Article Relationship between sperm DNA damage, induced acrosome reaction and viability in ICSI patients

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1 RBMOnline - Vol 15. No Reproductive BioMedicine Online; on web 20 June 2007 Article Relationship between sperm DNA damage, induced acrosome reaction and viability in ICSI patients Dr Ozmen began studying obstetrics and gynaecology in 1998 at the University of Ankara. After graduating from the residency programme in 2003, he continued working on reproductive medicine at the same centre. He is now undertaking a research doctorate on assisted reproduction at the University of Lübeck in Germany under the supervision of Professor Safaa Al-Hasani. His main interests are cryopreservation and micromanipulation of embryos, sperm DNA damage and endoscopic reproductive surgery. Dr B Ozmen B Ozmen 1,2, GS Caglar 1,3, F Koster 1, B Schopper 1, K Diedrich 1, S Al-Hasani 1,4 1 University of Schleswig-Holstein, Department of Gynecology and Obstetrics, Reproductive Medicine Unit, Campus of Luebeck, Luebeck-Germany; 2 University of Ankara, Department of Gynecology and Obstetrics, Center of Artificial Reproductive Techniques, Campus at Mamak, Ankara-Turkey; 3 Private Ankara IVF Center, Ankara-Turkey 4 Correspondence: sf_alhasani@hotmail.com Abstract The DNA damage in human spermatozoa is a relevant predictor of prognosis in male infertility, whereby increased sperm DNA damage impairs the outcomes of artificial reproduction. Theoretically, DNA damage should alter the special cellular functions of human spermatozoa, and lead to diminished acrosome reaction with reduced fertilization rates. Nevertheless, intracytoplasmic sperm injection (ICSI) has been reported to alleviate such negative outcomes due to DNA damage. This study investigated the relationship between DNA fragmentation and acrosome reaction as well as viability in ICSI patients. The study enrolled 42 men undergoing ICSI due to poor sperm parameters. The DNA fragmentation indexes (DFI) were 4 10% in 38% of the cases, and 10% in 19% of the cases. The results of both acrosome reaction and viability assays showed negative correlations with DFI values in all cases and especially in cases with fertilization rates <60% (P < 0.05). However, such correlations were not found in cases with fertilization rates >60%. There were no live deliveries in patients with high DFI levels (>10%). In conclusion, negative correlations were identified between increased DNA damage, and acrosome reaction and/or viability of human spermatozoa, especially in cases with reduced fertilization rates. Keywords: acrosome reaction, DNA fragmentation, intracytoplasmic sperm injection, sperm DNA damage, sperm viability Introduction 208 Parameters of basal diagnostic tests, conventional spermiogram and/or morphological criteria, have been reported to be inadequate as predictive markers in the prognosis of male infertility (World Health Organization, 1999). However, prognosis is directly related to the selection and specific timing of the right treatment of each individual infertile couple. Therefore, it is still unfair to claim an accurate prediction of prognosis in male infertility only by basal tests, and increased sensitivity along with specificity, is urgently warranted. Recently, other parameters that are unrecognizable by conventional sperm analysis were assumed valuable in prediction of prognosis (Ozaki et al., 2002). The DNA integrity of spermatozoa is one of these unrecognizable parameters that correlates well with semen quality (Sergerie et al., 2005). Increased DNA damage of human spermatozoa negatively alters the outcomes of assisted reproductive technology (Duran et al., 2002; Benchaib et al., 2003). Further, a negative correlation between human sperm DNA damage and fertilization rates has also been reported (Host et al., 2000; Huang et al., 2005). Nevertheless, application of intracytoplasmic sperm injection (ICSI) has been indicated to alleviate the negative consequences of sperm DNA damage on fertilization (Gandini et al., 2004) Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 Acrosome reaction is a key step of in-vivo fertilization, and is also a relevant and a good predictor of fertility potential of human spermatozoa (Makkar et al., 2003; Katsuki et al., 2005). Unfortunately, the relationship between acrosome reaction and sperm DNA damage, thus the potential role of increased DNA damage in fertilization failure, is still not clear. Theoretically, human sperm DNA damage might alter cellular functions, which could cause diminished acrosome reaction and reduced fertilization rates. If so, is there a special degree of DNA damage