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Laboratory Evaluation of Sperm Chromatin: TUNEL Assay

Article · January 2014 DOI: 10.1007/978-1-4419-6857-9_14

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Rakesh K Sharma Ashok Agarwal Cleveland Clinic Cleveland Clinic

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Rakesh Sharma and Ashok Agarwal

Abstract Routine semen analysis is unable to assess alterations in sperm chromatin organization such as DNA damage. Because fertility is based not only on the absolute number of spermatozoa but also on their functional capability, methods for exploring sperm DNA stability and integrity are being used to evaluate fertility disorders. A large number of direct and indirect tests that measure sperm DNA damage have been developed. This chapter focuses on one of those tests – the terminal deoxytransferase mediated deoxyuri- dine triphosphate (dUTP) nick end-labeling or TUNEL assay – which is increasingly being used in many laboratories.

Keywords 3PERMCHROMATINs45.%,ASSAYs3EMENANALYSISs,ABORATORYEVALUATION OFSPERMCHROMATINs4ERMINALDEOXYTRANSFERASEMEDIATEDDEOXYURIDINE triphosphate assay

One of the possible causes of infertility in men terminal deoxytransferase mediated deoxyuri- with normal semen parameters is abnormal sperm dine triphosphate (dUTP) nick end-labeling DNA. Fortunately, a number of sperm function assay, which is otherwise called TUNEL. This tests are available to assess sperm DNA integrity. test identifies in situ DNA strand breaks resulting One of the most commonly used tests is the from apoptotic signaling cascades by labeling the 3c-hydroxyl (3c-OH) free ends with a fluorescent label. The fluorescence, which is proportional to the number of strand breaks, can be measured either with microscopy or with flow cytometry. R. Sharma ( ) This chapter discusses the TUNEL assay in Andrology Laboratory and Center for Reproductive detail, including clinical protocols, clinical out- Medicine, Glickman Urological and Kidney Institute, comes, and future strategies aimed at optimizing OB-GYN and Women’s Health Institute, Cleveland Clinic, Cleveland, OH, USA this test and increasing its application as the test e-mail: [email protected] of choice in clinical andrology.

A. Zini and A. Agarwal (eds.), Sperm Chromatin: Biological and Clinical Applications in Male 201 Infertility and Assisted Reproduction, DOI 10.1007/978-1-4419-6857-9_14, © Springer Science+Business Media, LLC 2011 202 R. Sharma and A. Agarwal

Mechanisms of Sperm DNA Damage the 3c-OH end, and the fluorescence is propor- tional to the number of DNA strand breaks When sperm DNA is damaged, infertility, mis- (Fig. 14.1). This assay can be run either as a slide- carriage, and birth defects in offspring can occur based (fluorescent microscopy) (Fig. 14.2) or [1]. The main cause of sperm DNA damage is flow-cytometry assay [16] (Fig. 14.3, Table 14.3). oxidative stress [2–5]. Oxidative stress occurs TUNEL identifies what is termed as “real” or when levels of reactive oxygen species (ROS) actual DNA damage – that is, damage that has increase, when levels of antioxidant decrease, or already occurred – as opposed to “potential” both. A number of factors can lead to oxidative damage caused by exposing sperm to denaturing stress, including infection (viral or bacterial), conditions (Table 14.4). exposure to xenobiotics, and tobacco and alcohol All of the assays shown in Table 14.1 have a consumption. strong correlation with one another. Unfortunately, DNA fragmentation may also occur during sper- none of them are able to selectively differentiate miogenesis. During this process, torsional stress clinically important DNA fragmentation from increases, as DNA is condensed and packaged into clinically insignificant fragmentation. The assays the differentiating sperm head. Endogenous endo- also cannot differentiate the DNA nicks that nucleases (topoisomerases) may induce DNA frag- occur normally (physiological) from pathologi- mentation as a way of relieving this stress [6, 7]. cal nicking, nor can they evaluate the genes that Spermatozoa are transcriptionally and transla- may be affected by DNA fragmentation. These tionally inactive and cannot undergo conventional assays, including TUNEL, can only determine programmed cell death or “regulated cell death” the amount of DNA fragmentation that occurs called “apoptosis” but are capable of exhibiting with the assumption that higher levels of DNA some of the hallmarks of apoptosis including cas- fragmentation are pathological. pase activation and phosphatidylserine exposure on the surface of the sperm. This form of apopto- sis is termed as “abortive apoptosis” [8, 9]. Sperm Measurement of DNA Damage cells are able to repair some DNA damage during in Spermatozoa by TUNEL Assay spermatogenesis, but once they mature, they lose this innate ability [10, 11]. Therefore, posttestic- DNA damage can be measured using the ular sperm are more vulnerable to DNA damage. TUNEL assay by various protocols such as the Studies show that DNA damage is lowest in tes- following: ticular sperm and that it increases in epididymal 1. Biotin-d(UTP)/avidin system. and ejaculated sperm [12–15]. 2. BrdUTP/anti-Br-dUTP-FITC system. 3. Fluorescein isothiocynate labeled (FITC) dUTP system (In Situ Cell Detection kit, Measuring Sperm DNA Damage Catalog No. 11 684 795 910, Roche with TUNEL Diagnostics GmBH, Mannheim, Germany or Roche Diagnostics, Indianapolis, IN). Sperm DNA damage can be assessed with a num- 4. Apoptosis detection kit (Apo-Direct kit; ber of techniques that measure different aspects Catalog No. 556381; BD Pharmingen, San of DNA damage (Table 14.1). Each assay has its Diego, CA). own advantages and disadvantages (Table 14.2). We describe protocol #3 and #4 because they One of the most commonly used assays is the are commonly used tests for measuring sperm TUNEL assay. The quantity of DNA 3c-OH free DNA damage in sperm. The detection of sperm ends can be assessed in spermatozoa using this DNA fragmentation by flow cytometry and epif- assay in which the terminal deoxytransferease luorescence microscopy methods will also be (TdT) enzyme incorporates a fluorescent UTP at described. 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 203

Table 14.1 Basics of common sperm DNA integrity assays Basis of assay Measured parameter Direct assays TUNEL Adds labeled nucleotides to free % Cells with labeled DNA DNA ends Template independent Labels SS and DS breaks Comet Electrophoresis of single sperm cells % Sperm with long tails (tail length, DNA fragments form tail % of DNA in tail) Intact DNA stays in head Alkaline Comet Alkaline conditions, denatures all DNA Identifies both DS and SS breaks Neutral Comet Does not denature DNA Identifies DS breaks, maybe some SS breaks In situ nick translation Incorporates biotinylated dUTP at SS % Cells with incorporated dUTP DNA breaks with DNA polymerase I (fluorescent cells) Template-dependent Labels SS breaks, not DS breaks Indirect assays DNA break detection FISH Denatures nicked DNA Amount of fluorescence propor- Whole genome probes bind to tional to number of DNA breaks SS DNA SCD Individual cells immersed in garose % Sperm with small or absent halos Denatured with acid then lysed Normal sperm produce halo Acridine orange flow cytometric Mild acid treatment DFI – the percentage of sperm with assays (e.g., SCSA, SDFA) denatures DNA with SS or DS breaks a ratio of red to (red + green) Acridine orange binds to DNA fluorescence greater than the DS DNA (nondenatured) main cell population fluoresces green SS DNA (denatured) fluoresces red Flow cytometry counts thousands of cells Acridine orange test Same as above, hand-counting % Cells with red fluorescence of green and red cells DFI DNA fragmentation index; DS double-stranded; FISH fluorescence in situ hybridization; SCD sperm chromatin dispersion test; SCSA sperm chromatin structure assay; SDFA sperm DNA fragmentation assay; SS single-stranded; TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling From Zini and Sigman [17], with permission

First, the semen specimen is collected: (b) The sample should be collected in private in (a) Ideally, the sample should be collected after a room near the laboratory. If not, it should a minimum of 48 h and not more than 72 h be delivered to the laboratory within 1 h of of sexual abstinence. The name of the collection. patient, period of abstinence, date, and time (c) The sample should be obtained by masturba- and place of collection should be recorded tion and ejaculated into a clean, wide-mouth on the form accompanying each semen plastic specimen cup. Lubricants should not analysis. be used to facilitate semen collection. 204 R. Sharma and A. Agarwal

