Measuring apoptosis in human spermatozoa: a biological assay for semen quality?

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1 FERTILITY AND STERILITY VOL. 74, NO. 2, AUGUST 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Measuring apoptosis in human spermatozoa: a biological assay for semen quality? G. Jurjen E. Oosterhuis, M.D., a Andre B. Mulder, M.D., Ph.D., b Ellen Kalsbeek-Batenburg, B.Sc., b Cornelis B. Lambalk, M.D., Ph.D., c Joop Schoemaker, M.D., Ph.D., c and Istvan Vermes, M.D., Ph.D. b Medisch Spectrum Twente Hospital Group, Enschede; and Academic Medical Center Vrije Universiteit, Amsterdam, The Netherlands Received November 16, 1999; accepted February 10, Reprint requests: Istvan Vermes, M.D., Ph.D., Medisch Spectrum Twente Hospital Group, Department of Clinical Chemistry, P.O. Box , NL 7500 KA Enschede, The Netherlands (FAX: ; i.vermes@wxs.nl). a Department of Obstetrics and Gynecology, Medisch Spectrum Twente Hospital Group. b Department of Clinical Chemistry, Medisch Spectrum Twente Hospital Group. c Institute for Endocrinology, Reproduction and Metabolism, Academic Medical Center Vrije Universiteit /00/$20.00 PII S (00) Objective: [1] To determine whether apoptosis can be measured in ejaculated spermatozoa by flow cytometry using the Annexin V assay, which measures expression of phosphatidylserine on the outer leaflet of the cell membrane, or the TUNEL (terminal deoxynucleotidyl transferase mediated dutp [deoxy-uridine triphosphate] nick end labeling) assay, which measures occurrence of DNA strand breaks and [2] to correlate the outcome with routine semen variables and the hypoosmotic swelling (HOS) test. Design: Pilot study and clinical trial. Setting: Large teaching hospital and fertility center. Patient(s): Men whose semen was studied for various reasons. Main Outcome Measure(s): Percentage of apoptotic spermatozoa by two different assays, percentage of necrotic spermatozoa, concentration and motility of spermatozoa, and outcome of the HOS test. Result(s): Apoptosis can be measured in spermatozoa by flow cytometry using the Annexin V assay and the TUNEL assay. Twenty percent of spermatozoa were apoptotic according to both assays. A significant inverse correlation was seen between phosphatidylserine expression (Annexin V assay) and sperm concentration (r 0.389; P.05) and motility (r 0.289; P.05). A highly significant inverse correlation was seen between DNA double-strand breaks (TUNEL assay) and sperm concentration (r 0.629; P.0001). Conclusion(s): Flow cytometry can easily and reliably detect phosphatidylserine expression on the outer leaflet of the cell membrane and DNA strand breaks, both of which are hallmarks of apoptosis. About 20% of ejaculated spermatozoa are apoptotic, and the concentration of spermatozoa is lower in men with more apoptotic spermatozoa. (Fertil Steril 2000;74: by American Society for Reproductive Medicine.) Key Words: Annexin V assay, apoptosis, male factor infertility, spermatozoa, TUNEL assay Various tests are used to investigate the quality of human sperm, and the World Health Organization recently described criteria by which semen may be deemed adequate for potential fertility (1). Although conventional analysis of semen gives considerable information, its limitations in predicting the ability to achieve pregnancy are well acknowledged; other, more reliable technologies are still needed. In the past few years, attention has been paid to the significance of physiologic cell death, which occurs through during normal spermatogenesis (2, 3). Despite considerable effort, little information is available on the biological significance of apoptosis in spermatozoa or its possible role in male infertility. This lack of knowledge stems in part from the difficulties of studying the process of apoptosis in spermatozoa in vitro. In an attempt to investigate the role of apoptosis in the quality of human semen, we studied two apoptosis assays. During the process of apoptosis, one of the earliest events is disturbance of the asymmetry of the bilayer of the cell membrane. Phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane (4). The exposure of PS on the outside of the cell surface provides a chance to detect cells that are in an early stage of apoptosis. These cells can be detected 245