that limits the function of spermatozoa in inducing acrosome reaction and reduced fertilization ability? This study was performed to investigate the relationship between calcium ionophore-induced acrosome reaction and viability and DNA damage or fragmentation detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dudp nick-end labelling (TUNEL) assay in patients undergoing ICSI with poorquality semen samples. Materials and methods Study group The study group consisted of 42 couples scheduled to undergo ICSI treatment in the Department of Gynecology and Obstetrics, University of Schleswig-Holstein, and Campus of Luebeck. Couples with genetic disorders and those requiring testicular sperm extraction or micro-epididymal sperm aspiration were excluded from the study. An informed consent was obtained from all couples at the initiation of the treatment cycle, and the scientific committee of the Gynecology and Obstetrics department and the local scientific research team of reproductive medicine approved this study. Interventions All female partners underwent ovarian stimulation with a multidose antagonist (cetrorelix; 0.25 mg/day; Cetrotide, Serono International SA, Geneva, Switzerland) fixed protocol that was initiated on day 6. Both human menopausal gonadotrophin (HMG) (Menogon; Ferring Arzneimittel GmbH, Kiel, Germany), and recombinant FSH (Gonal- F; Serono International S.A., Geneva, Switzerland) were used for ovulation induction. Ovulation was induced by intramuscular injection of 10,000 IU of human chorionic gonadotrophin (Choragon; Ferring Arzneimittel GmbH, Kiel, Germany). Oocyte retrieval procedure was performed under ultrasound guidance at h after triggering. Semen samples were collected by masturbation after 3 days of sexual abstinence. Routine semen analysis using light microscopy was carried out to evaluate sperm concentration, motility and morphology. Morphological assessments of sperm samples were performed according to strict criteria (Menkveld et al., 1990). The swim-up technique was used for the preparation of semen samples. Prepared sperm samples were then divided into three equal parts and ICSI procedure carried out with one part. Of the remaining two parts, one was used for the TUNEL assay and the other for sperm viability with ionophore-challenged acrosome reaction. Embryo transfers were performed on day 2 in accordance with the German Embryo Protection Law. Embryos were graded as grade 1, 2 or 3 prior to the embryo transfer (Steer et al., 1992) and a quality score was obtained by multiplication of the grade of each embryo with its number of blastomeres. Methodology for TUNEL assay Air-dried sperm samples were used to determine DNA breaks with the TUNEL assay (Cell Death Detection Kit; Roche Biochemicals, Mannheim, Germany) following the manufacturer s specifications with minor modifications. Subsequent to the drying of sperm-containing slides, samples were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) at room temperature. Slides were then rinsed with PBS (ph 7.4) and permeabilised with 2% Triton X-100. The terminal deoxynucleotidyl transferase (TdT) fluorescentlabelled nucleotide mix was added to the slides and the labelling reaction was carried out for 1 h in the dark at 37 C in a humidified chamber. Thereafter, slides were rinsed twice in PBS and then counterstained with 10 mg/ml 4,6 diamidino-2- phenylindole (DAPI). Controls were included in every sample; for negative control, TdT was omitted in the nucleotide mix. The positive controls were prepared by incubating the sperm cells for 20 min at room temperature with 50 units/ml DNase I (Boehringer Mannheim, Mannheim, Germany). Total numbers of spermatozoa and percentages of cells with fragmented DNA were determined by analysing each microscope field using both light and fluorescence microscopy. At least 500 cells were counted in each sample. Each spermatozoon was identified to contain either normal (blue nuclear fluorescence due to DAPI) or fragmented DNA (intense green nuclear fluorescence). The final percentage of spermatozoa with fragmented DNA is referred to as DNA fragmentation index (DFI) performed by TUNEL assay (%). Assessment of viability and status of acrosome reaction After 3 h in the capacitation medium for induction of acrosome reaction, calcium ionophore A23187 was added to the capacitated sperm suspensions (1 h). Sperm were coincubated with Hoechst for detection of viability. The number of live and dead spermatozoa and their acrosome status were evaluated according to the methods described by Cross et al. (1986). (For a review of methods, see Cross and Meizels, 1989.) Hoechst (stock solution: 1 mg/ml) dissolved