Table 14.2 Advantages and disadvantages of various DNA integrity assays Direct assays Pros Cons TUNEL Can perform on few sperms Thresholds not standardized Expensive equipment not required Variable assay protocols Simple and fast Not specific to oxidative damage High sensitivity Special equipment required Indicative of apoptosis (flow cytometer) Correlated with semen parameters Associated with fertility Available in commercial kits COMET High Sensitivity Labor intensive Simple and inexpensive Not specific to oxidative damage Correlates with seminal parameters Subjectiveness in data acquired Small number of cells required No evident correlation in fertility Can perform on few sperm Lack of standard protocols Alkaline: identifies all breaks Requires imaging software Neutral: may identify more Variable assay protocols clinically relevant breaks Alkaline: may identify clinically unimportant fragmentation May induce breaks at “alkaline-labile” sites In situ nick translation Simple Unclear thresholds Indirect assays Less sensitive DNA break detection FISH Can perform on few sperm Limited clinical data SCD Easy, can use bright-field microscopy Limited clinical data Acridine orange flow Many cells rapidly examined Expensive equipment required cytometric assays Most published studies Small variations in lab conditions affect reproducible results Calculations involve qualitative decisions Manual acridine orange test Simple Difficulty with indistinct colors, rapid fading, heterogeneous staining 8-OHdg analysis High specificity Large amount of sample required Quantitative Introduction of artifacts High sensitivity Special equipment required Correlated with sperm function Lack of standard protocols Associated with fertility FISH fluorescence in situ hybridization; SCD sperm chromatin dispersion test; TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling From Zini and Sigman [17], with permission

(d) Coitus interruptus is not acceptable as a (f) Any unusual collection or condition of the means of collection because it is possible that specimen should be noted on the report the first portion of the ejaculate, which usu- form. ally contains the highest concentration of spermatozoa, will be lost. Moreover, cellular and bacteriological contamination of the Protocol #3: In Situ Death Detection kit sample and the acid pH of the vaginal fluid (Roche Diagnostics, Indianapolis, IN) adversely affect sperm quality. (e) The sample should be protected from extreme Reagents and Equipment temperatures (not less than 20°C and not (a) Flow cytometry tubes (12 × 75 mm) more than 40°C) during transport to the (b) Pipettes and pipette tips (1,000, 100, and laboratory. 50 PL) 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 205

Fig. 14.1 Schematic of the TUNEL assay

Fig. 14.2 Fluorescence microscopic staining with Fig. 14.3 Representative histogram showing (a) TUNEL TUNEL and propidium iodide. TUNEL-positive sperm negative and (b) TUNEL positive sample stain green and TUNEL-negative samples stain red

(c) Serological pipettes (2 and 5 mL) 2. Violet vial (Label solution): It consists of a (d) Sperm counting chamber (MicroCell; nucleotide mixture in a reaction buffer of ×1 Concep tion Technologies, San Diego, CA) concentration and has 5 × 550 PL aliquots. (e) Paraformaldehyde in phosphate-buffered 3. Benchtop centrifuge. saline, pH 7.4 4. Flow cytometer. (f) Ethanol (70%) 5. Phase and epifluorescence microscope.

In Situ Death Detection Kit Assay Principle 1. Blue vial/cap (Enzyme solution): It contains The cleavage of genomic DNA during apoptosis the terminal deoxynucleotidyl transferase leads to both single-strand breaks (nicks) and (TdT) enzyme solution. It is ×10 concentra- double-stranded, low-molecular-weight DNA tion and contains 5 × 50 PL aliquots. fragments. These DNA strand breaks can be 206 R. Sharma and A. Agarwal

Table 14.3 TUNEL positivity using flow cytometry or fluorescence microscopy technique Sample TUNEL positive (%) Reference size Flow cytometry Microscopy Muratori et al. [18] 140 11.07 ± 8.00 – (0.79–42.64) Muratori et al. [19]4315 Lopes et al. [20] 150 – 14.5 ± 1.5 (0.5–75) (swim up) Barroso et al. [21] 10 – 11.7 ± 7 (low motility sample) Donnelly et al. [22] 25 – 35 (15–60); 18 (7–45) (gradient) Gandini et al. [23] 52 – 11 (infertile) and ~2.5 (fertile) Oosterhuis et al. [24] 34 20 ± 15 (1.3–64) – Sergerie et al. [25] 97 15% controls – Ramos and Wetzels [26] 11 – 10.0 (controls) Zini et al. [27] 40 – 25.4 (infertile) and 10.2 (fertile) Duran et al. [28] 119 – 7.3 ± 3.5 (pregnancy) vs. 13.9 ± 10.8 (no pregnancy) (gradient separation) Sakkas et al. [29] 68 20.7(1.0–71.7) – Shen et al. [30] 60 ~15 – Weng et al. [9] 34 – 10 (patient) and 7 (control) with high motility vs. 33 (patient) and ~25 (control) in low motility samples Benchaib et al. [31] 108 – 12–15 (abnormal samples) and 6–7 (normal after gradient separation) Carrell et al. [32] 21 – ~38.4 (miscarriages) Erenpreisa et al. [33] 6 – 10–40 (range) (methanol:ethanol fixed) Erenpreiss et al. [34] 18 10.5 (4–27) (frozen) Lachaud et al. [35] 7 – 12.5 ± 2.2 (0 h; washed); 7.6 ± 1.1 (0 h; gradient); 1.7 ± 0.8 (0 h; swim up) Tesarik et al. [36] 18 – 8.9 ± 3.7 (patient) and 8.7 ± 3.6 (control) Greco et al. [37] 18 – 23.6 ± 5.1 (in ejaculates) vs. 4.8 ± 3.6 (testicular sperm) Sergerie et al. [38] 73 40.6 (patients) vs. 13.0 – (controls) Sergerie et al. [38] 113 40.9 ± 14.3 (patients) – and 13.1 ± 7.3 (controls) Sergerie et al. [38] 15 22.44 ± 29.48 (patients) – and 13.1 ± 17.56 (controls) Stahl et al. [39] 24 11 (2.5–31) (control) – Sepaniak et al. [40] 108 – 25.9 (nonsmokers) and 32 (smokers) Chohan et al. [41] 67 – 19.5 ± 1.3 (infertile) and 11.1 ± 0.9 (fertile) De Paula et al. [42] 77 – 8.6 ± 3.6 (patient) and 5.4 ± 2.7 (control) Aoki et al. [43] 79 40.8 ± 4.9 (low P1/P2) and 21.6 ± 1.7 (normal P1/ P2) and 28.3 ± 3.1 (high P1/P2) Spermon et al. [44] 22 – 21.0 (8.0–66.0) (pretreatment) and 25.0 (10–47) (posttreatment) Dominguez et al. [45] 66 39.7 ± 23.1 15.3 ± 10.3 Sakamoto et al. [46] 15 – 79.6 ± 13.6 prevaricocele and 27.5 ± 19.4 (postvaricocele) P1 protamine-1; P2 protamine-2 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 207 breaks 1. Electrophoresis of single sperm cells fragments form tail 2. DNA 3. stays in head Intact DNA Alkaline Comet 1. Alkaline conditions, denatures all DNA 2. Identifies both DS and SS breaks Neutral Comet 1. Does not denature DNA 2. Identifies DS breaks, maybe some SS % sperm with long tails (tail length, of in tail) DNA Direct Subjective not Clinically useful reference values established SS or DS breaks fluoresces green DFI – the percentage of sperm with a ratio red to green) fluorescence greater than the main cell + (red population Indirect Objective pregnancies 1. Mild acid treatment denatures DNA with 1. Mild acid treatment denatures DNA 2. Acridine orange binds to DNA (nondenatured) 3. Double-stranded DNA (denatured) fluoresces red Single-stranded DNA 4. counts thousands of cells cytometry Flow free breaks independent nucleotides to ends DNA % Cells with labeled DNA Objective Good Good Poor TUNELlabeled Adds 1. 2. Template 3. Labels SS and DS SCSA Comet damage is the method of choice? Why TUNEL methods for DNA Cost Inexpensive Expensive Inexpensive What is measured of assayType Ease of assayInstrumentationNature of assay Direct Reference alues labs run this assay Many cytometry Flow of samplesType TUNEL kit available Repeatability of Ranges from 10 to 30%assay to be shipped reference Lab Samples have Fresh or frozen Only in reference or designated labs of decreased >30% DFI indicative Robust, cytometry Flow labs perform this assay few Very Fresh or frozen Manual, no assay kits available Microscopy Fresh Principal Table 14.4 Table 208 R. Sharma and A. Agarwal identified by labeling the free 3c-OH termini with Positive control: Incubate fixed and permea- modified nucleotides in an enzymatic reaction bilized cells with DNase I (3–3,000 U/mL in (Fig. 14.1). 50 mM Tris-HCl, pH 7.5, 1 mg/mL BSA) for This occurs in two stages: (1) Labeling of 10 min at 25°C to induce DNA damage. DNA strand breaks with TdT, which catalyzes (g) Resuspend the pellet in 50 PL of the staining the polymerization of labeled nucleotides to free solution for 1 h at 37°C in the dark and mix 3c-OH DNA ends in a template-independent man- them. ner (TUNEL reaction) and (2) Fluorescein iso- (h) After staining, rinse twice in PBS/BSA (1%) thiocynate (FITC)-dUTP is incorporated into and resuspend in 200–500 PL PBS/BSA nucleotide polymers, and it can be directly (1%). detected and quantified by fluorescence micros- (i) The samples can be directly analyzed under a copy or flow cytometry. fluorescence microscope or by flow This kit is designed to be a precise, fast, and cytometry. simple nonradioactive technique to detect and Note: The kit is stable at −15 to −25°C. quantify the number apoptotic cells. It is specific Note: The enzyme solution (TdT) must be kept as it labels DNA strand breaks generated during on ice and should be discarded after use. apoptosis, which enables the test to discriminate Note: The samples can be counterstained with between apoptotic and necrotic cells. 0.5 Pg/mL of propidium iodide to provide background DNA staining. Sample Preparation (a) Wash the semen aliquot containing 2 × 106 spermatozoa by centrifuging at 800 g at room Protocol #4: APO-DIRECT™ kit temperature for 5 min with phosphate-buff- (BD Pharmingen, Catalog # 556381) ered saline. (b) After removing the seminal plasma, wash the Principal pellet twice in PBS with 1% bovine serum Fragmented DNA can be detected with a reaction albumin (BSA). catalyzed by exogenous TdT and refereed as end (c) Suspend the pellet in 100 PL of PBS/BSA labeling. The assay kit consists of two parts: Part (1%) and fix in 100 PL of 4% paraformalde- A (Component No. 6536AK) that must be stored hyde in PBS (pH 7.4) for 1 h at room tem- at 4°C and part B (Component No. 6536BK) that perature by vortexing. must be stored at −20°C (Table 14.5). (d) Resuspend the pellet in 100 PL of PBS and 1. Sample preparation permeabilize with 100 PL of 0.1% Triton–X (a) Following liquefaction, load a 5-PL 100 in 0.1% sodium citrate in PBS for 2 min aliquot of the sample on a Microcell slide in ice. Repeat two washes in PBS/BSA (1%). chamber for manual evaluation of concen- (e) Preparation of the staining solution tration and motility. Check the concentra- One pair of tubes (vial 1: Enzyme solution, tion of sperm in the sample. Adjust it to (50 PL) and vial 2: Label solution (550 PL)) 2–3 × 106/mL. is sufficient for staining ten samples. The (b) Suspend the cells in 3.7% (w/v) para- TUNEL reaction mixture is prepared by add- formaldehyde prepared in PBS (pH 7.4). ing 50 PL of enzyme solution to 450 PL of (c) Place the cell suspension on ice for label solution to give a total volume of 30–60 min. 500 PL. (d) Centrifuge to pellet the cells at 300 g for (f) Preparation of negative and positive 7 min. Discard the supernatant and sus- controls pend the pellet in 1 mL of ice-cold 70% Negative control: Incubate fixed and permea- (v/v) ethanol at −20°C until use. Cells bilized cells with 50 PL of label solution can be stored at −20°C several days (without TdT). before use. 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 209