2 by the calcium-dependent binding of annexin V (5, 6). In this study, we describe a test that detects expression of PS on spermatozoa by using staining with fluorescein isothyocyanate (FITC) labeled annexin V in conjunction with flow cytometry. By using additional labeling with the vital dye propidium iodide (PI), viable, apoptotic, and necrotic sperm populations can be distinguished simultaneously. The most characteristic feature of apoptosis, but one that occurs relatively late phenomenon in the apoptosis cascade, is activation of an endogenous endonuclease that generates numerous DNA double-strand breaks in chromatin. As a second technique for detecting apoptosis, we measured DNA fragmentation by using terminal deoxynucleotidyl transferase to incorporate FITC-labeled deoxynucleotides; this method, known as the TUNEL (terminal deoxynucleotidyl transferase [TdT] mediated dutp [deoxyuridine triphosphate] nick end labeling) assay, is also based on flow cytometry. To elaborate the diagnostic possibilities of these tests, the results are compared with both the results of the conventional semen parameters, i.e. the concentration and motility, and the hypoosmotic swelling (HOS)-test. MATERIALS AND METHODS Semen Samples Semen samples from 102 patients who visited our infertility clinic were collected after masturbation and allowed to liquefy at room temperature. All tests were started within 2 hours after collection of the samples. All experiments were conducted under the rules of the Declaration of Helsinki and were approved by the ethics subcommittee for research involving human subjects of Medisch Spectrum Twente Hospital Group, Enschede, The Netherlands. Routine Semen Analysis Routine screening was performed as described by the World Health Organization (1). Morphology of the spermatozoa was not determined in all semen samples and was therefore not taken into account in the analysis of the results. Motility of the spermatozoa was measured by using World Health Organization criteria; a drop of semen was placed on a clean glass slide that was heated to 37 C and then covered with a coverslip. The preparation was examined at a magnification of 400 by using a phase-contrast microscope. Motility was measured by determining the movements of at least 100 spermatozoa that were categorized as follows: categories A and B, spermatozoa with propulsive movements; category C, spermatozoa moving in place; and category D, immotile spermatozoa. The spermatozoa in categories A and B were considered motile spermatozoa. All tests were done twice. Hypoosmotic Swelling Test (HOS) The HOS test was performed as described by Jeyendran et al. (7) and as reported elsewhere (8). Before the test, 1% of the spermatozoa were noted to have coiled tails. A total of 0.5 ml of hypoosmotic solution (0.735 g sodium citrate. 2H 2 O, g fructose, and 100 ml H 2 O) and ml semen were gently mixed and incubated for minutes at 37 C. At least 100 random spermatozoa were then examined at magnification of 400. Spermatozoa were considered HOS positive if they showed signs of swelling, as described by Jeyendran et al. (7). The percentage of sperm swelling was then calculated. Annexin V/PI Binding Assay The technique used for the Annexin V assay and its characteristics are described elsewhere (9). To remove in vivo bound Annexin V, 200- L semen samples were washed twice with 1 mmol/l Ethylene Glycerol Tetraacetic Acid (EGTA)/Hepes A buffer (137 mmol/l NaCl, 2.68 mmol/l KCl, 10 mmol/l Hepes, 1.7 mmol/l MgCl 2, 25 mmol/l glucose; ph, 7.4) and once with Hepes A buffer without EGTA. One million spermatozoa were incubated in Hepes A buffer with 2.5 mmol/l Ca 2 that contained FITC-labeled Annexin V at a final concentration of 0.1 mg/ml [w/v] (APOPTEST; FITC, Nexins Research BV, Hoeben, The Netherlands) and PI at a final concentration of 1.3 g/ml [w/v] (Sigma, Deisenhofen, Germany). A negative control sample was analyzed by using Hepes A buffer without Ca 2. After exactly 15 minutes, the spermatozoa were analyzed in a flow cytometer (EPICs Profile II; Coulter Electronics, Krefeld, Germany). A minimum of 10,000 spermatozoa were examined for each test. The sperm population was gated by using forward-angle light scatter; side-angle light scatter was used to exclude electronic noise and debris. The FITC-labeled Annexin V positive sperm cells were measured in the FL1 channel and the PI-labeled sperm cells were measured in the FL2 channel of the flow cytometer. All tests were run in duplicate. The TUNEL Assay The characteristics of the TUNEL assay have been described elsewhere (10). The assay was performed for the most part according to the method described by Gavrieli et al. (11). One million washed spermatozoa were fixed with 4% [w/v] paraformaldehyde for 30 minutes at room temperature. The cells were washed once with Hepes A buffer. After permeabilization with 0.2% [v/v] Triton X-100 for 2 minutes on ice, the samples were washed twice with Hepes A buffer. A positive control sample was analyzed by incubating the spermatozoa with 50 U DNase (RNase Free; Boehringer Mannheim, Mannheim, Germany) in 0.2% [w/v] MgCl 2 /0.1% [w/v] CaCl 2 solution for 10 minutes at room temperature. Samples were then washed twice with Hepes A buffer. 246 Oosterhuis et al. Apoptosis in spermatozoa Vol. 74, No. 2, August 2000

