile population. et al. 2007; Bungum et al. 2007; Evenson and Wixon, 2008; The PPV and NPV are strongly associated with the Lin et al. 2008; Zini and Sigman, 2009a,b) that are beyond prevalence of the disease (Sergerie et al. 2005a). the scope of this chapter. Accuracy of the TUNEL test is The goal of a good sperm DNA test is to identify men not affected by the type of treatment [in vitro fertilization with male factor infertility (i.e. those with a condition (IVF) or ICSI] but by the type of assay (TUNEL versus that is contributing to the infertility problem of the SCSA) (Zini and Sigman, 2009b). In the following couple) rather than to classify them as possibly fertile or sections, we have discussed the association of sperm subfertile. The predictive value of assessing sperm DNA DNA damage as assessed by the TUNEL test and its damage can vary depending on the sperm DNA test and relation with the findings in the context of ART. the cutoff level that is used. This is reflected in various studies that have used various cutoff levels in predicting Association of DNA Damage with pregnancy loss using the SCSA or TUNEL assay and the Sperm Parameters odds ratio in predicting pregnancy loss (Evenson et al. 1999; Duran et al. 2002; Henkel et al. 2003, 2004; Sergerie Standard semen parameters, such as sperm concentration, et al. 2005a). Reporting positive and negative predictive motility and morphology, are negatively correlated with levels of sperm DNA fragmentation whether measured by values along with the prevalence of the disease (pregnancy, COMET (Irvine et al. 2000) TUNEL (Bench et al.; 1996; CHAPTER 48  TUNEL Assay for the Assessment of Sperm Chromatin Damage 473

Zini et al. 2001a,b) or SCSA (Chohan et al. 2006). Although by the 80% Percoll density centrifugation was the degree of correlation is different, they consistently show negatively related to the sensitivity by TUNEL assay that sperm from patients with abnormal sperm (Chohan et al. 2006; De Iullis GN et al. 2009) concentration, motility and morphology have increased • DNA damage was associated with a lower percentage levels of DNA damage. Unlike semen parameters, which of apoptosis in normozoospermic samples and are highly variable over time within individuals, measures the percentage of atypical sperm head forms of sperm DNA damage have been shown to be stabile over (p < 0.0006) (Gandini et al. 2000) time within an individual with the TUNEL assay (Sergerie • Paternal smoking increases sperm DNA damage as et al. 2005a,b). measured by TUNEL technique and reported to Cohn-Bacrie et al. (2009) conducted a prospective increase childhood cancer (Sun et al. 1997; Potts et al. study in more than 1,600 couples to determine the 1999). correlations between semen parameters including those • Sperm with low motility carry higher loads of DNA assessed using computer-aided sperm analysis and the damage as shown by TUNEL and comet assays fragmentation rates as determined by TUNEL (Irvine et al. 2000) (Table 48.2). In their study, they found that one-third of • A highly significant negative correlation between their patients had TUNEL rate of more than 30%. DNA sperm DNA fragmentation and concentration, total damage was associated with the following parameters: sperm count, and morphology before and after

• Patient age preparation of sperm on density gradient (Borini et al. S e c

• Length of abstinence (positive correlation; p < 0.006) 2006) t i

• Total motile sperm count (inverse correlation; p < 0.0001) o n

• Rapid progression (inverse correlation; p < 0.0001) Lack of Association between the TUNEL Assay 2 • Vitality (negative correlation; p < 0.0001) • Percentage of atypical forms (positive correlation; and the Following Variables p < 0.0006) No correlation was reported (Cohen-Bacrie et al. 2009) • Abnormal necks (positive correlation; p < 0.016) between sperm DNA damage and following parameters. • Coiled tails (positive correlation; p < 0.0001) • Number of motile and atypical spermatozoa per Others have reported an association of DNA damage ejaculate as measured by TUNEL to be associated with a number • Progressive motility after one hour and amplitude of of semen parameters. lateral head displacement • High nuclear density chromatin from sperm selected • Number of spermatozoa with abnormal basal piece

Table 48.2: Selected diagnostic properties of studies on sperm DNA damage and pregnancy after IVF and ICSI using TUNEL (Zini and Sigman, 2008)

Study n % DNA Sensitivity Specificity PPV NPV OR 95% CI Damage IVF Host et al., 2000 175 30 0.34 0.79 0.77 0.37 1.92 0.92, 4.04 Henkel et al., 2003 208 69 0.35 0.81 0.81 0.35 2.24 1.09, 4.58 Huang et al., 2005 217 19 0.22 0.83 0.50 0.57 1.30 0.66, 2.56 Borini et al., 2006 82 16 0.17 0.89 0.85 0.23 1.66 0.33, 8.28 Benchaib et al., 2007 84 10 0.07 0.86 0.50 0.32 0.46 0.11, 2.00 Frydman et al., 2008 117 44 0.58 0.68 0.64 0.35 2.97 1.39, 6.32