Table 14.5 Components of the Apo-direct kit Component No. Size (mL) Description Color code 51-6551AZa 25 PI/RNase staining buffer (5 Pg/mL PI, 200 Pg/ Amber bottle mL RNase) 51-6549AZa 0.50 Reaction buffer (contains cacodylate acid) Green cap (dimethylarsenic) 51-6550AZa 100 Rinsing buffer (contains 0.05% sodium azide) Red cap 51-6548AZa 100 Wash buffer (contains 0.05% sodium azide) Blue cap 51-6555EZb 0.40 FITC-dUTP (0.25 nmol/reaction; contains Orange cap 0.05% sodium azide) 51-6553LZb 5 Negative control cells (contains 70% vol./vol. Clear cap ethanol) 51-6552LZb 5 Positive control (contains 70% vol./vol. ethanol) Brown cap 51-6554EZb 0.038 TdT enzyme (10,000 U/mg) (20 Pg/mL in 50% Yellow cap vol./vol. glycerol solution) aComponent No. 6536AK to be stored at 4°C bComponent No. 6536BK to be stored at −20°C

Table 14.6 Preparation of staining solution for the TUNEL test Staining solution 1 assay (PL) 6 assays (PL) 12 assays (PL) Reaction buffer (green cap) 10.00 60.00 120.00 TdT enzyme (yellow cap) 0.75 4.50 9.00 FITC-dUTP (orange cap) 8.00 48.00 96.00

Distilled H2O 32.25 193.00 387.00 Total volume 51.00 306.00 612.00

2. Staining Protocol 3. Staining Solution (single assay) (a) Resuspend the positive (6552LZ) and (a) Prepare the staining solution by mixing negative (6553LZ) control cells supplied the appropriate amounts of the staining in the kit by swirling the vials. Remove reagents (Table 14.6). 2-mL aliquots of the control cell suspen- (b) Incubate the sperm in the Staining sions (approximately 1 × 106 cells/mL) Solution for 60 min at 37°C. and place in 12 × 75 mm centrifuge tubes. (c) At the end of the incubation time, add Centrifuge the control cell suspensions 1.0 mL of Rinse Buffer (6550AZ) (Red for 5 min at 300 × g and remove the 70% cap) to each tube and centrifuge each tube (v/v) ethanol by aspiration, being careful at 300 × g for 5 min. Remove the superna- to not disturb the cell pellet. tant by aspiration. (b) Resuspend each tube of control and sam- (d) Repeat rinsing with 1.0 mL of Rinse ple tubes with 1.0 mL of Wash Buffer Buffer, centrifuge, and then remove the (6548AZ) (Blue cap) for each tube. supernatant by aspiration. Centrifuge as before and remove the (e) Resuspend the cell pellet in 0.5 mL of the supernatant by aspiration. PI/RNase Staining Buffer (6551AZ). (c) Repeat the Wash Buffer treatment. (f) Incubate the cells in the dark for 30 min at (d) Resuspend each tube of the control cell room temperature. pellets in 50 PL of the Staining Solution (g) Analyze the cells in PI/RNase solution by (prepared as described below). flow cytometry. 210 R. Sharma and A. Agarwal

In addition to the negative and positive Fluorescence Microscopy controls provided with the kit, it is also The sperm suspension is counterstained with 4,6 important to include the negative and diamidoino-2-phenylindole (DAPI), 2 Pg/mL in positive sperm control samples. vecta shield (Vector, Burlingame, CA) or propid- s Negative control: In this the TdT enzyme ium iodide (5 PL). A minimum of 500 spermato- is omitted from the reaction mixture. zoa per sample are scored under 40× objective of s Positive control: DNA damage is the epifluorescence microscope. For the green induced by adding 100 PL of DNase I signal (FITC), an excitation wavelength in the (1 mg/mL) for 1 h at 37°C. range of 450–500 nm (e.g., 488 nm) and detec- Note: The volume of staining solution needed tion in the range of 525–565 nm are adequate can be adjusted based on the number of (green). The number of spermatozoa per field tubes prepared and multiplying with the stained with DAPI (blue) or PI (red) is first component volumes needed for one assay. counted and then the number of cells emitting Note: Mix only enough staining solution necessary green fluorescence (TUNEL-positive) is counted; to complete the number of assays prepared. and the numbers are expressed as percentage of Note: The staining solution is active for approxi- total count of the sample (Fig. 14.2). mately 24 h at 4°C. Note: If the sperm density is low, decrease the amount of PI/RNase Staining Buffer to Protocol for Shipping Semen 0.3 mL. Samples for TUNEL Test Note: The cells must be analyzed within 3 h of staining. The cells may begin to deterio- Semen samples can be shipped to labs that rate if left overnight before the analysis. perform the TUNEL assay. Following liquefac- tion, the sperm count should be checked using these steps: Measurement of Sperm DNA Damage 1. Fixation protocol: (a) Suspend the sperm cells (2–3 × 106 cells/ Flow Cytometry mL) in 3.7% (weight/vol.) paraformalde- A minimum of 10,000 events are examined for hyde prepared in PBS (pH 7.4). each measurement at a flow rate of about 100 (b) Place the cell suspension on ice for events/s on a flow cytometer (fluorescence acti- 30–60 min. vated cell sorting [FACS]) (Becton and Dickinson, (c) Centrifuge the cells for 5 min at 300 × g San Jose, CA). The excitation wavelength is and discard the supernatant. 488 nm supplied by an argon laser at 15 mW. Green (d) Adjust the cell concentration to 2–3 × 106 fluorescence (480–530 nm) is measured in the FL-1 cells/mL in 70% (vol./vol.) ice cold channel and red fluorescence (580–630 nm) in the ethanol. FL-2 channel. Spermatozoa obtained in the plots (e) Store the cells in 70% (v/v) ethanol at are gated using a forward-angle light scatter (FSC) −20°C until use. The cells can be stored at and a side-angle light scatter (SSC) dot plot to gate −20°C several days before use. out debris, aggregates, and other cells different (f) Label the cryovials with the sample infor- from spermatozoa. TUNEL-positive spermatozoa mation (i.e., date, name, type of sample, in the population are measured after converting the volume, etc.). data into a histogram (Fig. 14.3). The percentage of (g) At the time of shipping, place cryovials in positive cells (TUNEL-positive) are calculated on a the cryoboxes, place these in adequate 1,023-channel scale using the appropriate flow amount of dry ice and ship it by overnight cytometer software (FlowJo Mac version 8.2.4) courier. (FlowJo, LLC, Ashland, OR) as described by us (h) Enclose the list of the samples being earlier [47] (Fig. 14.3). shipped. 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 211

