IN VITRO FERTILIZATION

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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. IN VITRO FERTILIZATION Influence of deoxyribonucleic acid damage on fertilization and pregnancy Ralf Henkel, Ph.D., a Marjam Hajimohammad, a Thomas Stalf, Ph.D., b Christiaan Hoogendijk, M.Sc., c Claas Mehnert, M.Sc., b Roelof Menkveld, Ph.D., c Holger Gips, M.D., b Wolf-Bernhard Schill, M.D., a and Thinus F. Kruger, M.D. c Justus Liebig University and Institute for Reproductive Medicine, Giessen, Germany; and Tygerberg Hospital and University of Stellenbosch, Tygerberg, South Africa Received May 1, 2003; revised and accepted September 3, Supported by research grant no. I/ of the Volkswagen Stiftung, Hannover, Germany. Reprint requests: Ralf Henkel, Ph.D., Center for Dermatology and Andrology, Gaffkystrasse 14, D Giessen, Germany (FAX: ; ralf.henkel@derma.med. uni-giessen.de). a Center for Dermatology and Andrology, Justus Liebig University, Giessen, Germany. b Institute for Reproductive Medicine, Giessen, Germany. c Reproductive Biology Unit, Tygerberg Hospital and University of Stellenbosch, Tygerberg, South Africa /04/$30.00 doi: /j.fertnstert Objective: To investigate sperm DNA damage in relation to fertilization and pregnancy. Design: Prospective study. Setting: The Institute of Reproductive Medicine, Giessen, Germany. Patient(s): Semen collected from 249 patients attending the IVF program. Main Outcome Measure(s): The percentage of terminal deoxynucleotidyl transferase-mediated dudp nick-end labeling- (TUNEL-), Fas-, and annexin-v-positive sperm and the proportion of green-fluorescing sperm in the acridine orange stain was determined and correlated with sperm concentration, motility, fertilization, and pregnancy. Result(s): Significant correlations with the concentration of motile sperm were only found for the acridine orange stain (before and after sperm separation) and for the TUNEL assay (after sperm separation). Moreover, patients whose sperm had a high percentage of DNA fragmentations showed significantly lower pregnancy rates (TUNEL assay: 19.05% vs %; acridine orange stain: 24.58% vs %). The apoptosis parameters (annexin V binding and Fas expression) showed no statistically significant differences. Conclusions: Our data clearly demonstrate that DNA fragmentation, as determined by the TUNEL assay, is predictive for pregnancy in IVF. This implies that spermatozoa with DNA fragmentation can still fertilize an oocyte but that when paternal genes are switched on, further embryonic development stops, resulting in failed pregnancy. It seems that, at least in the patients we analyzed, apoptosis in the sperm does not play a role for fertilization. This would imply that DNA fragmentation in human spermatozoa is caused by external factors, such as reactive oxygen species, rather than by apoptosis. (Fertil Steril 2004;81: by American Society for Reproductive Medicine.) Key Words: DNA damage, apoptosis, DNA fragmentation, DNA integrity, pregnancy failure, human spermatozoa It is well known that the fertilization process depends on a whole sequence of functional parameters of the sperm (1) and the oocyte (2, 3). In recent years, scientists and clinicians attempting to understand the fertilization process better and to improve fertility diagnostics have become interested in apoptosis and DNA integrity. Apoptosis is the controlled disassembly of cells from within (4) and is characterized by changes in the phospholipid content of the plasma membrane outer leaflet. A very early sign of apoptosis is the translocation of the negatively charged phospholipid phosphatidylserine, which is normally located on the inner leaflet of the plasma membrane (5), from the inner to the outer side of the plasma membrane. This phospholipid is thus exposed on the external membrane surface (6). Another factor that has been shown to be involved in sperm apoptosis is Fas (7), a type I cell-surface protein that belongs to the tumor necrosis factor/nerve growth factor receptor family and mediates apoptosis (8). Binding of Fas ligand (FasL) to Fas induces apoptosis (9). Because Sertoli cells express FasL, this system might be involved in the regulation of spermatogenesis with regard to a limiting number of sperm cells that can be supported by the Sertoli cell (7, 10). Intimately involved in the deliberate disassembly of cells into so-called apoptotic bodies are the cytosolic aspartate-specific proteases 965

