Absence of Fas protein detection by flow cytometry in human spermatozoa

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1 FERTILITY AND STERILITY VOL. 81, NO. 4, APRIL 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Absence of Fas protein detection by flow cytometry in human spermatozoa Andrea Castro, M.Sc., a Daniela Parodi, B.S., a,d Ignacio Morales, M.D., b Marcia Madariaga, B.S., a Rafael Rios, M.D., c and Rosita Smith, M.Sc. a Institute of Maternal and Child Research, School of Medicine, University of Chile, San Borja-Arriarán Clinical Hospital, Santiago, Chile Received April 14, 2003; revised and accepted August 13, Supported by FONDECYT No Reprint requests: Rosita Smith, M.Sc., Institute of Maternal and Child Research, University of Chile, P.O. Box 226-3, Santiago, Chile (FAX: ; rsmith@med.uchile.cl). a Institute of Maternal and Child Research, School of Medicine, University of Chile. b Urology Department, Barros-Luco Trudeau Hospital, National Health Service, Santiago, Chile. c Endocrinology Department, San Borja- Arriarán Clinical Hospital, National Health Service, Santiago, Chile. d PROGRESAR Fellowship /04/$30.00 doi: /j.fertnstert Objective: To investigate the expression of Fas protein on the surface of ejaculated spermatozoa of normozoospermic and nonnormozoospermic men. Design: Prospective study. Setting: University infertility clinic. Patient(s): Twenty-three volunteer normozoospermic men (controls) and 43 men undergoing infertility evaluation (cases). Intervention(s): Analysis of ejaculated spermatozoa by indirect immunofluorescence of Fas protein by flow cytometry. Main Outcome Measure(s): Comparison of flow cytometric analysis of autofluorescence, control tests (secondary antibody and isotype control), and experimental tests (anti-fas monoclonal antibody) in the spermatozoa of ejaculated samples. Result(s): No expression of Fas protein was found on the surface of ejaculated spermatozoa of controls and cases. Conclusion(s): The Fas molecules are not present in substantial amounts in the ejaculated spermatozoa of normozoospermic and nonnormozoospermic men. Therefore, our results do not support the abortive apoptosis theory. (Fertil Steril 2004;81: by American Society for Reproductive Medicine.) Key Words: Fas protein, flow cytometry, human spermatozoa Recent studies have demonstrated that apoptosis is the underlying mechanism of germ cell death during normal spermatogenesis and that is a major mechanism in regulating spermatogenesis of various mammalian species, including humans. Substantial evidence suggests that apoptosis is under supracellular control in the testis that involves the Fas/FasL system (1 10). Fas (CD95, APO-1) is a 45-kDa type 1 transmembrane receptor protein, which belongs to the tumor necrosis factor/nerve growth factor receptor family (11). The specific interaction of Fas with its cognate ligand, the Fas ligand (FasL), initiates the recruitment of specific transduction signaling molecules at the intracellular domain of Fas, known as the death domain, leading to the initiation of the apoptotic pathways (12). In several mammalian species, including humans, both Fas and FasL are expressed in the seminiferous epithelium of the testes (2, 5, 7, 10, 13). It is generally accepted that Fas is expressed in germ cells and FasL in Sertoli cells. However, some controversies exist regarding the exact localization of these proteins. Several authors have reported the expression of Fas protein in the apoptotic spermatocytes and spermatids (2, 5, 7), and also in Sertoli cells (9, 10, 14). Others authors have detected FasL in Sertoli cells (2, 5, 7), both in Sertoli and germ cells (9, 14), or in neither of them (13). FasL has been suggested to activate the membrane-bound Fas receptors and thereafter the apoptotic cascade(s) in the germ cells. However, additional regulation of apoptosis by the Fas system may also exist because FasL has also been found in germ cells, in Fas, and in Sertoli cells. The presence of Fas and FasL on spermatozoa has been reported in humans (15, 16) and rodents (13), respectively. Reportedly, Fas protein is increased in men with an altered 1019