(i) Ensure that the quantity of ice is sufficient TUNEL assay includes specific detection of free to last 2–3 days in case of an unexpected DNA ends (“nicks”) by enzymatic incorporation delay in delivery. of marked nucleotides. Therefore, an important question is whether these adaptations are adequate Reference Ranges of Sperm Damage to allow reagents to access the highly compacted We have established sperm DNA damage refer- sperm DNA without inducing damage. Owing to ence ranges using the protocol described for the a lack of standardization in methods, it is difficult apoptosis detection kit (protocol #4). Unprocessed to determine if the variations in the findings are or “raw” liquefied seminal ejaculates were used, real (related to biology) or due to differences in and healthy donors of proven and unproven fertil- methods. Therefore, an important question is ity were included. A receiver operating charac- whether the treatments used to prepare the sperm teristic (ROS) curve was used to establish the may themselves induce DNA damage. cutoff values (Fig. 14.4). 1. Accessibility of the DNA. Normal sperm DNA damage: <20% Reagents used in the TUNEL test such as ter- Abnormal DNA damage: >20% minal deoxynucleotidyl transferase (TdT) can be a limiting factor. If the protamine bound Sensitivity and Specificity chromatin is resistant to nucleases, it would be The sensitivity of the TUNEL test was 64% with resistant to enzymes such as TdT. However, specificity of almost 100%. This cutoff value is improperly stabilized protamine and histone specific to our program; other centers should bound DNA can be expected to be accessible establish their own lab cutoffs, as this will vary to TdT as TdT can access DNA breaks in with the methodology, assay reagents, staining nucleosomal DNA. Therefore, the TUNEL steps, and patient population (Fig. 14.4). assay can be expected to reveal this type of chromatin structure. 2. Sperm preparation. Factors Affecting TUNEL Assay Results It is important to understand that the results will be different if the test is performed before The methods used for DNA damage assessment or after sperm preparation. This will also were originally developed and validated for the influence the predictive potential of assisted investigation of DNA in somatic cells. The reproductive technology (ART) success. The TUNEL assay has been shown to be discrimi- native for clinical pregnancy using either raw semen or cohorts of spermatozoa prepared by density-gradient centrifugation for clinical use [28, 48]. 3. Presence of dead cells. Interpretation of the results can be confounded by the presence of dead cells as in the case of tests performed on unprocessed semen. Dead cells contain fragmented DNA, and this may bias the overall results. 4. Number of cells examined. A large number of spermatozoa (approxi- mately 400) must be counted for accuracy. If a lower number is counted, the confidence limits will increase. Counting by flow cytometry is Fig. 14.4 Receiver operating characteristic (ROC) curve showing cutoff of 19.26%, sensitivity of 64%, and speci- faster and more accurate and robust than ficity of 100% counting with optical microscopy. In fact, flow 212 R. Sharma and A. Agarwal

cytometry results were shown to be 2.6 fold ilar cutoff value (19.2 vs. 20%) to those reported higher than the results from fluorescence by Sergerie et al. [38]. microscopy [45]. These values are similar to those reported 5. Inter-and intraobserver as well as the inter- and earlier (20%; [31] and 24.3%; [49]; and 24%; intraassay variations. [38]). These values are much lower than the Establishing inter- and intraobserver as well threshold established for SCSA (t30%). Both as inter- and intraassay variations is extremely sensitivity and specificity are associated with important [47]. For example, the total varia- intrinsic performance of the TUNEL assay. tion among a set of healthy normal men However, the PPV and NPV are strongly associ- (control) would tend to be much less than the ated with the prevalence of the sample [38]. total variability among a set of infertile Standardized methods that allow researchers patients, so even if separate observers to compare results from different laboratories are provided a similar degree of interobserver needed. It is important to understand and control variability in the two populations with respect for changes in sperm chromatin that occur after to their absolute difference in assigned ejaculation and to distinguish between genuine TUNEL values, the results would look more and artifactual variations caused by a lack of impressive in the more variable patient sam- reagent access to DNA. Standardized protocols ple, since the variance components are com- and appropriate external quality controls are pared to total variability. necessary to implement findings worldwide. For 6. Other factors. useful clinical cutoff limits, it is also necessary Similarly, in establishing the cutoff or normal that the test can distinguish between affected and threshold values as well as the sensitivity, unaffected individuals. In this context, correla- specificity, NPV and PPV, it is important to tions by themselves are not adequate and other determine whether a test will be utilized as a means of interpreting results such as predictive screening/diagnostic test or to predict an value, likelihood ratios, odds ratios, ROS cutoff established end point. Sensitivity is important value are more valuable [50]. and must be high for a test to be used for screening or diagnostic purposes so that it can be offered to a large population. However, Future of TUNEL Assay specificity becomes critical for a test to be offered as a predictive marker of a defined end The literature suggests that this test is a safe and point. Positive and negative predictive values effective means of measuring sperm DNA dam- are dependent on the prevalence of infertility age. Additional research needs to be generated to in the tested population, so they will be differ- further fine tune the lower thresholds and mini- ent in populations where the percentage of mize the variations in the methodology. The fertile subjects may be higher [47]. highly specialized and compacted nature of sperm Sergerie et al. [38] examined the threshold chromatin makes it less permeable and less sensi- values of TUNEL in 47 men with proven fertility tive to allow the terminal enzyme (TdT) to access and in 66 infertile men. The infertile men had the DNA stand breaks deep within the sperm higher mean level of DNA damage than the nucleus. This particular issue may be one factor proven fertile men (40.9 ± 14.3% vs. 13.1 ± 7.3%) that contributes to variation in results. To over- (P < 0.001). The area under curve was 0.93 for a come this challenge, a recent study [51] has used 20% cutoff; the specificity was 89.4% and the Dithiothreitol (DTT) to expose the chromatin for sensitivity was 96.9%. The positive and negative 45 min prior to the fixation step. This simple predictive values were 92.8 and 95.5%, respec- additional step significantly improved the signals tively. Our study [47] with 194 infertile patients generated by the spermatozoa. Furthermore, the and a control population consisting of men with TUNEL methodology was refined to include a proven and unproven fertility showed a very sim- vitality stain (Live/Dead Fixable Dead cell stain) 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 213 that remained associated with the spermatozoa different stages of maturation: implications in the during fixation and processing of the TUNEL diagnosis and treatment of male infertility. Hum Reprod. 2001;16:1912–21. assay, thereby allowing both DNA integrity and 3. Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, vitality to be simultaneously detected in the Sharma RK, Meyer A, Nelson DR, Thomas AJ, Jr. same flow-cytometry assay. This modification Negative effects of increased sperm DNA damage in allows the assay to be more sensitive and robust. relation to seminal oxidative stress in men with idio- pathic and male factor infertility. Fertil Steril. Measuring viability in the spermatozoa tested 2003;79:1597–1605. for DNA damage by TUNEL is critical, as this 4. Aitken RJ, Sawyer D. The human spermatozoon-not may help further improve the predictive value of waving but drowning. Adv Exp Med Biol. 2003; this test. 518:85–98. 5. De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, Hedges A, Nixon B, Aitken RJ. DNA damage in human spermatozoa is highly correlated Conclusions with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2’-deoxyguanosine, a marker of oxidative stress. Biol Reprod. 2009;81:517–24. Sperm DNA integrity is essential for the accu- 6. Sakkas D, Mariethoz E, Manicardi G, et al. U. Origin rate transmission of genetic information. Sperm of DNA damage in ejaculated human spermatozoa. chromatin is a highly specialized and compact Rev Reprod. 1999;4:31–7. structure that is essential for protection and 7. Marcon L, Boissonneault G. Transient DNA strand breaks during mouse and human spermiogenesis new transmission of the human genome. A large insights in stage specificity and link to chromatin number of tests are available to assess different remodeling. Biol Reprod. 2004;70:910–8. aspects of sperm DNA integrity, but there is no 8. Sakkas D, Seli E, Bizzaro D Tarozzi N, Manicardi consensus on the optimal technique or appropri- GC.. Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during sper- ate clinical cutoff levels. We review the use of matogenesis. Reprod Biomed Online 2003;7:428–32. TUNEL test by flow cytometry and fluorescence 9. Weng SL, Taylor SL, Morshedi M, Schuffner A, microscopy as used by different laboratories, its Duran EH, Beebe S, Oehninger S. Caspase activity advantages and challenges and highlight further and apoptotic markers in ejaculated human sperm. Mol Hum Reprod. 2002;8:984–91. improvements to make it more robust. This test 10. Matsuda Y, Tobari I, Maemori M, Seki N. Mechanism has the potential of being offered to a select of chromosome aberration induction in the mouse egg group of infertile patients presenting with idio- fertilized with sperm recovered from postmeiotic pathic infertility or in cases where oxidative germ cells treated with methyl methanesulfonate. Mutat Res. 1989;214:165–80. stress may be an underlying issue. The use of 11. Aitken RJ. Founders’ Lecture. Human spermatozoa: this test can be cost-effective in establishing the fruits of creation, seeds of doubt. Reprod Fertil Dev. DNA integrity of the sperm in selected cases of 2004;16:655–64. male infertility by any fertility testing facility 12. Steele EK, Lewis SE, McClure N. Science versus clinical adventurism in treatment of azoospermia. with access to flow cytometry before consider- Lancet. 1999;13;353:516–7. ing other more expensive ART procedures. 13. O’Connell M, McClure N, Powell LA, Steele EK, Further research is needed to create a platform Lewis SE. Differences in mitochondrial and nuclear for andrology labs and other testing centers to DNA status of high-density and low-density sperm fractions after density centrifugation preparation. use with this test in measuring sperm DNA Fertil Steril. 2003;79:754–62. damage. 14. Lewis SE, O’Connell M, Stevenson M, Thompson- Cree L, McClure N. An algorithm to predict preg- nancy in assisted reproduction. Hum Reprod. 2004;19:1385–94. References 15. Suganuma R, Yanagimachi R, Meistrich ML. Decline in fertility of mouse sperm with abnormal chromatin 1. Aitken RJ, De Iuliis GN. On the possible origins of during epididymal passage as revealed by ICSI. Hum DNA damage in human spermatozoa. Mol Hum Reprod. 2005;20:3101–8. Reprod. 2010;16:3–13. 16. Sailer BL, Jost LK, Evenson DP. Mammalian sperm 2. Ollero M, Gil-Guzman E, Lopez MC, et al. 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Luke Simon and Sheena E.M. Lewis

