Human Sperm DNA Fragmentation: Correlation of TUNEL Results As Assessed by Flow Cytometry and Optical Microscopy

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1 Original Article Human Sperm DNA Fragmentation: Correlation of TUNEL Results As Assessed by Flow Cytometry and Optical Microscopy David Domínguez-Fandos, 1,2 María Isabel Camejo, 1,2 José Luis Ballescà, 3 Rafael Oliva 1,2 * 1 Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona, Barcelona, Spain 2 Genetics Service, Hospital Clınic i Provincial, Barcelona, Spain 3 Institut Clınic of Gynaecology, Obstetrics and Neonatology, Hospital Clınic i Provincial, Barcelona, Spain Received 28 August 2007; Accepted 28 September 2007 This article contains supplementary material available via the Internet at jpages/ /suppmat. Grant sponsor: Ministry of Science and Technology (Plan Nacional de ID); Grant numbers: BMC , BFU ; Grant sponsors: Fondos FEDER, IDIBAPS, Fondo of Health Research (Ministerio de Sanidad y Consumo); Grant number: V-2003-REDC07A-O. Present address of Marıa Isabel Camejo: Department of Organisms Biology, University of Simon Bolıvar, Caracas, Venezuela *Correspondence to: Rafael Oliva, Human Genetics, Faculty of Medicine, University of Barcelona, Casanova 143, Barcelona, Spain roliva@ub.edu Published online 30 October 2007 in Wiley InterScience ( wiley.com) DOI: /cyto.a International Society for Analytical Cytology Abstract An association between DNA fragmentation in sperm determined by the terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate (dutp) nick end labeling (TUNEL) assay and the incidence of reproductive failure has been reported, either using flow cytometry or optical microscopy. However, the results obtained using each of these two approaches are different. Since there is a relative lack of studies standardizing these two approaches, the direct comparison of the results described in the different articles is difficult at present. To allow the comparison of the TUNEL results obtained using flow cytometry and optical microscopy, we applied these two approaches in a total of 66 human sperm samples. A positive correlation is detected in the TUNEL results as measured by flow cytometry and optical microscopy (Spearman; r = 0.720, P ). The percentage of TUNEL-positive spermatozoa assessed by flow cytometry is 2.6 times higher than that detected in optical microscopy (39.7% % versus 15.3% %). Although there is a good correlation of the TUNEL results obtained by flow cytometry and optical microscopy, the percentages obtained with either technique are different. Therefore, the TUNEL results described in the present work should be valuable to compare the results described in many independent articles, using either optical microscopy or flow cytometry. ' 2007 International Society for Analytical Cytology Key terms DNA fragmentation; TUNEL; flow cytometry; microscopy; infertility; sperm IT is known that an increased DNA fragmentation is present in the sperm cells of infertile patients (1 4). In addition, an altered DNA integrity in spermatozoa leads to poorly assisted reproduction results (2,5 17). The selection of spermatozoa to perform ICSI is presently based on its morphology and motility, but these parameters do not give information about DNA integrity. Spermatozoa with damaged DNA may also result in increased risk of anomalies in newborns and increased risk of childhood cancer (18 21). Therefore, laboratory techniques to evaluate DNA integrity are important toward a better management of the infertile patients. The etiology of these strand breaks may involve aberrant recombination, defective chromatin packaging, abortive apoptosis, and oxidative stress (22). There are several approaches available to evaluate DNA integrity in human spermatozoa (23 25). One of the most widely used techniques is the terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate (dutp) nick end labeling (TUNEL) assay. Using this approach, the DNA fragmentation is assessed through the labeling with dutp of the double- and single-stranded DNA breaks present in spermatozoa (26 28; and also references cited in Table 2). The TUNEL assay has been considered as a measure of the DNA strand breaks and as a biological assay for sperm quality at the assisted reproduction laboratories (5,6,9,10,28 31). However, there are some factors that may result in variation in the Cytometry Part A 71A: , 2007