ICSI Host et al., 2000 61 59 0.57 0.38 0.58 0.36 0.79 0.28, 2.25 Henkel et al., 2003 54 48 0.68 0.63 0.79 0.50 3.67 1.12, 12.0 Huang et al., 2005 86 57 0.64 0.50 0.55 0.60 1.80 0.76, 4.27 Borini et al., 2006 50 60 0.71 0.75 0.90 0.45 7.36 1.67, 32.4 Benchaib et al., 2007 218 17 0.19 0.87 0.72 0.37 1.55 0.70, 3.41 Abbreviations: CI = confidence interval; ICSI = intracytoplasmic sperm injection; IVF = in vitro fertilization; NPV = negative predictive value; PPV = positive predictive value; OR = odds ratio; TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling 474 Manual of Assisted Reproductive Technologies and Clinical Embryology or circular flagella Discriminating Power of the TUNEL Assay • Semen volume and sperm concentration Natural or Intrauterine Insemination • Semen pH • Morphology of head, acrosome and intermediate In the context of natural conception, a higher percentage piece of DNA fragmentation was seen as detected by TUNEL • Variation of sperm agglutination factors (microscopic evaluation) in infertile patients than in • Presence of leukocytes and polynuclear cells in the normozoospermic controls (Gandini et al. 2004). ejaculated sperm Similarly, none of the semen samples with more than • Patient’s age 12% sperm DNA fragmentation initiated a pregnancy following intrauterine insemination (Duran et al. 2002). Association of TUNEL Test with ART Variables The discriminating value of the TUNEL test in intrauterine insemination cycles was seen where a limited • DNA damage measured by TUNEL assay was number of oocytes (one or two) were available compared negatively related to both fertilization and embryo with IVF (Duran et al. 2002). cleavage rate after IVF (Sun et al. 1997) • A negative correlation between DNA damage was IVF and/or ICSI seen with both fertilization and embryo cleavage rate after IVF, suggesting that sperm with DNA damage The studies that have assessed the association between will fertilize less efficiently and impair embryonic DNA damage and ART outcome have produced mixed 2

n results (Tables 48.3 and 48.4).

o development (Sun et al. 1997; Borini et al. 2006) i

t • Sperm from infertile men with higher percentage of • Discordant views are reported in literature c

e concerning the correlation of the TUNEL assay with S DNA fragmentation failed to decondense after ICSI and fertilize (Sakkas et al. 1996; Lopes et al. 1998; Host DNA damage, particularly in an IVF setting (Sun et et al. 2000) al. 1997; Lopes et al. 1998; Host et al. 2000) • In groups with low (< 10%) or high (> 10%) DNA • In ART, the pregnancy rate was lower in IVF patients damage, a highly significant correlation was observed when the TUNEL positive sperm test was above the following ICSI between the clinical pregnancy rate cutoff value of 36.5% [calculated from a receiver (45% vs. 10%) and the pregnancy losses rate (no operating characteristic curve (ROC) curve] than biochemical pregnancies or miscarriages, 0% vs. when at 35% or below the cutoff value (Henkel et al. 625%) (Borini et al. 2006) 2004) • Patients with female partners who experienced • In one study, there was no correlation of TUNEL assay recurrent pregnancy loss showed a significant increase (high or lower degree of fragmentation) with IVF in sperm DNA fragmentation (Carrell et al. 2003). outcome in terms of pregnancy (Benchaib et al. 2003) • In a meta-analysis, no correlation was seen between Lack of Association of TUNEL Assay with sperm DNA damage and the fertilization rate during IVF or ICSI using TUNEL assay (Li et al. 2006). This ART Variables is expected since the embryonic genome is not • After IVF, no correlation was seen between the clinical expressed until the four-cell stage to eight-cell stage, pregnancy rate and pregnancy loss rate in groups and this fertilization may not be dependent on the with less than 10% or more than 10% DNA damage sperm DNA integrity (Braude et al. 1988) (Borini et al. 2006). Similarly, no correlation was seen • The DNA fragmentation rate was significantly lower between sperm DNA fragmentation and the in ICSI when pregnancy was obtained. No fertilization rate in ICSI (Borini et al. 2006) pregnancies were reported when the DNA • Sperm concentration, progressive motility and fragmentation was more than 20% (Benchaib et al. normal sperm morphology did not influence the 2003) clinical pregnancy rate (number of patients with fetal • No significant difference was seen in pregnancy rates heartbeat divided by the number of treatments) and after IVF or ICSI between patients with high DNA pregnancy loss rate (number of biochemical levels and low DNA levels of DNA fragmentation pregnancies and spontaneous miscarriages divided using a TUNEL cutoff value of 15% (Benchaib et al. by the number of -human chorionic gonadotropin 2007) positive patients) following IVF or ICSI (Borini et al. • When using the TUNEL assay, clinical pregnancy 2006) rates following IVF but not ICSI decreased significantly for patients with a high degree of sperm CHAPTER 48  TUNEL Assay for the Assessment of Sperm Chromatin Damage 475

Table 48.3: Selected diagnostic properties of studies on sperm DNA damage and pregnancy loss in IVF and ICSI using TUNEL (Zini and Sigman, 2008)

Study n Pregnancy Abnormal Sensitivity Specificity PPV NPV Odds Ratio Loss (%) Test (test %) (95% CI) IVF Borini et al., 2006 82 6 11 0.91 0.94 0.50 0.99 60 (0.18–144708) Benchaib et al., 2007 84 15 15 0.50 0.91 0.50 0.91 10.0 (0.87–114.8) Frydman et al. 2008 117 19 32 0.64 0.75 0.37 0.90 5.25 (1.31–21.11)

ICSI Borini et al., 2006 50 25 25 0.97 0.99 0.97 0.99 2700 (0.38–2 x 107) Benchaib et al., 2007 218 12 15 0.38 0.88 0.30 0.91 4.54 (0.89–23.38) Abbreviations: ICSI = intracytoplasmic sperm injection; NPV = negative predictive value; PPV = positive predictive value; OR = odds ratio

remains to be firmly established. Further studies utilizing Table 48.4: Distribution of sperm DNA fragmentation by TUNEL in S e

standardized protocols and established thresholds for c

1,633 ejaculates (Cohen-Bacrie et al., 2009) t i

sperm DNA damage are needed in order to make the o Fragmentation (%) No. of Patients (%) n

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