in Dulbecco s phosphate-buffered saline (DPBS) was added to a final concentration of 1 μg/ml and coincubated with spermatozoa for 10 min. Samples were washed free of unbound stain by centrifugation twice at 900 g for 5 min through 4 ml of 2% (w/v) polyvinylpyrrolidone-dpbs; the supernatant was removed and the pellets were resuspended in 50 μl DPBS. Spermatozoa were mounted as smears on glass slides and air dried in an incubator at 39 C. They were fixed with methanol for 30 s. The slides were then washed with a stream of distilled water for 2 min. After drying, spermatozoa were incubated with 30 μg/ml fluorescein isothiocyanate Pisum sativum (FITC-PSA) in DPBS for 30 min and washed again with a stream of distilled water for 10 min. In order to avoid fading of the fluorescence, the slides were mounted with SlowFade Antifade Kit (Invitrogen Corporation, Germany). Spermatozoa 209

3 210 were examined using a Zeiss Axiovert 135 epifluorescence microscope (Zeiss, Jena, Germany; magnification 1000). For each slide, 200 spermatozoa were scored. The disappearance of labels (negative staining) of the acrosomes was scored as acrosome reacted. Statistics All available data were entered and analysed using the Statistics Package for Social Sciences for Windows 9.0 (SPSS Inc., Chicago, IL, USA). In statistical analysis, Kruskal Wallis, Mann Whitney U-, Spearman Rank and Kendall Tau tests were used for correlations of parameters and comparisons. The criterion for statistical significance was set at P < Results Semen samples of all subjects were found to be abnormal according to the spermiogram guidelines of the WHO and/or strict morphological criteria (Menkveld et al., 1990). The mean DFI value of the subjects in the study was 2.5 ± 6.9%. However, the DFI values were found to be above 10% in 19% (eight patients), and between 4% and 10% in 38% (16 patients) of the cases. In the remaining cases (18 patients), DFI values lower than 4% were recorded. There was no difference in descriptive characteristics comprising the mean age of female partner and male partner, cycle stimulation duration, and oocyte quality among these subgroups in regard to DFI value (data not shown). A negative correlation was observed between DFI values and the percentage of acrosome-reacted spermatozoa (AR) (P = 0.001, R = 0.49) (Table 1 and Figure 1). Further, DFI values were also negatively correlated with the percentage of viable AR spermatozoa (alar) (P = 0.005, R = 0.43) (Table 1 and Figure 1). Mean viability decreased as the DFI value of spermatozoa increased when assessed in the three DFI subgroups of spermatozoa <4, 4 10, and >10% (all P < 0.05; Figure 1). Unsurprisingly, the percentage of viable spermatozoa correlated positively with the percentage of AR (P < 0.001, R = 0.67). No correlation was observed between the percentages of progressive straight motile spermatozoa and the results of TUNEL assay (Table 1). However, the mean total sperm concentration was found to be lower and the mean percentage of spermatozoa with abnormal morphology was found to be higher in cases with DFI levels >10% (Table 1). Likewise, apparently higher DFI levels were also detected in cases with fertilization rates lower than 60%, although the difference did not reach statistical significance (Table 2). In regard to the results of AR and viability assays, there was no difference among cases with fertilization rates lower than or greater than 60% (Table 2). On the contrary, morphology and number of cryopreserved embryos were favourable in cases where fertilization rates were above 60% (both P < 0.05; Table 2). The percentages of progressive motility and of normal morphology were shown in groups with fertilization rates above and below 60% (Table 2). Surprisingly, the percentage of motile spermatozoa was found to be higher in the group with fertilization rate lower than 60%, which reduced the prognostic value of the percentage of motile spermatozoa. On the contrary, the percentage of spermatozoa with normal morphology was found to be higher in the group that underlined the predictive value of this spermiogram parameter. It is also interesting that the mean age of female partner was found to be significantly younger in cases with fertilization rates higher than 60% (P < 0.05; Table 2). The negative correlations of DFI with the percentage of AR and alar were not apparent in cases with fertilization rates lower than 60% but remained in cases with fertilization rates above 60% similar to former correlations observed in assessment of the whole group (P = 0.008, R = 0.61 and P = 0.002, R = 0.65). Contrary to the results of TUNEL assay, the parameters of traditional