2 semen profile, thus supporting the theory of an abortive apoptosis, which could account for the presence of Faslabeled spermatozoa in the ejaculate (16). The aim of this study was to investigate the presence of the Fas protein in the membrane of ejaculated spermatozoa from men with varicocele, a history of cryptorchidism, or idiopathic alteration of their semen parameters. MATERIALS AND METHODS Subjects Semen samples were obtained by masturbation after 3 4 days of sexual abstinence from 66 patients attending the Human Reproduction Unit, Institute of Maternal and Child Research, San Borja-Arriarán Clinical Hospital. After 30 minutes of liquefaction, samples were analyzed for sperm concentration, morphology, progressive motility, and vitality according to World Health Organization (WHO) guidelines (17). Each man underwent an andrological examination, and special attention was paid to the presence of clinical varicocele and a history of cryptorchidism. Normozoospermic healthy men were studied as controls (n 23), and patients with varicocele (n 22), cryptorchidism (n 6), or with idiopathic abnormal seminal parameters (n 15) served as cases. The circulating serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were within the normal ranges in all the patients. The study was approved by the Institutional Review Board at the University of Chile Medical School. After the semen analysis, the samples were processed for Fas immunofluorescence by flow cytometry within 1 hour after ejaculation. Antibodies The presence of Fas receptor on the surface of the spermatozoa was detected with an IgG1 mouse anti-fas primary monoclonal antibody (DX2 clone, Code No. M3554) and an FITC-conjugated secondary antibody F(ab ) 2 fragment (rabbit antimouse IgGk) (Code No. F 0313). The isotype control used in each experiment was a mouse IgG1k (Code No. X 0931). All the antibodies were from Dako Corporation (Carpinteria, CA). Indirect Immunofluorescence For each experiment we used 0.3/ cells. After the initial wash in human tubal fluid (HTF) plus 4% of serum substitute supplement (SSS) (Irvine Scientific, Santa Ana, CA), the cells were resuspended in the same medium and incubated for 45 minutes at 37 C with the primary antibody at a concentration of 2 g antibody/ cells. Following the incubation period, the cells were washed in 1 ml of HTF plus 4% SSS, resuspended in 50 L of the secondary antibody diluted 1/50 in HTF plus 4% SSS, and incubated for an additional period of 30 minutes at 34 C. After two washes in 1 ml of HTF plus 4% SSS, the cells were resuspended at /ml of the same medium and stained with 1 g/ml of propidium iodide. In each analysis, the control samples included autofluorescence, a secondary antibody control, and an isotypematched control. Antibodies were added at saturating concentrations as previously determined by using the same protocol in peripheral leukocytes (positive control). Flow Cytometry Fluorescence analyses were performed using a Becton Dickinson FACSCalibur Flow Cytometer, CellQuest software, and standard settings: Fluorescence 1 (FL1) logarithmic detector 677 V and Fluorescence 3 (FL3) logarithmic detector 505 V. The forward scatter and side scatter properties were used to discriminate for size and granularity, respectively, in the flow cytometer. For each sample, the analysis was performed separately in the propidium iodide negative (viable) and positive (nonviable) subpopulations, and was based on the measurement of 10,000 20,000 cells. RESULTS To study the Fas protein expression in a selected group of patients we performed a detailed clinical history and physical examination in all patients. The control group consisted of normozoospermic healthy donors attending the in vitro fertilization clinic at our institute, who did not show any andrological abnormality on clinical examination. The cases consisted of men who had varicocele, cryptorchidism history, or were idiopathic oligozoospermic. The patients with varicocele had normal (n 15) or abnormal semen parameters (n 7; two with oligozoospermia, four with teratozoospermia, and one with oligoasthenoteratozoospermia). All the patients who had a history of cryptorchidism showed abnormal semen parameters (three with oligozoospermia and three with oligoasthenoteratozoospermia). The group of patients with idiophatic abnormal seminal parameters included: oligozoospermia (n 2), asthenozoospermia (n 1), teratozoospermia (n 2), oligoasthenozoospermia (n 3), asthenoteratozoospermia (n 2), and oligoasthenoteratozoospermia (n 5). The standard semen parameters in controls and patients are listed in Table 1. To investigate the presence of Fas protein on the surface of spermatozoa from controls and cases, we first discriminated between spermatozoa and cellular debris using the distribution of cellular size and granularity in the flow cytometer. Figure 1A shows the spermatozoa in region 1 (R1). In addition, we performed an individual analysis on the propidium iodide negative (viable, R2) and positive (nonviable or damaged cells, R3) subpopulations of spermatozoa. We performed this analysis because of autofluorescence and the possible occurrence of higher nonspecific antibody binding to damaged cells Castro et al. Absence of Fas protein in human sperm Vol. 81, No. 4, April 2004