2 (caspases) (4), which have also been detected in their active form in human spermatozoa (11). Because of these apoptotic events, DNA is fragmented and the cells disassembled (12). However, sperm DNA fragmentation occurs in every ejaculate and can also be induced by reactive oxygen species (ROS) (13, 14). It also seems that patients who undergo treatment with assisted reproductive procedures, especially with intracytoplasmic sperm injection (ICSI), have a significantly higher risk of embryonic death because sperm with fragmented DNA might fertilize an oocyte (15). Therefore, the role of paternal DNA in early human embryo development cannot be ignored. Recently, Sun et al. (16) and Lopes et al. (17) found a correlation between DNA fragmentation and fertilization rate after IVF and ICSI. Others later confirmed these results (18, 19). Moreover, it can also be speculated that fragmented DNA might be a cause of early embryo death (20). Janny and Ménézo (21) found a strong relationship between cleavage and blastocyst formation rate, and Shoukir et al. (22) demonstrated a lower blastocyst formation rate after ICSI compared with IVF. Tesarik et al. (23) presented data showing that paternally derived abnormalities can manifest as early as in the zygote and can lead to embryos that cannot even reach the blastocyst stage or that do not implant in the uterus. However, the question of what causes such pregnancy failures remains: is DNA fragmentation the result of sperm-cell apoptosis, or is it initiated by ROS? Most studies investigating the influence of apoptosis on fertilization and pregnancy have made use of the presence of endogenous nicks in ejaculated spermatozoa, as identified by terminal deoxynucleotidyl transferase-mediated dudp nickend labeling (TUNEL) assay (13) or the Comet assay (24), which can be indicative of an apoptotic process (25). The TUNEL assay evaluates a rather late stage of apoptosis (DNA fragmentation) and detects both apoptotic and necrotic cells (26, 27). Early phases of apoptosis are characterized by disturbed membrane function, namely by an asymmetric distribution of phospholipids, which occurs before a progressively disturbed integrity of the membrane (6), and the expression of Fas (7, 8), and cannot be evaluated by the TUNEL assay. Therefore, in this study, we investigated DNA fragmentation by means of the TUNEL assay and early signs of apoptosis by means of annexin V binding, which, depending on Ca 2, has a high affinity to phosphatidylserine (28). In addition, the expression of Fas was detected by a non celldeath-inducing anti-fas antibody (anti-cd95), and DNA integrity was studied by the acridine orange stain. Our aim was to gain deeper insight into fertilization and pregnancy failure due to DNA damage. MATERIALS AND METHODS Semen samples were taken from 249 randomly selected patients undergoing standard IVF treatment at the Institute for Reproductive Medicine of the University of Giessen, Germany and examined for DNA fragmentation by means of the TUNEL assay (n 167) and for DNA integrity with acridine orange staining (n 234). Moreover, early markers of apoptosis annexin V binding (n 212) and Fas expression (n 223) were investigated. These data were correlated to sperm concentration, motility, fertilization, and pregnancy. Sperm concentration and motility were determined according to World Health Organization criteria (29). Patients who underwent microsurgical epididymal sperm aspiration/testicular sperm extraction or cryopreservation of the ejaculate were excluded from the study. Ovarian stimulation was performed according to a standardized short protocol with a GnRH analogue (Suprecur; Hoechst, Frankfurt, Germany) with hmg (Menogon; Ferring, Kiel, Germany). Ovulation was induced by administration of 10,000 IU hcg (Predalon 5.000; Organon, Oberschleissheim, Germany), and follicles were aspirated transvaginally according to standard techniques 35 hours after hcg administration. To flush the follicles, human tubal fluid (HTF) medium according to Quinn et al. (30) supplemented with 5 IU/mL heparin (Roche, Basel, Switzerland), 20 mmol/l N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES; Sigma, St. Louis, Missouri), and 0.2% serum albumin (Behring, Marburg, Germany) was used. Motile spermatozoa were isolated with a standard swim-up procedure from washed spermatozoa in HTF medium containing 0.2% serum albumin. Co-culture of the oocytes with spermatozoa (approximately 100,000 sperm per oocyte) was performed in HTF medium supplemented with 0.2% serum albumin at 37 C under an atmosphere of 5% CO 2. After 18 hours, oocytes were checked for fertilization. We assessed DNA fragmentation and apoptosis by means of TUNEL assay, acridine orange staining, Fas expression, and annexin V binding, and correlated these data with fertilization and pregnancy rates in IVF. An aliquot of those ejaculates used for insemination was washed with HTF medium containing 1% human serum albumin (HTF-HSA). The sample was then subdivided into aliquots, and the tests were performed according to standard protocols. TUNEL Assay For the TUNEL assay, a detection kit (Apoptosis Detection System Fluorescein; Promega, Mannheim, Germany) was used. Sperm suspensions were centrifuged for 10 minutes at 300 g and 4 C. The supernatant was discarded, and the remaining pellet was washed in phosphate-buffered saline (PBS), ph 7.4, (Oxoid, Basingstoke, Hampshire, United Kingdom). A droplet of this sperm suspension was smeared onto pretreated slides (Superfrost; Menzel, Braunschweig, Germany), air-dried, and fixed by immersion in freshly prepared 4% methanol-free formaldehyde in PBS for 25 minutes at 4 C. The slides were then washed in fresh PBS for 5 minutes at room temperature, treated with 0.2% Triton X-100 in PBS for 5 minutes, and rinsed twice in PBS for 966 Henkel et al. DNA damage and fertilization in vitro Vol. 81, No. 4, April 2004