2 Table 1. Statistical Descriptors of the Different Basic Semen Parameters and Results of the Tunel Assay Under the Different Conditions MEAN STANDARD DEVIATION MINIMUM MAXIMUM NUMBER OF SAMPLES Basic patient and seminal parameters Age of patients (yr) Normal morphology (%) Sperm count (10 6 sperms/ml) Motility a (rapid progressive, %) Motility b (slow or sluggish, %) Motility c (nonprogressive, %) Volume (ml) TUNEL-positive sperm (%) Optical microscopy Observer Observer Cytometry (%) TUNEL results among the different laboratories, such as the use of different methodological variants (32 38). One of the important alternatives in the TUNEL assay is to measure the results either using fluorescence microscopy or flow cytometry. However at present, there is a relative lack of studies standardizing these two approaches. Therefore, so far it is difficult to compare the results of the different articles published (Table 2). The aim of the present study has been to evaluate the effect in the number of detected positive TUNEL spermatozoa, by comparing the evaluation of the sample by fluorescence microscopy versus flow cytometry. MATERIALS AND METHODS Sample Collection and Sperm Preparation Sixty-six sperm samples (ejaculates) were collected from patients undergoing routine semen analysis for infertility in the Institute of Gynecology, Obstetrics and Neonatology from the Hospital Clinic from Barcelona. The samples were collected by masturbation in sterile containers, after at least 3 days of sexual abstinence and were allowed to liquefy. After 30 min of semen liquefaction, standard semen parameters (normal morphology, sperm count, motility, and volume) were measured according to the World Health Organization guidelines using a computer-assisted semen analyzer (CASA, Photolux) and a Makler counting chamber (Sefi Medical Instruments, Hainfa, Israel; Table 1). Before participating in this project, each donor signed an informed consent. The project was approved by the bioethics committee of the hospital. TUNEL Assay TUNEL was assayed as previously described (32,39 42) using the In Situ Death Detection Kit AP; TUNEL kit (Roche Diagnostic Corp., Indianapolis, IN). A semen aliquot containing spermatozoa was washed by centrifugation, 800g at room temperature for 5 min with phosphate-buffered saline (PBS; BioMérieux, Marcy l Etoile, France). After removal of the seminal plasma, the pellet was washed twice in PBS with 1% bovine serum albumin (BSA; BioRad, Hercules, CA). Then the pellet was suspended in 100 ll of PBS/BSA 1% and fixed in 100 ll of 4% paraformaldehyde in PBS (ph 7.4) for 1 h at room temperature and in agitation. The pellet was suspended in 100 ll of PBS and permeabilized with 100 ll of 0.1% Triton X-100 in 0.1% sodium citrate in PBS for 2 min in ice. The permeabilization solution was removed by centrifugation, and two additional washes were performed with PBS/ BSA 1%. The pellet was resuspended in 50 ll of the staining solution for 1 h at 378C in the dark and mixing them each 15 min. The staining solution contained terminal deoxytransferase (TdT). The negative and the positive control were performed, respectively, by omitting the TdT enzyme following the kit instructions and by preincubating fixed and permeabilized sperm samples with DNase I (40 IU/mL) for 10 min at room temperature to produce DNA breaks. After the staining, two washes with 1% BSA in PBS were performed, and the spermatozoa were resuspended in 200 ll of PBS/0.1% BSA. One aliquot of 10 ll (containing sperm cells) was smeared on a microscope slide for posterior analyses by optical and fluorescence microscopy. The rest of the sample was diluted with PBS and analyzed by flow cytometry. Forty-five samples were stained with 5 ll of propidium iodide (PI) before analyzing them by flow cytometry. Aliquots used for microscopy determination were not stained with PI, since fluorescein (present in the Roche s kit) has the same wavelength excitation of PI. Fluorescence Microscopy measurements For microscopy evaluation, the slides were viewed using an optical and fluorescence Olympus BX50 microscope. The percentage of spermatozoa with fragmented DNA was determined simultaneously by analyzing each microscope field using both light microscopy to count the total number of spermatozoa and fluorescence microscopy using wide interference blue filter to excite the fluorescent dye (fluorescein), producing an excitation between 460 and 490 nm and an emission [515 nm. Fluorescein is typically excited by the 488-nm line, and the emission is collected at 530 nm. The spermatozoa with intense green fluorescence representing positive TUNEL cells were counted. For each slide, about 400 spermatozoa 1012 Sperm TUNEL by Flow Cytometry and Microscopy

3 Figure 1. TdT (terminal deoxynucleotidyl transferase)-mediated dutp nick-end labeling (TUNEL) assay of spermatozoa. Forward-angle light scatter (FSC) versus side-angle light scatter (SSC) dot plots obtained by flow cytometry are represented in the top. The gates used to select the events for subsequent fluorescein isothiocyanate (FITC) analysis were determined depending on the event position in the dot plot, related with its FSC versus SSC, and this is different from sample to sample. Frequency distribution histograms (events versus FITC label) of spermatozoa stained with TUNEL are represented in the bottom. (A) negative control. The window M1 was adjusted arbitrarily to obtain about 1% TUNEL-positive events. (B) Positive control (spermatozoa treated with DNAse I) with 98.48% TUNEL-positive sperm. (C) Example of a sample from a patient with a high level of TUNEL-positive cells (40.99%). (D) Example of a sample from a patient with a low proportion of TUNEL-positive cells (9.86%). The horizontal line (M1) indicates spermatozoa that