3 TABLE 1 Seminal parameters of the patients. Normal forms (%) Progressive motility (%) The Fas expression studied by immunofluorescence and flow cytometry did not show any difference between the mouse antihuman Fas and the isotype monoclonal antibody in both spermatozoa subpopulations analyzed (Fig. 1B), indicating no expression of Fas protein on the surface of ejaculated spermatozoa. As positive controls we used peripheral blood mononuclear cells (PBMC), which were independently analyzed in subpopulations of lymphocyte and monocyte as shown in R1 and R2, respectively (Fig. 2A). We found a positive reaction with the mouse antihuman Fas monoclonal antibody in 100% of monocytes and in a subpopulation of 50% of the lymphocytes, but there was no positive reaction with the isotype monoclonal antibody (Fig. 2B). DISCUSSION Sperm (mill/ml) Sperm vitality (%) Controls (n 23) Mean SD Range Cases (n 43) Normo- (n 15) Mean SD Range Oligo- (n 7) Mean SD Range Astheno- (n 1) Mean SD Range Terato- (n 6) Mean SD Range Oligoastheno- (n 3) Mean SD Range Asthenoterato- (n 2) Mean SD Range Oligoasthenoterato- (n 9) Mean SD Range , Note: The prefixes in the table precede zoospermia. Castro. Absence of Fas protein in human sperm. Fertil Steril Recent studies have demonstrated that programmed cell death occurs spontaneously in the cycle of the seminiferous epithelium (18). Induced germ cell apoptosis occurs at specific stages of the spermatogenic cycle, and the existence of a supracellular control involving the Fas/FasL system of germ cell death during spermatogenesis has been documented (3, 5 7, 19, 20). The role of the Fas/FasL system in the regulation of testicular germ cell apoptosis has been demonstrated by several authors, mainly based on the Fas/FasL system expression in testicular tissue (1 3, 5, 7, 8, 10, 14) and on the in vitro blockade of induced apoptosis under serum- and hormone-free conditions (7, 8, 19), with anti-fas or anti- FasL antibodies (5, 7), disruption of FasL by antisense oligonucleotide treatment (5), and with a universal caspase inhibitor (7). Additional evidence about the participation of Fas/FasL system in germ cell apoptosis includes in vivo radiation exposure (6), treatments with Sertoli cell toxicants (5, 6), and withdrawal of endogenous testosterone (10). In the present study, we report the absence of Fas expression on the surface of human spermatozoa from normozoospermic and nonnormozoospermic men. Our results are not concordant with previous reports and question the proposed abortive theory of the apoptotic process during spermatogenesis (15, 16, 21). The abortive theory postulates that some forms of infertility associated with the presence of abnormal semen parameters might result from an aborted program of germ cell death. In this case, the apoptotic process is incomplete and fails to eliminate germ cells, even though the apoptotic program in the testes has been initiated (15, 16, 21). The abortive apoptosis suggests that the apoptotic mechanisms have misfunctioned, have been overridden, or have not been completed due to the release of the quasi-apoptotic spermatozoa into the seminiferous tubule (15, 16, 21). We studied Fas expression using spermatozoa in fresh semen samples from healthy normozoospermic men (controls) without andrological abnormalities and from patients with varicocele, a history of criptorchidism, or idiopathic alteration of the semen parameters (cases). In previous reports, Fas expression on the surface of human spermatozoa was compared between normozoospermic men and men presenting different degrees of alterations in their semen parameters (16). Fas expression in normozooospermic men reached up to 10%, but in subjects with alterations of sperm number, motility, or morphology, it reached up to 50% of the spermatozoa per sample, indicating that in these patients an abortive apoptosis had taken place (16). Fas expression found by Sakkas et al. (16) in oligoasthenoteratozoospermia and oligoteratozoospermia samples (1% 50%), may be explained because they did not study selected subpopulations of men, which could account for the high variation in the percentage of Fas positive ejaculated sperm. According to our results, no expression of Fas protein was found on the spermatozoa from a considerable number of normozoospermic controls and in patients with a normal or FERTILITY & STERILITY 1021