3 TABLE 1 Sperm concentration, motility, and concentration of motile spermatozoa in the ejaculate and after sperm separation by means of standard swim-up technique. Sperm concentration (10 6 /ml) Motility (% global motility) Concentration of motile spermatozoa (10 6 /ml) Ejaculate Medium after sperm preparation with swim-up Note: Data are presented as mean SD. n 249. another 5 minutes at room temperature. Excess liquid was removed by tapping the slides. The procedure was done according to the detection kit manufacturer s instructions. After final rinses, excess water was drained off, a drop of anti-fade solution (Molecular Probes, Eugene, Oregon) was added, the samples were covered with cover slips, and 300 randomly selected spermatozoa were immediately analyzed with an epifluorescence microscope (Zeiss, Oberkochen, Germany) at 1000 magnification. The percentage of greenfluorescing sperm (TUNEL positive) was determined. Negative controls without TdT enzyme were prepared for each batch of analyzed slides. Acridine Orange Staining Staining with the metachromatic dye acridine orange is a method for determining single-stranded DNA. This dye intercalates in the DNA and fluoresces green with doublestranded and red with single-stranded DNA. In brief, depending on the sperm concentration in the ejaculate, L of ejaculate was diluted 1:5 with HTM medium, washed, and centrifuged for 10 minutes at 500 g twice. The supernatant was discarded, and the pellet was smeared onto a glass slide and air-dried. Slides were then fixed overnight in Carnoy solution (66% ethanol, 33% glacial acid), washed with distilled water, and stained for 5 10 minutes at room temperature with a freshly prepared acridine orange solution (10 ml 0.1% acridine orange in distilled water, 40 ml 0.1 mol/l citric acid, 2.5 ml 0.3 mol/l Na 2 HPO 4 ). Finally, slides were washed carefully in distilled water, mounted with glycerol/pbs (9:1), and 300 randomly selected sperm were analyzed with an epifluorescence microscope. The percentage of green-fluorescing sperm (double-stranded DNA) was calculated. Annexin V Binding To determine the sperm s binding ability to annexin V, the sperm suspension in HTF-HSA with sperm (total number) was diluted 1:10 with PBS, washed, and centrifuged for 10 minutes at 500 g. The supernatant was discarded and the remaining pellet resuspended in 195 L binding buffer (10 mmol/l HEPES/NaOH, 140 mmol/l NaCl, 2.5 mmol/l CaCl 2, ph 7.4). Next, 5 L annexin V labeled with fluorescein isothiocyanate (FITC) (Annexin V FITC Kit; Bender MedSystem Diagnostics, Vienna, Austria; catalog no. BMS306FI) was added, and the samples were incubated in the dark at room temperature for 60 minutes. Then 800 L binding buffer was added, and the samples were centrifuged for 10 minutes at 500 g. Finally, three quarters of the supernatant was removed, the pellet was resuspended in the remaining liquid, a sample was smeared on a slide and covered with a cover slip, and 300 randomly selected spermatozoa were analyzed at 1000 magnification with an epifluorescence microscope. The percentage of green-fluorescing sperm (annexin V positive) was calculated. Fas Expression of Spermatozoa A sperm suspension with spermatozoa was diluted with 3 ml PBS and centrifuged for 10 minutes at 500 g. The supernatant was discarded, and the pellet was resuspended in 20 L anti-cd95 FITC (1:50 dilution) (ALX ; Alexis Deutschland, Grünberg, Germany) in 50 mmol/l phosphate buffer, 100 mol/l KCl, 5% glycerol, 0.2% bovine serum albumin, and 0.04% NaN 3, ph 7.4, and incubated for 45 minutes on ice. After subsequent washing in PBS, sperm were smeared onto a glass slide, the sample was covered with a cover slip, and 300 randomly selected spermatozoa were analyzed at 1000 magnification with an epifluorescence microscope. The percentage of green-fluorescing sperm (Fas positive) was calculated. Statistical Evaluation All statistical calculations were performed with the appropriate tests after checking normal distribution of the data with statistical software (MedCalc ; MedCalc Software, Mariakerke, Belgium). RESULTS Table 1 lists summary statistics of sperm concentration and motility in the ejaculate and after sperm preparation. The mean ( SD) results of the TUNEL assay, acridine orange stain, annexin V binding, and Fas expression of spermatozoa FERTILITY & STERILITY 967