3 TABLE 1 Concentration, motility, HOS test results, presence of DNA strand breaks (TUNEL assay), PS expression (annexin V binding), and cell viability (PI labeling) of semen samples. Sperm characteristic No. of semen samples analyzed a Mean SD Minimum Median Maximum Concentration ( 10 6 spermatozoa/ml) Motility category a b (%) HOS-positive spermatozoa (%) TUNEL-positive spermatozoa (%) Annexin V positive/pi-negative spermatozoa (%) Annexin V positive/pi-positive spermatozoa (%) Note: HOS hypoosmotic swelling; PI propridium iodide; PS phosphatidylserine; TUNEL terminal deoxynucleotidyl transferase mediated dutp (deoxyuridine triphosphate) nick end labeling. a In some cases, some variables were not analyzed because of lack of biological material. Fifty microliters of TUNEL mix (TdT and FITC-labeled dutp in a 1:9 ratio) (Boehringer Mannheim) were added. To the negative control, no dutp was added. The samples were incubated for 60 minutes at 37 C and washed twice with Hepes A buffer. They were then resuspended in 500 L Hepes A buffer. For each determination, at least 5,000 spermatozoa were examined by using flow cytometry. The sperm population is gated by using forwardangle light scatter, and side-angle light scatter dot plot to exclude electronic noise and debris. The FITC-labeled dutp-positive spermatozoa were measured in the FL1 channel of the flow cytometer. Statistical Analysis Data analyses were performed by using the nonparametric Mann-Whitney U test to evaluate differences in data type and distribution. The Spearman rank correlation test was used to calculate the correlation coefficient between the relative number of TUNEL-positive spermatozoa and the cell concentration. The Pearson correlation coefficient was used for all other correlation studies. Differences were considered significant if the probability of their occurrence by chance was.05 (paired Student t-test). RESULTS Conventional Semen Analysis Results of the routine semen analysis are shown in Table 1. The investigated semen samples had a sperm concentration of /ml (mean SD); 47% 20% of the spermatozoa appeared to be motile, and 79 11% of the spermatozoa were HOS positive. Apoptosis as Indicated by PS Expression (Annexin V assay) A typical example of cells stained with FITC-labeled Annexin V and PI is shown in Figure 1. As many as 20% 10% apoptotic cells (Annexin V positive but PI-negative) and 19% 7% secondary necrotic spermatozoa (positive for both Annexin V and PI) were found (Table 1). A significant inverse correlation was seen between the percentage of Annexin V positive spermatozoa and both the concentration of spermatozoa (r 0.389; P.05) and the motility of the sperm cells (r 0.289; P.05). The percentage of apoptotic cells did not correlate significantly with the outcome of the HOS test (r 0.045; P.719). Apoptosis as Measured by DNA Double- Strand Breaks (TUNEL assay) The TUNEL assay used to determine cells with DNA double-strand breaks was reliable. Negative control samples showed a signal in less than 3% of the cells, and in positive control samples, more than 85% of the cells showed a signal. Figure 2 shows a typical example of cells stained with FITC-labeled dutp. In the investigated semen samples, 20% 15% of the spermatozoa showed DNA fragmentation (Table 1). A strong significant inverse correlation was seen between the percentage of TUNEL-positive cells and the concentration of spermatozoa (r 0.629; P.0001) (Fig. 3). The percentage of TUNEL-positive spermatozoa was inversely but not significantly correlated with the results of the HOS test (r 0.344; P.054). No correlation was seen between the number of TUNELpositive spermatozoa and the percentage of spermatozoa that showed PS expression. DISCUSSION No relation exists between the outcome of conventional semen analysis, including spermatozoa concentration and motility, and pregnancy in assisted reproduction if sperm variables are above certain minimum levels (12, 13). Con- FERTILITY & STERILITY 247

4 FIGURE 1 (A) Forward-angle light scatter/side-angle light scatter dot plot. (B) Dot plot of double staining with fluorescein isothyocyanate labeled Annexin V and propridium iodide (PI). The lower left quadrant contains viable, nonapoptotic cells, which exclude PI and are negative for fluorescein isothyocyanate labeled Annexin V. The lower right quadrant shows apoptotic cells, which exclude PI but bind Annexin V. The upper right quadrant represents secondary necrotic cells, which bind Annexin V as well as PI. We studied two simple assays for measuring apoptosis in fresh ejaculated spermatozoa that might prove to be adequate tools for further determination of the quality of semen. The two assays are based on flow cytometry, which allows measurement of apoptosis at