Abstract Sperm DNA damage is associated with poorer assisted reproductive treatment (ART) outcomes including reduced fertilization rates, embryo quality, and pregnancy rates and higher rates of spontaneous miscarriage and childhood diseases. It shows promise as a more robust biomarker of infertility than conventional semen parameters. Among the sperm DNA testing methods, the alkaline comet assay is a sensitive, reliable, and powerful tool to detect even low levels of DNA damage within individual sperm. The present chapter provides an overview of the use of the alkaline comet assay in sperm. This includes the need for standardization of the alkaline comet assay protocol and its present strengths and weaknesses. Since sperm DNA damage is often the result of increased oxidative stress in the male reproductive tract, primarily formed due to an imbalance between reactive oxygen species generation and antioxidant depletion, a novel addition to the comet assay to measure oxidized bases is explored. The potential use of antioxidant therapy to protect against such damage is also described. Finally, the diagnostic and prognostic values of sperm DNA damage measures in determining the assisted reproductive technology (ART) success are discussed.

Keywords 3PERM COMET ASSAY s 3PERM $.! s !SSISTED REPRODUCTIVE TREATMENT s#OMETASSAYs/XIDATIVESTRESSINMALEREPRODUCTION

The Need for Novel Diagnostic and Prognostic Tests S.E.M. Lewis ( ) Centre for Public Health, Queen’s University of Belfast, Room 208, Institute of Clinical Science, Belfast, Male infertility is implicated in more than 40% of Northern Ireland, UK couples presenting for treatment with assisted e-mail: [email protected] reproductive technology (ART). Conventional

A. Zini and A. Agarwal (eds.), Sperm Chromatin: Biological and Clinical Applications in Male 217 Infertility and Assisted Reproduction, DOI 10.1007/978-1-4419-6857-9_15, © Springer Science+Business Media, LLC 2011 218 L. Simon and S.E.M. Lewis semen analysis continues to be the only routine quality and function (reviewed by Saleh and test to diagnose male infertility. However, semen Agarwal [6]). They have been shown to cause analysis cannot discriminate between the sperm DNA fragmentation in the reproductive tract as of fertile and infertile men [1]. Recent evidence well as damage in ejaculated sperm [7]. High has suggested that instability in the genomic levels of ROS have also been reported in the material of the sperm nuclei is a more robust seminal plasma of infertile men [8]. Sperm are parameter in measuring the fertility potential of vulnerable to the oxidative-stress-mediated dam- sperm, either in vivo or in vitro. For a test to be age, due to their structure with a high proportion useful diagnostically or prognostically, it must of polyunsaturated fatty acids in their plasma have a threshold value that provides a discrimina- membranes [9]. As sperm cannot repair such tory power above or below the threshold value damage, sperm DNA has evolved to protect itself with little overlap between groups of fertile and by compact packaging of the sperm DNA by infertile men and couples with ART success and protamines [10, 11]. failure. However, neither the routine semen anal- The exact mechanisms by which ROS induces ysis nor the available sperm DNA tests yet meet DNA damage are poorly understood, However, these standards (reviewed in references [2, 3]). ROS-induced sperm DNA damage is exemplified The primary function of the sperm is to deliver by DNA cross-links, frameshifts, production of the paternal genome to the oocyte. Recent studies base free sites, chromosomal rearrangements and have shown a number of sperm nuclear abnor- DNA base-pair oxidation [12–14]. It is also well malities such as DNA strand breaks, Y chromo- known to cause strand breaks, with the levels of some microdeletions, alterations in chromosome ROS correlated with increased percentage of sin- number, distorted epigenetic regulation and gle and double-strand damage in sperm [15–17]. sperm’s environmental milieu during epididymal ROS-mediated DNA damage is also seen in the transport and ejaculation. Factors such as formation of modified bases, which are often increased oxidative stress or low levels of anti- converted into strand breaks and considered to be oxidants may have implications on male repro- important biomarkers for oxidative DNA damage ductive health [4]. As the structural organization [18]. Finally, ROS cause gene mutations such as of the sperm chromatin is also essential for the point mutations and polymorphism [19, 20]. normal function of the sperm [5], character ization Seminal plasma is contaminated with ROS of sperm DNA quality has gained importance. In [21, 22] primarily produced by leukocytes and recent years, comet assay, TUNEL, SCSA, and defective sperm [23]. The presence of elevated SCDA or Halo assay, in situ nick end labeling levels (>1 × 106/mL) of leukocytes in the semen is have been studied extensively to analyze sperm defined as leukocytospermia [24] and is associ- chromatin integrity. Each of these tests deter- ated with increased levels of ROS, leading to mines different aspects of DNA integrity, but to sperm DNA damage [25]. Cytoplasmic droplets date, combining all the studies available in meta- are also associated increased ROS generation and analysis shows that these tests lack the statistical poor sperm quality [26, 27]. power and diagnostic potential necessary to incorporate them into routine clinical use. Environmental and Lifestyle Hazards