are positive for TUNEL technique. were evaluated by two different viewers (200 each) and the average was performed. Flow Cytometry measurements For flow cytometry evaluation, a minimum of 10,000 events were examined for each measurement at a flow rate of about 200 events/s on a flow cytometer (FACS Calibur; Becton Dickinson). Data were processed using CELLQUEST (Becton Dickinson) and WinMDI (Windows Multiple Document Interface for Flow Cytometry; Copyright Joseph Trotter) software. Green fluorescence (TUNEL-positive cells) was measured using a nm bandpass filter. Spermatozoa obtained in the plots were gated by using forward-angle light scatter (FSC), and side-angle light scatter (SSC) dot plot to gate out debris, aggregates, and other cells different from spermatozoa. Then, TUNEL-positive spermatozoa in these populations were measured in the corresponding histogram (Fig. 1). Forty-five of the 63 samples analyzed by cytometry were stained with 5 ll of PI. In these samples, the events were gated not only based on the FSC/SSC plot but also based on the PI staining. Statistical Analysis Statistical analysis were performed by using SPSS version 11.5 software (SPSS Corp., Chicago, IL), and statistical tests have been evaluated by using at least a significance level of To identify significant correlations between variables, a Spearman s test was performed in all cases, except where specifically indicated otherwise. Lines in the dispersion plots were calculated by regression. Data in tables are expressed as mean SD. Both the nonparametric Wilcoxon-matched pairs test and the Student t-test were performed to compare the mean of the different conditions assay. These tests have been evaluated by using at least a significance level of RESULTS Basic Sperm Parameters The mean and distribution of the basic sperm and semen parameters in the samples analyzed are shown in Table 1. The detailed sperm parameters corresponding to each of the samples included in the study can be found in the Supplementary Table 1. Normal morphology, sperm count, and motility correlated with each other (Spearman; P \ 0.005; data not shown). Other basic sperm parameters did not correlate. TUNEL Assay As Assessed by Microscopy The overall incidence of DNA fragmentation in 52 sperm samples was measured by microscopy by two independent observers. The mean percentage measured by the two observers was 15.8% 11.0% and 14.6% 11.0%, respectively (Table 1). The results obtained by microscopy between two different observers correlated (Spearman; r , P \ 0.001; Fig. 2A). Cytometry Part A 71A: ,

4 Figure 2. Correlation among the TUNEL results obtained. (A) Linear correlation of TUNEL-positive sperm count by microscopy between two different observers (Spearman; r , P \ 0.001). (B) Linear correlation of TUNEL-positive sperm count by microscopy and flow cytometry (Spearman; r , P \ 0.001). TUNEL Assay As Assessed by Cytometry and Correlation with the Results Obtained Using Microscopy The TUNEL results corresponding to each of the samples included in the study can be found in the Supplementary Table 1. The results obtained without PI staining to gate samples correlated very well with gating with PI (Spearman; r , P \ 0.001; data not shown). However, the means were different when comparing the TUNEL results obtained gating only by FSC/SSC ( ) and those using the PI option ( ; P\ 0.001; Wilcoxon-matched pairs test and Student t-test). However, we decided to consider only the results obtained without PI, because most articles in the field do not use PI either (5,28,37,40,43,44) and only in a few articles PI is used (24,38,45). The overall incidence of DNA fragmentation in 63 sperm samples measured by cytometry without PI was 39.7% 23.1% (Table 1). The Spearman correlation between the TUNEL-positive sperm as assessed by microscopy and cytometry was (or when cytometry gated by PI; P \ 0.001; Fig. 2B). Also the mean of TUNEL results obtained with microscopy and cytometry was compared. The % of TUNEL-positive sperm was significantly 2.6 times higher when measured by cytometry as compared to the same samples measured by microscopy. In the case of the assessment of the TUNEL results assessed by cytometry when gating with PI, the values obtained were 2.1 times higher than those obtained using microscopy (P \ 0.001; Wilcoxon-matched pairs test and Student t-test; Fig. 2B). Correlation Between the Results of TUNEL Assay and Basic Sperm Parameters The results of the TUNEL assay as assessed by the two different techniques correlated with rapid progressive and slow motility (Spearman; P \ 0.05; Fig. 3). Other basic sperm parameters did not correlate with TUNEL assay. DISCUSSION In this work we have compared the results of the TUNEL assay, as assessed by cytometry and optical microscopy, in the same set of samples and we have determined that they correlate well but that the mean results obtained using cytometry are 2.6 times higher than those obtained using microscopy (Table 1 and Fig. 2). It is now clear that the presence of DNA breaks in the sperm samples of patients may determine their reproductive