the single-cell level as well as automatic analysis of thousands of cells in a few seconds. This advantage of flow cytometry in apoptosis research cannot be overstated. Although it has the advantage of al- FIGURE 2 Frequency distribution histograms of spermatozoa stained with fluorescein isothyocyanate labeled dutp without using TdT (A; negative control); with DNase, which digests DNA (B; positive control); and with the use of TdT (C). The horizontal line indicates apoptotic spermatozoa that are positive for fluorescein isothyocyanate labeled dutp. sequently, new techniques have been proposed to increase the discriminating power of semen analysis. The importance of cell death in normal spermatozoa is currently a subject of great interest. Some investigators have found that transgenic animals, in which genes for proteins involved in the control of apoptosis are deleted or overexpressed, showed increased germ cell apoptosis, leading to oligozoospermia and infertility (14 16). In spermatozoa that were thawed after cryostorage, Annexin V binding has been shown to correlate with motility (17). 248 Oosterhuis et al. Apoptosis in spermatozoa Vol. 74, No. 2, August 2000

5 FIGURE 3 Inverse correlation between TUNEL-positive spermatozoa and sperm count (r 0.629; P.0001). lowing in situ examination of cells, light microscopy permits examination of a few hundred cells at the most. Moreover, bias can be introduced; each cell has to be examined visually, and cells could be counted twice. Flow cytometry permits examination of thousands of cells in a few seconds, thereby enormously increasing precision. We compared the results of the two tests with the findings of routine semen analysis to assess the relation between apoptosis and routine semen variables. As expected, the relative number of spermatozoa that express PS (Annexin V positive cells) and the relative number of cells that show DNA double-strand breaks (TUNEL-positive cells) strongly correlated with lower sperm concentrations. This means a low sperm concentration might be caused by a high rate of apoptosis. We have reason to believe that this apoptotic process takes place just before or just after ejaculation because the TUNEL assay, which measures apoptosis at a relatively late stage of the process, is more strongly correlated with sperm concentration than was the Annexin V assay, which measures apoptosis during an early stage of the process. Apoptosis can take place in a few hours or even minutes (18). In addition, the expression of PS, a feature seen in an early stage of apoptosis, was also significantly correlated with lower sperm motility. Again, we hypothesize that this assay can detect cells that are early in their apoptotic process, which induces decreased motility, but it cannot detect decreased concentration as well as the TUNEL assay does. No correlation is seen between the relative number of spermatozoa that express PS and the number that show DNA strand breaks. This finding can be explained by the fact that expression of PS and presence of DNA strand breaks occur during different phases of the apoptotic process. The inverse correlation between the percentage of TUNELpositive cells and the percentage of HOS-positive cells showed a trend toward significance (P.054). We have shown before that sperm count is significantly correlated with the percentage of HOS-positive cells (8). We would therefore have expected the number of apoptotic cells to correlate significantly and inversely with the percentage of HOSpositive spermatozoa. The low number of semen samples adequate for determining TUNEL (n 34) is probably due to the lack of statistical significance. We conclude that the use of flow cytometry to measure apoptosis in semen is a novel and reliable approach to study the functional viability of spermatozoa. Our data show that measurement of PS expression and testing for the presence of DNA strand breaks in spermatozoa may be suitable biological screening tests for detecting apoptotic sperm cells in humans. The additional value of measuring apoptosis to test for male infertility should be evaluated in a prospective study in relation to the occurrence of pregnancy or fertilization, a study which we are currently undertaking. Intervention by manipulating apoptosis might ultimately result in improvement of semen characteristics. References 1. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 3rd ed. Cambridge, UK: Cambridge University Press, Hikim AP, Wang C, Lue Y, Johnson L, Wang XH, Swerdloff RS. Spontaneous germ cell apoptosis in humans: evidence for ethnic differences in the susceptibility of germ cells to programmed cell death. J Clin Endocrinol Metab 1998;83: Erkkila K, Hirvonen V, Wuokko E, Parvinen M, Dunkel L. 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