4 FIGURE 1 Representative dot plots of the flow cytometry analysis of Fas expression on spermatozoa by indirect immunofluorescence. (A) The distribution of granularity vs. size of events acquired in the flow cytometer and the events considered as spermatozoa population for the cytometric analysis are shown in region 1 (R1 in the left histogram). The damaged or nonviable spermatozoa that incorporate propidium iodide (high fluorescence intensity-fl3) are shown in R3 (right histogram) and were separated for analysis from the viable spermatozoa (low fluorescence intensity-fl3) as shown in region 2 (R2 in the right histogram). (B) Spermatozoa were processed by immunofluorescence as described in Materials and Methods. The analysis of the viable spermatozoa is shown in the upper histograms and of the nonviable spermatozoa in the lower histograms. A comparison of the corresponding autofluorescence (negative control), secondary control (negative control), isotype control and anti-fas, shows no differences between them. Castro. Absence of Fas protein in human sperm. Fertil Steril abnormal semen profile. Moreover, our patients exhibited common andrological disorders, such as varicocele and a history of cryptorchidism, and, therefore, the criteria used to select our patients cannot explain the observed differences in the results obtained. Regarding the methodology employed, indirect immunofluorescence, the main differences between the work of Sakkas et al. (15, 16) and the present study are the use of the anti-fas antibody DX2 clone from a different manufacturer, the use of an isotype-matched antibody, and the inclusion in this study of a positive control of Fas expression. The use of the same clone but from a different manufacturer was not trivial in our study because when we used the DX2 clone and its corresponding isotype antibody from two different manufacturers (Pharmingen and Becton Dickinson), we found nonspecific binding for both antibodies, mainly on sperm from abnormal semen samples (not shown). Therefore, in the present work, the clone DX2 from Dako, which did not show nonspecific binding, was used. Furthermore, certain evidence that demonstrates that autofluorescence and nonspecific binding of antibodies are higher in nonviable or damaged cells (22). Therefore, in the 1022 Castro et al. Absence of Fas protein in human sperm Vol. 81, No. 4, April 2004

5 FIGURE 2 Representative dot plots of the flow cytometry analysis of Fas expression on mononuclear peripheral leukocytes by indirect immunofluorescence. (A) The distribution of granularity vs. size of events acquired in the flow cytometer and the events considered as subpopulations of lymphocyte and monocyte for the cytometric analysis are gated in R1 and R2, respectively. (B) Cells were processed by immunofluorescence as described in Materials and Methods. Only the viable cell analysis is shown. The upper histograms show the lymphocyte analysis and the lower histograms show the monocyte analysis. A comparison of the corresponding autofluorescence (negative control), secondary control (negative control), isotype control and anti-fas, indicates that monocytes and a subpopulation of lymphocytes are positive for Fas. Castro. Absence of Fas protein in human sperm. Fertil Steril present study, we conducted a separate analysis in these subpopulations. In relation to the positive control, the percentage of Fas expression shown in cell subpopulations of monocyte and lymphocyte was in accordance with that reported in the literature (23). The analysis by flow cytometry in peripheral blood from normal controls showed Fas protein in less than 10% of the B-lymphocytes and up to 30% of the T-lymphocytes. In contrast, in shorter surviving leukocytes, such as monocytes and granulocytes, the Fas protein