4 TABLE 2 Correlation of the results from the TUNEL assay, acridine orange stain, annexin V binding, and Fas expression with the concentration of motile spermatozoa before and after sperm separation. Concentration of motile sperm Before sperm separation After sperm separation TUNEL assay (n 167) r r P.3627 P.0293 Acridine orange stain (n 234) r r P.0015 P.0021 Annexin-V-binding n 212 r r P.8326 P.8876 Fas-expression n 223 r r P.6282 P.7421 Note: Significant correlations were found for the acridine orange stain before and after sperm preparation and for the TUNEL assay after sperm separation. before sperm separation were 34.1% 17.1%, 14.1% 10.8%, 19.8% 13.1%, and 19.8% 14.3%, respectively. The correlation of motile sperm concentration with the markers of early apoptosis (annexin V binding and Fas expression) were not significant, either before sperm separation or afterwards. However, significant positive correlations with the concentration of motile spermatozoa were found for the acridine orange stain before and after sperm preparation and for the TUNEL assay after the swim-up (Table 2). Regarding fertilization and pregnancy, our data showed no or only weak direct correlations (Table 3). As expected, Fas expression and annexin V binding of the spermatozoa showed quite similar results and were highly significantly correlated (r ; P.0001). On the other hand, the difference between the percentage of TUNEL-negative sperm and green-fluorescing sperm in the acridine orange stain was highly significant. A statistically significant difference was also found for the percentage of green-fluorescing sperm in the acridine orange stain between those patients whose wives became pregnant and those whose wives did not (median pregnant: 14.0; median not pregnant: 10.0; P.0423). However, for the difference between good-fertilizing (fertilization rate 50%) and poor-fertilizing (fertilization rate 50%) semen samples, no significance was found (median pregnant: 9.83; median not pregnant: 12.58; P.0547). After the performance of receiver operating characteristic (ROC) curve analyses with pregnant yes or no as discriminator and application of calculated cut-off values for further differentiations (Table 4), significant differences between TUNEL-negative ( 36.5% green-fluorescing sperm) and TUNEL-positive sperm ( 36.5% green-fluorescing sperm) were revealed for the pregnancy rate (34.65% vs %; P.0344) as well as between acridine orange positive ( 12% green-fluorescing sperm) and acridine orange negative sperm ( 12% green-fluorescing sperm) (37.93% vs %; P.0156) (Fig. 1A and B). For the percentage of patients with good fertilization rates ( 50%), no significant differences could be calculated (TUNEL assay: P.5134; acridine orange stain: P.2928) (Fig. 1A and B). Thus, the probability for the female partners of becoming pregnant is almost twice as high, or 50% higher at the same fertility potential, if the percentage of TUNEL-positive sperm in the husband s ejaculate is low ( 36.5%) and the percentage of chromatin-intact sperm is high ( 12% greenfluorescing sperm in acridine orange stain). For the other parameters, annexin V binding (fertilization: P.6042; pregnancy: P.0990) and Fas expression (fertilization: P.6106; pregnancy: P.2803), no significant differences could be found (Fig. 1C and D). TABLE 3 Correlation of different parameters to fertilization in vitro and pregnancy. Fertilization Pregnancy TUNEL assay (n 167) r r P.5234 P.2102 Acridine orange stain (n 234) r r P.1120 P.0423 Annexin V binding (n 212) r r P.8859 P.9693 Fas expression (n 223) r r P.4047 P.5274 Note: Among the parameters tested, only the acridine orange test shows a significant correlation to pregnancy. DISCUSSION For a number of years scientists have been interested in the involvement of apoptosis in spermatogenesis and its impact on male fertility. It has been shown that an early and massive wave of programmed cell death of spermatogenic cells is necessary for the control and development of normal spermatogenesis (10, 31). On the other hand, severe testicular germ-cell apoptosis reflects a low success of assisted reproduction techniques and procedures (32). In the testis, apoptosis in Sertoli cells and germ cells seems to be regulated differently. Whereas FSH withdrawal increased DNA fragmentation without any effect on caspase activity in primary spermatocytes and spermatids, no effect was observed in Sertoli cells. On the other hand, testosterone withdrawal led to a significant increase in caspase activity and DNA fragmentation in Sertoli cells but not in germ cells (33). 968 Henkel et al. DNA damage and fertilization in vitro Vol. 81, No. 4, April 2004