spermiogram, such as percentage of motility and percentage of normal morphology were found to be significantly associated with the results of acrosome reaction and viability assessments (P < 0.05; Figure 2). For instance, when cases were assessed in regard to a 90% threshold value of spermatozoa viability, the percentage of progressive motile spermatozoa and normal morphology were positively associated with the viability of spermatozoa (P = 0.02, R = 0.44 and P = 0.04, R = 0.31) (Figure 2). Particularly, the percentage of progressive motile spermatozoa and normal morphology were also positively correlated with the acrosome status and the percentage acrosomereacted spermatozoa (P = 0.04, R = 0.30 and P = 0.04, R = 0.21) (Figure 2). Embryo grades were not different for cases with respect to DFI values, or for cases having fertilization rates lower and higher than 60% (data not shown). The results of the assessment of viability or acrosome reaction were similar both for conception and non-conception cases (data not shown). However, all cases that resulted in live births had a DFI value lower than 10%. The only couple that conceived with >10% DFI was a biochemical pregnancy giving a 12.5% (1/8) pregnancy rate per cycle in all cases (Table 1). On the contrary, the biochemical pregnancy, clinical pregnancy and live birth rates per cycle among patients with DFI levels lower than 10% were 32% (11/34), 29.4% (10/34) and 21.2% (7/33) (Table 1). One patient in the pregnant group with DFI level lower than 10% was lost from the follow-up due to her private doctor choice after 12 weeks of gestational age and confirming clinical pregnancy by transvaginal ultrasonography. In the other three cases, one was a biochemical pregnancy whereas the two remaining cases resulted in early pregnancy loss. The DFI values of these two cases were 4.5 and 9%. Oocyte quality in the cases with high DFI levels (>10%) was not different from the cases with low DFI levels (<4% and 4 10%) (data not shown). Discussion Sperm DNA damage is a crucial paternal factor that directly interferes with the fertility potential of infertile couples (Henkel et al., 2003). Negative correlations have been indicated between increased DNA damage in human spermatozoa and percentage of progressive motility or percentage of normal morphology (Huang et al., 2005; Sergerie et al., 2005). Increased sperm DNA fragmentation

4 Table 1. Sperm parameters and outcome of intracytoplasmic sperm injection according to DNA fragmentation index. Parameter DFI value (%) P-value <4 (n = 18) 4 10 (n = 16) >10% (n = 8) DFI value (%) 1.6 ± ± ± 6.5 Sperm concentration ( 10 6 /ml) 55.4 ± ± ± 30.2 <0.05 c Motile spermatozoa (%) 53.2 ± ± ± 23.3 NS Normal morphology (%) 8.5 ± ± ± 6.1 <0.05 b,c Viability rate (%) 91.9 ± ± ± 8.7 <0.05 a,b AR (%) 94.7 ± ± ± 8.5 <0.05 a,b alar (%) 85.0 ± ± ± 11.8 <0.05 a,b Fertilization rate (%) 54.2 ± ± ± 24.2 NS Pregnancy rate/per cycle (%) d <0.05 Values are mean ± SD unless otherwise stated; alar = viable acrosome-reacted spermatozoa; AR = acrosome-reacted spermatozoa; DFI = DNA fragmentation index performed by TUNEL assay; NS = not statistically significant; Motile spermatozoa = progressive straight and straight motile spermatozoa (WHO criteria a and b). a <4% DFI group was statistically different from 4 10% DFI group. b 4 10% DFI group was statistically different from >10% DFI group. c >10% DFI group was statistically different from other two groups. d Pregnancy rate/per cycle comprised both biochemical and clinical pregnancies. Figure 1. Variables in all cases according to DNA fragmentation index (DFI) values: DFI <4%, n = 18; DFI 4 10%, n = 16; and DFI >10%, n = 8. * denotes a statistically significant difference; P <

5 Table 2. Variables according to fertilization rates among all cases. Variable Cases with Cases with P-value fertilization rate fertilization rate <60% (n = 26) >60% (n = 16) Age of female partner (years) 33.4 ± ± 2.5 <0.05 Motile spermatozoa (%) 53.2 ± ± 26.2 <0.05 Normal morphology (%) 5.2 ± ± 7.8 <0.05 Viability (%) 90.4 ± ± 10.8 NS DFI (%) 6.5 ± ± 6.1 NS AR (%) 86.1 ± ± 7.4 NS Number of cryopreserved embryos (n) 0.5 ± ± 3.8 <0.05 Values are mean ± SD unless otherwise stated; AR = acrosome-reacted spermatozoa; DFI = DNA fragmentation index performed by TUNEL assay; Motile spermatozoa = progressive straight and straight motile spermatozoa (WHO criteria a and b); NS = not statistically significant. Figure 2. The parameters of spermiogram and percentage with normal morphology according to results of viability and acrosome reaction assay: sperm viability <90%, n = 16; sperm viability <90%, n = 26; acrosome-reacted spermatozoa <85%, n = 18; acrosome-reacted spermatozoa >85%, n = 24. * denotes a statistically significant difference; P <