was constitutively expressed (23). Several studies suggest that the subpopulation of Fas-positive lymphocytes represents activated lymphocytes that must be eliminated for the attenuation of secondary autoimmune reactions contributing to the maintenance of self-tolerance (12). It is interesting to mention that the function of the FasL protein in rodents testis has been strongly questioned, based upon the fact that this protein is generally believed to be expressed in Sertoli cells, where, like in humans, it may play an immune protective role and serve as a physiological regulator of germ cell apoptosis. However, another study demonstrated that the attribution of FasL expression to Sertoli cell is erroneous because the protein is only displayed on mature spermatozoa (13). FERTILITY & STERILITY 1023

6 The occurrence of the apoptotic process in spermatocytes and spermatids has been documented by several authors in the adult testis of human and other mammalian species. These reports include the parallel analysis of different approaches, such as the apoptotic morphological characteristics by transmission electron microscopy, deoxyribonucleic acid (DNA) fragmentation by Tdt-mediated dutp nick end labelling (TUNEL), and Fas expression by immunohistochemistry (3, 7, 8, 10, 18, 24 26). Moreover, there is evidence of increased apoptosis in spermatocytes and spermatids in human spermatogenic defects, and after the in vivo and in vitro withdrawal of androgen or survival factors (7, 8, 10, 18, 24). In previous studies in human testicular biopsies, the proportion of apoptotic Fas-positive germ cells evaluated was so scarce as 2 and 4 per 100 nuclei of Sertoli cells in normal tissues and incomplete postmeiotic arrest, respectively (2, 3). Therefore, it is difficult to explain the high Fas expression previously described (16) considering the scarce expression of testicular Fas-positive germ cells and the close correlation between the apoptotic markers and Fas expression on testicular germ cells, especially considering that part of the apoptotic cells are phagocytated by Sertoli cells (3). Different apoptotic markers, such as some morphological characteristics, DNA fragmentation, annexine V binding, and caspase expression, have been reported to be correlated with each other, indicating that an apoptotic process may be present in the human spermatozoa (20, 26 29). However, correlation between Fas and other apoptotic markers has not been demonstrated until now. A recent study by Sakkas et al. (15) found that TUNEL positivity and apoptotic markers do not always exist simultaneously; however, semen samples with a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas expressing cells. Moreover, in a recent investigation, sperm DNA fragmentation did not have a significant relationship with other characteristics suggestive of apoptosis, such as nuclear shape, chromatin packing, or presence of translucent vacuoles in cytoplasmic residues, but was correlated with ultrastructural features indicative of an impaired motility (30). Therefore, sperm DNA fragmentation would also indicate abnormalities compatible with a lower degree of maturation, as has been previously suggested (28 30). The apoptosis-like phenomena in the spermatozoa could be the final result of various pathologies, which could be triggered by changes in hormone levels or by abnormal exposure to death-promoting signals, such as reactive oxygen species. The presence of DNA fragmentation, annexine V binding, and caspase protein expression together in the human spermatozoa suggest that an apoptosis process has taken place. However, whether this apoptosis process is initiated in the testes or along the male genital tract is unknown. Our results suggest that the Fas/FasL system, which is involved in the apoptotic regulation of the testicular germ cells, is not operating on the ejaculated spermatozoa. References 1. Eguchi J, Koji T, Nomata K, Yoshii A, Shin M, Kanetake H. Fas-Fas ligand system as a possible mediator of spermatogenic cell apoptosis in human maturation-arrested testes. Hum Cell 2002;15(1): Francavilla S, D Abrizio P, Rucci N, Silvano G, Properzi G, Straface E, et al. Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis. J Clin Endocrinol Metab 2000; 85(8): Francavilla S, D Abrizio P, Cordeschi G, Pelliccione F, Necozione S, Ulisse S, et al. 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