Causes of Sperm DNA Damage It has recently been reported that male fertility declines with age, even though spermatogenesis In recent years, the generation of reactive oxy- continues [28]. An increase in male age has been gen species (ROS) has been widely studied in associated with increased genetic and chromo- the male reproductive tract and reported to be a somal defects [29, 30]. Men over 37 years have concern because of their toxic effects on sperm been shown to three times more sperm DNA 15 Basic and Clinical Aspects of Sperm Comet Assay 219 damage then men aged <37 years [31, 32]. Male very little is known about their effect on sperm germ cells are particularly vulnerable to environ- DNA [48]. There is a very strong and significant mental chemicals and xenobiotics that cause correlation between smoking and genetic defects DNA damage [33]. Studies also show the adverse in the sperm [49, 50]. Smoking increases oxida- impact of some occupations to increase the tive stress, which results in depletion of antioxi- sperm DNA damage, for example among coke dants in the seminal plasma, thereby inducing oven workers [34]. Oh et al. [35] concluded that oxidative DNA damage to the sperm [15] and there are elevated levels of DNA damage among mutagenic adducts [51]. Recent studies have also waste incineration workers, when compared with suggested a possible link between cell-phone use men from similar origin. Further, men working associated with electromagnetic radiations and in the factories with organic molecules such as sperm DNA damage [52–56]. Finally, physical styrene show a significant amount of increase factors such as mild scrotal heating [57] and radio in sperm DNA damage [36]. Similarly, men frequencies [55] have also been proven to dimin- working in the insecticide and pesticide indus- ish sperm DNA integrity. tries have higher levels of sperm DNA damage [37, 38]. A further hazard for sperm DNA is by phar- The Comet Assay: What Does macological exposure to drugs. This has become It Measure? very common as molecular medicine advances, especially in the field of cancer. Chemothera- For a sperm DNA test to be clinically useful, (a) it peutic drugs are genotoxic to the male germ should measure both single- and double-strand cells. A well-known example for such an inter- breaks, as both may be important and the oocyte vention is the cyclophosphamide [39, 40] in has limited ability to repair fragmented paternal animal model. Hellman et al.’s [41] cyclophos- DNA, (b) it should measure the level of DNA phamide treatment resulted in a five fold increase fragmentation in each sperm, as an ejaculate is in DNA damage. Environmental exposure of xen- known to show a high degree of variation, (c) the obiotics cannot be avoided in our contemporary methodology should be appropriate for cell lysis lifestyle because these pollutants are present in and DNA decondensation for full extent of our food, water, and air. Studies have shown the damage to be determined, (d) the test must have association between environmental estrogens strong predictive capacity for pregnancy outcome and derived compounds and male infertility and little overlap between fertile and infertile samples. through elevated sperm DNA damage [42]. Among the tests currently available, the alkaline Environmental pollutants such as organochlo- comet assay addresses the first three above-mentioned rides [43] and smog [44] also have the ability to issues but useful thresholds have not been estab- induce DNA damage. Bennetts et al. [45] lished yet to validate the assay. showed that estrogenic compounds such as Initially, the comet assay [58] was designed to 2-hydroxyestradiol induce redox cycling activi- characterize the structure of the nucleus. However, ties and concomitant sperm DNA damage. when electrophoresis of DNA strands after alka- These examples support the belief that expo- line denaturation came into existence in 1988 by sure to xenobiotics has powerful impacts on Singh et al., the detection of DNA damage within sperm DNA and sperm functions, leading to the nucleus became a possibility. Collins et al. [59] male infertility. suggested that the migrated comet tail after elec- Lifestyle choices also play an important role trophoresis consists of fragments originated from in male infertility. For example, smoking and relaxation of supercoiled loops and single-stranded consumption of alcohol and caffeine have been DNA formed under alkaline conditions. Some associated to the increase in nuclear DNA damage studies suggest that double-strand DNA breaks of the white blood cells [46, 47]; on the contrary, alone may be detected under neutral conditions 220 L. Simon and S.E.M. Lewis