potential. There are also many procedures to measure DNA breaks directly or indirectly (46 49). There are many articles clearly demonstrating the importance of the TUNEL results in determining many aspects of fertility (Table 2). Some studies have given a threshold value as a predictor of the quality of sperm and the assisted reproduction outcomes (Table 2; 7,9,10,14,24,30,38,47). Among all methods available, there have been different articles comparing the results of the TUNEL assay to other methods to assess DNA integrity (25). However, it is also clear from observation of Table 2 that TUNEL results in the different articles are usually reported either using optical microscopy or cytometry, but not using both methods (Table 2). Since the results obtained using both methods are different (Table 1 and Fig. 2), the comparison of the results described in the different articles published to date (Table 2) was very difficult. The mean of TUNEL-positive spermatozoa in our study was 15.3% 10.3% when assessed by microscopy. These results are in very good agreement with previously reported studies in fresh sperm, also assessed by microscopy (25,58; Table 2). Moreover, the mean result when assessed by flow cytometry was 39.7% 23.1% (32.8% 23.3% if gated with PI) Sperm TUNEL by Flow Cytometry and Microscopy

5 Figure 3. Linear correlations among TUNEL results and motility of the samples. TUNEL-positive sperm by microscopy as well as by cytometry correlates with rapid progressive motility (Spearman; P \ 0.05). These results are also in good agreement with previous studies, also assessed by flow cytometry (36,37,43; Table 2). Therefore, it is important to standardize this assay to measure the DNA breaks in the sperm, since the variability of the technique does not currently allow its use as a distinctive identifier of the samples with impaired fertility (35). The use of different variations of the TUNEL technique could explain the different results described in the different studies (32). It would be important to further investigate whether the use of different commercial TUNEL kits could modify the TUNEL positivity. A wide variety of TUNEL kits is available at present: the dutp-fluorescein TUNEL technique (14,25,50), the dutp-biotin modified TUNEL technique (5,6,26,38,56), and the dudp-fluorescein technique (9). It would be interesting to compare the different assays differing in sensitivity with the Roche s kit used in the present study, because the sensitivity of the detection of DNA strand breaks in sperm could be different. The procedure used to gate the population of interest in the dot plots when evaluating the samples using flow cytometry is also different in the different published studies. Some authors use PI to gate the population of interest in the flow cytometry (24,38,45). We decided to gate the population of interest based on the use of FSC and SSC dot plot as described (5,28,37,40,43,44), but also assessed the effect of gating with PI. These different procedures to gate the population of interest also changed the obtained results slightly (but significantly). We adjusted arbitrarily the M1 window in the plots of the negative controls to include 1% of TUNEL-positive events (Fig. 1A). Oosterhuis et al. (28) adjusted this window to obtain \3% of positive spermatozoa in the negative control. The same authors in the same work also used the plot of the positive control to establish the threshold ([85%) to count the percentage of positive spermatozoa in the sample plots. Also, different data processing methods have been used based on the plots obtained (36 38,45). Thus, the different procedures to establish a threshold between positivity and negativity in spermatozoa may change the percentage of TUNEL positivity in the samples significantly. So, it could be important to reach a consensus to measure the samples in such a way, so that the results could be compared in the different studies. To our knowledge, only a previous article has compared the TUNEL results as assessed by cytometry to those obtained by microscopy, although in a small number of samples (n 5 13; 45). In agreement with our results, these authors also found that the results of TUNEL as assessed by cytometry correlated to those obtained by microscopy (r ; 45). However, the ratio of % of TUNEL positivity by cytometry and optical microscopy was not reported in this study. Therefore, we have used the individual value plot data from this article (45) and determined that the ratio of % of TUNEL positivity by cytometry and optical microscopy in this study was 1.6. This value determined in a small number of samples is lower than that determined in the present article (2.6 when gating without PI or 2.1 when gating with PI). The differences could be due to the small number of samples analyzed in the previous article but also due to slight variations in the TUNEL assay procedure and quantification methods, such as variations in the flow cytometry strategy or the subjectivity when counting labeled sperm cells by microscopy. Nevertheless, their results are consistent with ours in that flow cytometry results in higher TUNEL values as compared to microscopy. There is another article where a simultaneous evaluation using fluorescence microscopy and flow cytometry was carried out on the same populations. However, this was performed to ensure that the percentages of cells detected by flow cytometry accurately characterized each sample, and it has been impossible to compare their results with ours since they did not report the data (63). Despite the correlation of the two techniques, some authors consider that the performance of the TUNEL assay improves when it is carried out using flow cytometry method as compared with the microscopy protocols (24,26,28,64). The reason is that Cytometry Part A 71A: ,