5 TABLE 4 ROC curve analyses of the sperm parameters analyzed, with pregnancy as selection criterion. TUNEL assay Acridine orange stain Annexin V binding Fas expression Cut-off value Sensitivity Specificity Positive predictive value Negative predictive value Note: Data are expressed as %. In ejaculated spermatozoa, it has repeatedly been shown that DNA damage as determined by the TUNEL assay or the Comet assay is significant negatively correlated with fertilization and pregnancy in IVF (16, 18, 24), ICSI (17), and IUI (19). However, in most cases, the DNA damage has not been clearly characterized: DNA fragmentation can be a consequence of apoptosis (12), but it can also be induced by ROS (13, 14). Reactive oxygen species are normally produced in ejaculate by leukocytes (34) or the sperm cells themselves (35, 36) and are also associated with male infertility (37, 38). Thus, it is actually not quite correct to speak about apoptotic events when DNA fragmentation is determined. To the best of our knowledge, an investigation of fertilization and pregnancy in relation to clear markers of apoptosis (i.e., the externalization of phosphatidylserine or Fas expression) has not yet been performed. In this study, we found an expected strong correlation (r ; P.0001) between both apoptosis markers but no correlation with fertilization rates or pregnancy. However, our data clearly demonstrate that DNA fragmentation as determined by the TUNEL assay is predictive for pregnancy in IVF. Although no direct correlation between the percentage of TUNELpositive sperm and the fertilization rate and pregnancy was observed, patients with a high percentage of TUNEL-positive spermatozoa ( 36.5%) showed a significantly lower mean pregnancy rate than those patients with a low percentage of TUNEL-positive sperm ( 35.5%) (19.05% vs %). In contrast, no significant difference could be observed for the mean fertilization rates (63.49% vs %). On principle, a similar result could be observed for the DNA integrity as determined by the acridine orange stain (pregnancy: 37.93% vs %; fertilization: 61.21% vs %). Therefore, our results confirm observations by Sun et al. (16) and Host et al. (18), who showed a significant influence of DNA fragmentation on male fertility. This also suggests that sperm DNA fragmentation might be a cause for early embryo death (20). Although there are reports describing limited developmental potential (39) and nuclear abnormalities (40) of fragmented embryos, it is not known why these embryos are apoptotic. This could be due to DNA fragmentation in the embryo itself, the oocyte, or the spermatozoa. If the spermatozoa are predamaged and show DNA fragmentation, it could be assumed that oocytes might still be fertilized and the 2-pronuclei stage formed, but that when the paternal genes are switched on at the 4-cell stage, further embryonic development stops because of oxidation and fragmentation of the paternal DNA, even after blastocyst formation. This paternal influence on embryonic development seems to manifest even before blastocyst formation, resulting in developmental arrest and, thus, failed pregnancy (23). Our data would support this idea. Shoukir et al. (22) found a significantly lower blastocyst formation rate after ICSI compared with IVF. These authors state that the role of paternal DNA in early human embryo development cannot be ignored. Thus it seems that patients who undergo a treatment with assisted reproductive procedures, especially with ICSI, have a significantly higher risk of sperm with fragmented DNA fertilizing an oocyte, which might lead to early embryo death (15). In the human, Sakkas et al. (41) showed that failed fertilized oocytes injected with sperm from patients with a high percentage of endogenous DNA nicks in the sperm population contain more condensed spermatozoa. At this point, the question concerning the causes for the DNA fragmentation of spermatozoa arises; that is, whether the fragmentation is caused by apoptosis (12) or ROS, which can be generated either by leukocytes (34) or by the sperm themselves (42). Although notable percentages (approximately 20%) of sperm cells showing signs of apoptosis were found in this study, we did not find any correlation of fertilization and pregnancy to early markers of apoptosis (annexin V binding and Fas expression). On the other hand, Oosterhuis et al. (43) found similar percentages of ejaculated apoptotic sperm by annexin V binding that negatively correlated with sperm concentration and motility. This would contribute to the theory that apoptosis in mature spermatozoa is initiated during spermatogenesis as a consequence of altered germ-cell maturation, which might be associated with an up-regulation of Fas gene expression (44). Normally, FERTILITY & STERILITY 969