(pH 8–9) [60, 61], and in these studies the level of cells should not be excluded during analysis measurable DNA damage is low compared to the [70]. The DNA-specific fluorescent dyes are used alkaline comet assay. This is due to either the mea- for comet visualization. The most frequently surement of additional DNA damage by the alka- used fluorescent dyes are ethidium bromide, line condition or the relatively higher migration of propidium iodide, DAPI, SYBR Green I, and DNA strands under alkaline conditions [62]. benzoxazolium-4-quinolinum oxazole yellow The extent of DNA damage in individual cells homodimer [71]. Addition of an antifade reagent could be monitored by the use of image analyzing along with fluorescent dyes could significantly system. Presently, different commercial software reduce fluorescence quenching [72]. Nofluorescent packages are available to measure the comet dyes such as silver nitrate are also reported for parameters. A fully automated comet analyzing comet assay; however, the efficiency of the assay system has also been developed [63]. In the past, is reduced [73]. Excess of fluorescence dye could different methodologies were used to measure the increase the background intensity of the slides extent of DNA damage such as the proportion of thereby very low-molecular-weight DNA frag- cells with altered tail DNA migration, approaches ments could not be measured. Hence, standard- classifying comets into several categories based ization of the comet assay is required for accurate on the tail migration [64, 65]. However, these performance. approaches are generally limited to electropho- retic conditions. Hughes et al. [66] reported that the evidence for intact DNA is considered more Strengths of Comet Assay important in relation to fertility status than mea- surement of other comet parameters that could be The comet assay is one of the most sensitive tech- altered by the experiment conditions. niques available to measure DNA damage, and The commonly used comet parameters are according to Aravindan et al. [74], the results of percentage head DNA, percentage tail DNA, tail comet assay are also related to the results obtained length, and olive tail moment. The software sys- from the TUNEL assay. The alkaline comet assay tem analyzes the light intensities (fluorescence) could be used in all the cell types and also in the in the head relative to the tail to determine the sperm [75]. The assay requires only a few numbers percentage of DNA present in the head and tail. of cells; hence, the assay is possible in cases of The background light intensity is subtracted from oligospermia and testicular biopsy. The DNA head and tail intensities to get the actual value. damage data can be collected at the level of indi- Also, the sperm populations are known to be vidual cells, making the analysis efficient. The more heterogeneous, and the baseline values of removal of protamines and histones during the DNA damage of sperm population in an ejaculate assay reveals the total DNA damage in the cell. are substantially higher than those in somatic The range of DNA damage measured in sperm cells [67]. Although, few number of sperm could using the alkaline comet assay varies from 0–100% be analyzed in the comet assay, Hughes et al. [66] showing its capacity to identify sperm with much demonstrated that the analysis of 50 sperm is or little damage. A further advantage is that, unlike sufficient to provide a measurement of DNA the TUNEL and SCSA, which detect primarily damage of the total sperm population with a breaks in histone-associated chromatin, the comet coefficients of variation lower than 4%. assay has a broader use in detecting breaks in both The comet assay is highly sensitive to detect protamine- and histone-bound chromatin equally. extensive fragmented cell in the form of nonexis- tent heads or a large diffused tail termed as “ghost” or “clouds” or “hedgehogs” [68]. In such Weaknesses of Comet Assay cases, the comet image system cannot interpret the full extent of DNA damage [69]; therefore, it One disadvantage of the comet assay is that it is advisable to consider the ghosts as completely lacks standardized protocols, which makes it damaged cells. In sperm, such highly damaged difficult to combine the results from different 15 Basic and Clinical Aspects of Sperm Comet Assay 221 laboratories [76]. This should be resolved by The first variation relates to lysis conditions. agreement on an optimal protocol (see next sec- Absence of cytoplasm in sperm makes it difficult tion). The assay is criticized for the use of high to optimize lysis conditions compared to the pH conditions, which is known to denature the somatic cells. For example, in some labs, lysis of alkaline-labile sites measurable after electropho- plasma membranes is performed by incubating resis [77], making it difficult to discriminate cells with a buffer (usually containing Proteinase between endogenous and induced DNA breaks. K, Triton X-100, and high concentrations of However, labile sites may be considered as NaCl) for a short time (3 h), in others a long, even another form of potential damage, and some con- overnight period (18 h) [83–85]. sider this as a strength, in that an indication of As discussed previously, the sperm genomic existing and potential damage may be more DNA is more highly condensed than somatic important clinically. The assay is also criticized cells preventing the migration of the comet tail, for an underestimation of DNA damage that may so for use with sperm it requires the use of occur through entangling of DNA strands or the additional steps to decondense the tightly packed presence of proteins and cross-linked DNA DNA. A wide range of strong reagents (Proteinase strands, which could restrict the movement of K, Triton X-100, Dimethyl Sulfoxide, DTT, and DNA fragments during electrophoresis. In some LIS) have been used to remove protamines and protocols, incomplete chromatin decondensation histones [67, 83, 84, 86–88], but these agents will not allow all strand breaks to be revealed. may also induce damage. The presence of these Overlapping comet tails decrease the accuracy of different approaches prevents interlaboratory the assay, and few small tail fragments are lost or comparisons. too small fragments are difficult to be visualized. To reduce the level of laboratory-induced As in other DNA tests, strong reducing agents are damage and make the assay more reproducible, sometimes used to remove protamines, and they our group has replaced Proteinase K with DTT may increase what is perceived to be baseline and LIS and for a shorter duration of 3 h [85]. damage. Also, the assay requires a laborious Another difference between labs is the pH at process of analysis and shows high interla- which the assay is performed. Currently, electro- boratory variation and, hence, is not used clini- phoresis is carried out with wide range of buffers cally [78]. Owing to a labor-intensive and sensitive with pH ranging from pH 8.0 to 13.5 [66, 67, protocol, the assay requires skilled technicians for 84–86, 89]. Such a wide range of pH conditions accuracy. Finally, the available software to mea- again makes results difficult to compare, as the sure DNA damage cannot recognize “Ghost cells” extent of DNA migration is highly influenced by without head DNA and overlapping comet tails, the degree of alkali denaturation and the pH making the scoring difficult. However, most of value. these weaknesses can be corrected with appropri- A further confusion from “comet” studies ate protocols and training. comes from the lack of standardization of comet parameters described in different studies. There are several parameters used in comet studies. Need for Standardized McKelvey et al. [90] described it as “DNA Methodology for the Comet migration can be determined visually by the categorization of comets into different “classes” The comet assay is currently used primarily for of migration. The percentage of DNA in the tail genotoxic studies, although it is a test with great (percent migrated DNA), tail length and tail potential for ART [79]. For use with sperm, a moment (fraction of migrated DNA multiplied number of academic and methodological issues by some measure of tail length). Of these, tail need to be addressed, as there is no generally moment and/or tail length measurements are accepted protocol for the assay, even though the most commonly reported, but there is much international groups of scientists [53, 80–82] to recommend the use of per cent DNA in tail, have used it extensively. as this gives a clear indication of the appearance 222 L. Simon and S.E.M. Lewis of the comets and, in addition, is linearly related predictive value in assisted reproduction outcome to the DNA break frequency over a wide range has been assessed by few [86, 95]. of levels of damage. The approach or parameter In a recent study from our group [82] of 360 used must be clearly defined and, if not typical, couples having IVF or ICSI we reported that be justified.” sperm DNA damage is associated with poorer Hughes et al. [67] recommended the use of ART outcomes and promises to be a more robust percent tail DNA, as its coefficients of variation biomarker of infertility than conventional semen was less than 4%. Measurement of fifty comets parameters. We found significant inverse corre- from a single slide is reported to have a coeffi- lations between DNA fragmentation, fertiliza- cient of variation of less than 6% within a sperm tion rate, and embryo quality assessed by the population [67]. They also reported the reproduc- alkaline comet assay (to detect both double and ibility of the image analysis software with single strand breaks) following IVF treatment. repeated analysis of individual sample showed a A decrease in fertilization rates were observed coefficient of variation of less than 5.4%. as DNA damage of native semen and DGC Tice et al. [71] recommended the measure- sperm increased. Low DNA damage (0–20%) ment of tail length, percent tail DNA and tail showed a significantly higher fertilization rate moment, finding different results between tail compared with DNA damage >60%. Our work DNA and tail moment. However, Kumaravel supports that of Morris et al. [88] who also and Jha [91] did not find any statistical differ- reported a significant correlation between fertil- ence with olive tail moment and percentage tail ization and DNA damage when measured by the DNA to analyze the extent of DNA damage. neutral comet assay (measuring double-strand The percentage of tail DNA is reported to be breaks only). However, by contrast, no correla- directly proportional to the dosage of radiation tions were observed between fertilization rates and concentration of hydrogen peroxide. By and DNA fragmentation measured in alkaline contrast, the olive tail moment is highly influ- comet assay by Tomsu et al. [95]. enced by the study conditions, so it is not con- Our study [82] also showed a decrease in sistent between labs and, thus, not advisable embryo quality following IVF treatment, as DNA for use. fragmentation increased both in native semen and In summary, agreement on a standardized DGC sperm. The embryo quality showed a sig- protocol for the comet to necessary to compare nificant decrease, when DNA damage was greater results between groups. To reduce the additional than 60% in the native semen. The embryo cumu- DNA damage caused during the assay proce- lative score calculated according to Steer et al. dure, the duration of lysis, the composition of [96] was 15.5 in the group where sperm DNA the lysis buffer, the method of decondensation, fragmentation was <20% and was only 7.3 where the pH for unwinding, and electrophoresis con- sperm DNA fragmentation was >60% in DGC dition and parameters to be reported should be sperm. Similarly, Tomsu et al. [95] showed a neg- standardized. ative correlation between embryo quality and DNA fragmentation in both the native semen and the DGC sperm. However, Morris et al. [88] did Clinical Significance of DNA not find any association in embryo quality and Fragmentation Measured DNA damage. In contrast to associations follow- by the Comet Assay ing IVF, we did not find any correlation between sperm DNA damage and fertilization rate or The alkaline comet assay is proving to be a useful embryo quality when ICSI was used as a treat- diagnostic tool for male infertility. The clinical ment of choice [82]. importance of the comet assay in assessing male Using pregnancy as the outcome measure, infertility has been demonstrated by a number of Morris et al. [88] did not find an association authors [79, 92–94]. However, until recently, its between clinical pregnancy and sperm DNA 15 Basic and Clinical Aspects of Sperm Comet Assay 223 fragmentation measured by the neutral comet assay. Similarly, Tomsu et al. [95] in a small Clinical Significance of Existing study (n = 40) no associations were found. Strand Breaks Plus Adducts However, we found a significant difference in Measured by the Comet Assay DNA fragmentation of clinically pregnant and nonpregnant couples following IVF [82]. By con- To analyze modified bases in the sperm DNA, we trast, although couples undergoing ICSI who have used the prokaryotic repair enzyme (FPG) failed to achieve a clinical pregnancy tended to as an intermediate step during the alkaline comet have more DNA fragmentation but it was not assay, to introduce breaks at sites of modified statistically significant. bases [82]. We found inverse relationships between total DNA damage (existing strand breaks plus modified bases) and IVF and ICSI outcomes after conversion of modified bases to Further Uses of the Comet DNA strand breaks by FPG. There was a signifi- to Measure DNA Adducts cant increase in DNA damage after treatment with the DNA glycosylase FPG in both native A major cause of sperm DNA damage is oxida- and DGC samples. In the IVF patients, addition tive stress due to the generation of the ROS of the FPG enzyme showed a significant increase from contaminating leukocytes, defective in mean percentage of sperm DNA fragmentation sperm, and antioxidant depletion [23, 97]. FPG in nonpregnant compared with that from preg- is the commonly used bacterial repair enzyme nant couples (55 vs. 72) in the native semen and that could recognize and excise 8-OHdG and (42 vs. 56) in DGC sperm. Similarly, in ICSI other modified bases generated by ROS. This couples, when modified bases were included, FPG enzyme has been shown to possess affini- the percent DNA damage between pregnant and ties toward the various modified DNA bases nonpregnant couples was markedly different [98, 99]. The catalytic activity of FPG involves (63 vs. 80 in native semen, and 50 vs. 65 in DGC a three-step process: (a) hydrolysis of the sperm), in contrast to comet assay without FPG glycosidic bond between the damaged base and where it was not significant. the deoxyribose, (b) incision of DNA at abasic sites, leaving a gap at the 3c and 5c ends by phosphoryl groups, and (c) removal of termi- The Risks of Using Sperm nal deoxyribose 5c-phosphate from 5c terminal with Damaged DNA site to excise the damaged base showed by Kuznetsov [100]. Sperm DNA damage measured by SCSA, When a eukaryotic or prokaryotic base repair TUNEL, and alkaline and neutral comet assays enzyme or glycosylase is introduced as an inter- has been closely associated with all the stages of mediate step during the alkaline comet assay, the ART outcome such as fertilization, embryo qual- modified bases can be converted into single- ity, implantation pregnancy, and spontaneous strand breaks [101, 102]. Addition of base repair abortion [105, 106]. A limited amount of sperm enzymes can increase the sensitivity of the assay DNA damage can be repaired by the oocyte post by including the modified bases, resulting in fertilization, but above a threshold limit this pro- total DNA damage measured after the alkaline cess is either incomplete or inappropriate, result- comet assay [103]. Among the modified bases, ing in genetic mutations and may impact the 8-OHdG is the most commonly studied bio- viability of the embryo and the health of the off- marker and is often selected as a representative spring [107]. Men suffering from male infertility of oxidative DNA damage due to its high have high levels of sperm with DNA damage, specificity, potent mutagenicity, and relative which result in an negative impact on their ART abundance in DNA [33, 104] outcome [25, 108–112]. 224 L. Simon and S.E.M. Lewis