6 REFERENCE Table 2. Articles Describing TUNEL Results Assessed Either by Cytometry, by Microscopy, or Using Both Techniques SAMPLE SIZE % TUNEL POSITIVITY BY CYTOMETRY % TUNEL POSITIVITY BY MICROSCOPY a Lopes et al. (6) (0.5 75) (swim-up) Barroso et al. (32) (low motility) 1 1 (high motility) Donnelly et al. (50) (15 60); 18 (7 45) (gradient) Gandini et al. (51) (infertile) and 2.5 (fertile) Muratori et al. (45) ( ) (range) 5 28 (range) Oosterhuis et al. (28) (1.3 64) Sergerie et al. (52) (in controls) Ramos and Wetzels (53) (controls) vs (24 h with H 2 O 2 ); 9.4 vs (0, 50 Grays c radiation). Zini et al. (3) (infertile) and 10.2 (fertile) Duran et al. (9) vs. (pregnancy); (no pregnancy) (gradient) Sakkas et al. (40) ( ) Shen et al. (54) Weng et al. (33) (P) and 7 (C) (high motility) vs. 33 (P) and 25 (C) (low motility) Benchaib et al. (10) (abnormal samples) and 6 7 (normal) (gradient) Carrell et al. (55) (miscarriages) Erenpreisa et al. (56) (range) (methanol:acetone fixed) Muratori et al. (57) Erenpreiss et al. (24) (4 27) (frozen) Lachaud et al. (58) (0 h; washed); (0 h; gradient); (0 h; swim-up) Tesarik et al. (14) (P) and (C) vs (P) and (C) (late paternal) Greco et al. (34) (in ejaculates) vs (in testicular sperm) Sergerie et al. (36) (patients) and 13.0 (controls) Sergerie et al. (37) (patients) and (controls) Sergerie et al. (38) (patient) and (control) Stahl et al. (59) (2.5 31) in controls Sepaniak et al. (43) (nonsmokers) and 32 (smokers) (P \ 0.001) Chohan et al. (25) (infertile) and (fertile) de Paula et al. (60) (P) and (C); (P) and (C) (postfreeze) Aoki et al. (61) (low P1/P2); (normal P1/P2); (high P1/P2) Spermon et al. (62) ( ) (pretreatment); 25.0 (10 47) (postreatment) Present work a P, patient or intervention group; C, control. the TUNEL assay using cytometry allows rapid analysis of a large number of sperm in a short period of time and provides a highly sensitive means of defining sperm with fragmented DNA in a more objective, automatic, and precise manner. It is well known that the objective methods are highly reproducible and more sensitive for positive cells than the subjective methods. Flow cytometry as a laser-dependent method has a higher sensitivity for weak positive cells detection as compared to the human eye. The alternative of microscopy, although it has the advantage of allowing in situ examination, only allows the examination of a few hundred cells at most, it is more time consuming, and it is subject to higher observer bias since each cell has to be examined 1016 Sperm TUNEL by Flow Cytometry and Microscopy

7 visually in a qualitative subjective manner. However, despite the better sensitivity of flow cytometry, the most helpful method of DNA damage evaluation should be the one that has the better predictive value in reproduction outcome. So, the following step may be the standardization of DNA evaluation methods to apply them as useful pregnancy predictors. The different thresholds are quite variable ranging from 12 up to 25%. The variation in all these threshold values are likely to be due to the numerous methodological variants of the TUNEL technique as discussed earlier, but they also could be due to the pretreatment of the semen. In fact, one strategy to reduce sperm DNA fragmentation is the use of densitygradient centrifugation, swim-up, or glass wool filtration techniques to select spermatozoa. These procedures increase significantly the sperm DNA integrity as compared to the use of native semen (56,65 68). To use flow cytometry or microscopy is also an important choice, as it results in a large variation in the TUNEL positivity values (Table 2). So far the comparison of the TUNEL results described in articles has been difficult because of the relative lack of studies describing the correlation between both methods. The results reported in the present work should be very valuable in allowing better comparison of TUNEL studies. LITERATURE CITED 1. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, Irvine DS. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 1998;59: Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: Relationship with semen quality. J Androl 2000;21: Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA integrity. DNA denaturation and DNA fragmentation in fertile and infertile men. 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