6 FIGURE 1 Influence of DNA fragmentation, DNA integrity, and apoptosis markers (annexin V binding and Fas expression) on fertilization (black bars) and pregnancy (striped bars) in vitro after differentiation and separation of the patients into positive and negative groups according to the cut-off points calculated by ROC curve analysis. Determination of DNA damage by means of the TUNEL assay (A) and the acridine orange stain (B) shows a significant influence on pregnancy but not on fertilization. Insemination sperm from patients with a low percentage of TUNEL-positive sperm or a high percentage of sperm with double-stranded DNA resulted in almost twice the pregnancy rate (Fisher exact test) as in the others. The fertilization rate showed no difference. For parameters of apoptosis (annexin V binding and Fas expression; C and D, respectively), no difference could be calculated either for fertilization or for pregnancy. these sperm would be eliminated (45). However, if the removal mechanism fails or the sperm escape the removal mechanism (46), these earmarked sperm would contribute to poor sperm quality. Because in this study none of the apoptosis parameters correlated with DNA integrity or DNA fragmentation, an influence of apoptosis in ejaculated sperm on fertilization and pregnancy in IVF seems rather unlikely and makes other mechanisms for oxidization of DNA plausible. This would imply that DNA fragmentation in human spermatozoa is caused by external factors, such leukocytes or ROS, rather than by apoptosis. Reactive oxygen species produced by leukocytes and predamaged spermatozoa affect sperm functions at late stages (47 49). Because even low amounts of ROS are harmful to sperm DNA integrity (50), a causality between ROS and/or leukocytes in the ejaculate and DNA fragmentation should not be neglected. 970 Henkel et al. DNA damage and fertilization in vitro Vol. 81, No. 4, April 2004

7 Another hypothesis regarding the origin of fragmented DNA in sperm comes from animal experiments that showed that endogenous nicks are normally present at late stages of spermatogenesis (step spermatids) in rats and mice and are thought to have physiologic importance (51 53). Once chromatin packaging is completed, nicks normally are no longer observed. Thus, it seems that the presence of endogenous nicks is highest during transition from round to elongated spermatids. McPherson and Longo (51) postulated that chromatin packaging requires the enzyme topoisomerase II to create and ligate nicks to facilitate protamination during spermiogenesis. They propose that these nicks are necessarily involved in chromatin rearrangement during displacement of histones by the protamines. Consequently, endogenous nicks in ejaculated sperm would be indicative of incomplete maturation of spermatozoa during spermiogenesis, which would result in disturbed chromatin condensation due to underprotamination. In conclusion, this study shows that human sperm DNA damage as determined by DNA fragmentation with the TUNEL assay and DNA integrity as assessed by acridine orange stain, although still controversially discussed (54, 55), is predictive of pregnancy. At least in patients whose spermatozoa are good enough to undergo standard IVF procedure, DNA damage seems not to be a limiting factor for fertilization. However, it could clearly be seen that sperm DNA damage results in pregnancy failure and can therefore be one factor that might contribute to the gap between fertilization and pregnancy rates. On the other hand, the impact of apoptosis as determined by clear apoptotic markers (annexin V binding and Fas expression) on sperm fertilizing capacity seems rather low. This also means that a clear distinction should be made between apoptotic DNA fragmentation and DNA fragmentation caused by ROS produced either by leukocytes or by the sperm cells themselves. In addition, the possibility of a causal link between ROS and/or leukocytes in the ejaculate and DNA fragmentation should not be neglected. To understand the importance of apoptosis and the pathophysiology of ROS better, future work should focus even more on their impact on sperm function, because DNA-damaged sperm might increase the risk of genetic diseases in the offspring (56). Acknowledgments: The authors thank Ms. A. Hanschke, Center for Dermatology and Andrology, Justus Liebig University, for skillful technical assistance and Mrs. S. Henkel for excellent linguistic review. References 1. Oehninger S, Sueldo C, Lanzendorf S, Mahony M, Burkman LJ, Alexander NJ, Hodgen GD. A sequential analysis of the effect of progesterone on specific sperm functions crucial to fertilization in vitro in infertile patients. Hum Reprod 1994;9: Oehninger S, Veeck L, Franken D, Kruger TF, Acosta AA, Hodgen GD. 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