In recent years, sperm DNA damage has The effect of sperm DNA damage has been gained interest to understand the fertilization attributed to embryo development, particularly process to improve fertility diagnostics. The between four and eight cell stage of preimplanta- influence of DNA damage on fertilization rates tion development until which the embryonic in assisted reproduction is still controversial. genome is transcriptionally inactivated and the A number of papers have analyzed the possible paternal genome plays a significant contributory association between sperm DNA damage and fer- role in embryo function during the transcriptional tilization rates in vitro [16, 106, 111, 113–127]. activity [133]. Therefore, the effect of sperm But, many of these papers suggest that sperm DNA damage impacts more on pregnancy rates DNA damage does not affect fertilization rates than embryo quality [115]. [106, 111, 115, 117–120, 126, 127]. Sperm with Couples who failed to achieve a pregnancy damaged DNA are still capable of fertilization are known to have a higher mean level of DNA [93] but its effect is prominent in the later stages fragmentation than pregnant couple after IVF [128]. Sperm with abnormal chromatin packing treatment [105, 112, 115, 118–120, 122, 133, and DNA damage is showed to result in decon- 135]. This implies that sperm with DNA frag- densation failure, which results in fertilization mentation can still fertilize an oocyte but that failure [25]. It is also showed that that a signifi- when paternal genes are “switched on,” further cantly proportion of nondecondensed sperm in embryonic development stops, resulting in failed human oocytes has a higher DNA damage, com- pregnancy [121]. In contrast to these reports, no pared to decondensed sperm and higher degree of significant association between sperm DNA chromatin damage, this may prevent the initia- damage and clinical pregnancies has been tion or completion of decondensation, and may reported [88, 95, 114, 116, 123, 124, 126, 127, be an important factor leading to a failure in 136, 137]. Studies using animal models show fertilization [129]. A negative correlation between that oocytes and developing embryos can repair the proportion of sperm having DNA strand sperm DNA damage; however, there is a thresh- breaks and the proportion of oocytes fertilized old beyond which sperm DNA cannot be repaired after IVF is established [114]. [138]. They also reported that sperm with defec- Measurement of sperm DNA damage has been tive DNA can fertilize an oocyte and produce shown to have a significant negative effect on the high-quality early-stage embryos, but then, as developing embryo [130]. Poor sperm DNA qual- the extent of the DNA damage increases, the ity is associated with poor blastocyst develop- likelihood of a successful pregnancy decreases. ment and the failure to achieve a clinical Virro et al. [132] have shown that high levels of pregnancy. Sperm DNA damage has a significant sperm DNA damage significantly decrease the impact on embryo development [16, 95, 105, pregnancy rates and results in higher rate of 114, 126, 129, 131–133]. However, a number of spontaneous abortions. An increase in sperm studies have contradicted the influence of DNA DNA damage is associated with decreased damage on embryo development [106, 108, 109, implantation, thereby a decrease in pregnancy 112, 115–119, 122, 123, 126, 127]. Abnormalities rates [118]. By contrast, Bungum et al. [136] and in the embryo seen in vitro can be more directly Boe-Hansen et al. [137] showed a decrease in related to male factors because the results can be implantation rates with increase in DNA damage assessed without the interference of female fac- but no effect is seen on clinical pregnancies. tors such as uterine and endocrine abnormalities Frydman et al. [106] showed increase in DNA that may lead to miscarriage after embryo trans- damage not only decrease implantation and preg- fer [134]. The embryonic genome is activated on nancy rates but also increase spontaneous mis- day three, and its transcriptional products take carriage rates. Lin et al. [127] also observed an over from the regulatory control provided by increase in miscarriage rates with an increase in maternal messages stored in the oocyte [132]. DNA damage. 15 Basic and Clinical Aspects of Sperm Comet Assay 225

It is also shown that damage in the paternal genome could result in abnormalities occur during Two People but Just One postimplantation development [139]. Genetic Prognostic Test abnormalities in the paternal genome in the form of strand breaks are a significant cause of miscar- The quest for one perfect test to predict a out- riages [134]. Sperm DNA damage could likely be come with multifactorial input is particularly the cause of infertility in a large percentage of unachievable when this outcome involves not just patients [140]. However, these studies may not one individual but, in the case of ART, two causal, but simply associations between DNA partners. Since female factors such as age, occyte damage and reduced ART outcomes. Are the and embryo quality, and uterine competence all tests clinically useful? impact significantly on pregnancy, it is not surprising that if one test on the male partner is not acceptably strong. The current literature The Clinical Usefulness exemplifies how the controversies as to the use- of the Comet Test fulness of sperm DNA testing are exacerbated by flawed experimental design. Couples undergoing Two recent systematic reviews have shown that IVF treatment can be divided into those with the impact of sperm DNA damage on ART out- female, male, and unexplained infertility. A large comes decreases from IUI to IVF and is least proportion of couples undergoing IVF treatment useful in ICSI [3, 141]. In IVF, using TUNEL are due to female causes. and SCSA assays, the odds ratios is 1.57 (95% In many studies, couples with male, female, CI 1.18–2.07; p < 0.05). However, in our study and idiopathic infertility have been grouped using the alkaline comet [82] we obtained an together. In order to assess the clinical usefulness odds ratio of 4.52 (1.79–11.92) in native semen of a test for one partner of the infertile couple, the and 6.20 (1.74–26.30) in DGC sperm for clini- appropriate patient population should be identi- cal pregnancy following IVF, indicating its fied. Future studies should be designed to mini- promise as a prognostic test. Owing to the high mize the variation in these female factors. Only sensitivity of the test and level of damage then can we accurately determine the effects of observed when both strand breaks and modified sperm DNA and thereby maximize the usefulness bases were measured it was not possible to of the test. establish thresholds for our novel combined test. Following ICSI, the odds ratio for clinical preg- nancy was 1.97 (0.81–4.77) using native semen Protection of DNA from Damage and 2.08 (0.93-4.68) in DGC sperm showing less strength and supporting the combined odds In the male reproductive tract, oxidative stress is ratio of 1.14 from the meta-analyses by Collins due to the increase in the production of ROS, et al. [141] and Zini and Sigman [3]. This rather than the decrease in the seminal antioxi- supports the belief that ICSI bypasses genetic, dants. Owing to the lack of cytoplasm excluded as well as functional defects, but the results are during spermatogenesis, there is no self DNA counterintuitive. Given the many animal studies repair mechanism in the sperm; therefore, anti- showing adverse effects of DNA damage on the oxidants in the seminal plasma are essential to long-term health of offspring (reviewed by reduce the oxidative stress, and it is the only Aitken et al. [142]; Fernadez-Gonzalez et al. available mechanism for the sperm to protest [143]), we need to follow-up the children born against oxidative-stress-mediated DNA damage. by ISCI to make sure that this genetic heritage Naturally, the concentration of antioxidants in does not have long-term adverse effects of these seminal plasma is 10 times greater than in blood children’s health even if short-term success in plasma [144], and the presence of antioxidants terms of pregnancies is achieved. in the seminal plasma protects the functional 226 L. Simon and S.E.M. Lewis integrity of the sperm against the oxidative damage. This in turn would have a positive effect stress [145]. Several other studies showed the on male infertility [174]. Cryopreservation of role of antioxidants against ROS [21, 146–148]. sperm is known to increase the level of sperm However, some studies show limited protection DNA damage [93, 182–184]. of antioxidants against induced ROS [149]. Oxidative stress occurs when the level of ROS Low levels of antioxidants in semen are associ- exceeds the antioxidant protection resulting in ated with suboptimal semen parameters (Kao sperm DNA damage. Approximately, half of et al. [189]) and increased sperm DNA damage infertile men exhibit oxidative stress [185]. In light [150]. Oral administration of the antioxidants has of these considerations, future research to deter- been shown to significantly increase antioxidant mine the best regime of antioxidant therapy so be levels in the seminal plasma and an improvement pursued to find an effective treatment [186–188]. in the semen quality [151–155]. Specifically, anti- oxidant treatment to infertile patients by oral administration of vitamins significantly improved Conclusions and Future their sperm motility [152, 154, 156–158], sperm Recommendations concentration [12, 159, 160], and normal mor- phology of the sperm [152, 159]. Improvement in Clinical evidence shows the negative impact of semen parameters by administration of oral anti- sperm DNA fragmentation on reproductive out- oxidants were seen in volunteers as well as patients comes, and sperm from infertile men show higher [154, 161]. Studies by Lenzi et al. [162–164] levels of DNA fragmentation than the sperm of reported a protective function of antioxidants on fertile or donor men. Recent studies have shown semen quality due to a reduction of ROS and a that the use of alkaline comet assay to test sperm reduction in the lipid peroxidation of the mem- DNA fragmentation is a useful tool for male brane. By contrast, other studies have shown no infertility diagnosis and early predictor of ART significant effects of oral antioxidant treatment on outcomes. Below novel “comet” threshold values semen parameters [165–167]. The absence of of sperm DNA fragmentation in both native effects in these studies may be due to shorter semen and DGC sperm obtained from the alkaline duration of treatment [167, 168] and/or very low comet assay, there is evidence of infertility in vivo dosage of antioxidants used [169]. and in vitro. Therefore, it is beneficial to assess Administration of oral antioxidants had been sperm DNA fragmentation in couples presenting shown to significantly decrease sperm DNA with infertility problems and also in patients damage [12, 170–173] and to reduce sperm DNA undergoing ART. We encourage studies to ana- adducts [174] and the incidence of aneuploidy in lyze the impact of sperm DNA fragmentation sperm [175], thereby increasing the assisted and to validate the current protocol of the alka- reproductive success [176, 177]. line comet assay through large multicenter trials, Protection of sperm from DNA damage should using good quality control, with standardized also be monitored during sperm processing and protocols. cryopreservation when they are especially vul- nerable. The absence of antioxidant protection in these procedures has been shown to increase sperm DNA damage [15]. Zalata et al. [178] References showed that high-speed centrifugation and 1. Guzick DS, Verstreet JWO, Factor-Litvak P, et al. removal of sperm from the protective seminal Sperm morphology, motility, and concentration in plasma resulted in ROS-mediated DNA damage. fertile and infertile men. 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