6 has also been found to be correlated with reduced hyperactivation of spermatozoa and hampered penetration of zona-free hamster oocytes (Chan et al., 2001). Particularly, high rates of fertilization failure, even with ICSI, have been defined in cases with DNA-damaged spermatozoa (Lopes et al., 1998; Huang et al., 2005). High incidence of DNA fragmentation has been frequently observed among infertile couples with unexplained aetiologies and with recurrent pregnancy failures whereby reduced fertilization and high abortion rates are commonly expected (Host et al., 2000; Carrell et al., 2003). Further, altered assisted reproduction success and poor assisted outcomes in couples with increased sperm DNA damage were observed in the majority of the related studies (Duran et al., 2002; Benchaib et al., 2003). Calcium ionophore-induced acrosome reaction test, another prognostic marker in male infertility, has been reported to correlate well with in-vitro fertilization rates (Katsuki et al., 2005). The results of the induced acrosome reaction test are also intended to help avoid unnecessary IVF attempts by identifying patients with low-fertilizing capacity (Risopatron et al., 2001). Therefore, the test has a significant value in prediction of the outcomes of assisted reproduction technology (Makkar et al., 2003; Katsuki et al., 2005). In the present study, a significant correlation between increased sperm DNA damage and reduced acrosome reaction has been demonstrated clearly. The viability of spermatozoa was also negatively correlated with increased sperm DNA damage. All these results indicated that increased DNA damage in human spermatozoa seems to alter the viability and induced acrosome reaction. As a result, it can be presumed that one possible cause of reduced fertilization rates and poor outcomes of assisted reproduction in cases with increased sperm DNA damage might be reduced acrosome reaction. Special cut-off values of human sperm DNA integrity for improved success of assisted reproduction have been defined in regard to the implemented method (Henkel et al., 2003; Sergerie et al., 2005; Evenson, 2006). The DFI cut-off values in ICSI were reported to be as high as 36%, which is somewhat higher than the cut-off values defined for intrauterine insemination (IUI) and conventional IVF (Henkel et al., 2003; Evenson, 2006). These high cutoff values with ICSI suggest that ICSI procedure could overcome some degree of increased sperm DNA damage by skipping some physiological steps, in which special sperm functions are critically required (Lopes et al., 1998; Carrell et al., 2003). In light of the literature and the present study, it can be presumed that acrosome reaction might be one of these physiological steps, and might be altered by high DNA fragmentation, which is the main sign of increased DNA damage in human spermatozoa. The strict selection of sperm for ICSI has been claimed to cause such high DFI threshold values (Evenson, 2006). By this selection, it can be advocated that low DNA-damaged human spermatozoa are being chosen due to better motility and morphology in the ICSI procedure, which is also supposed to have a better induced acrosome reaction. Consequently, improved fertilization rates could be expected due to the selection of healthy spermatozoa for the ICSI procedure. It has been demonstrated that improved success could be obtained with high magnification ICSI in cases with increased DNA damage whereby a detailed morphological assessment of human spermatozoa has been performed (Hazout et al., 2006) Another topic of discussion is the origin of DNA anomalies or DNA damage in human spermatozoa, and its consequences or possible effects on assisted outcomes by altering special sperm functions. Three main origins have been indicated previously: (i) abnormal chromatin packing with underprotamination or protamine deficiency; (ii) apoptosis of human spermatozoa; and (iii) oxidative stress with overproduction of reactive oxygen species (ROS) (Evenson, 2006). Adverse environmental influences that lead to increased ROS production might also cause lowered acrosome reaction and sperm vitality rates due to possible damage to the sperm membrane. Notably, other origins of sperm DNA damage, unlike protamine deficiency of human spermatozoa, do not always preclude fertilization (Nasr-Esfahani et al., 2005). Therefore, determining the origin of DNA damage is another critical point. On the other hand, the aim of this study was only to show the correlation between acrosome reaction or viability and DNA damage of human spermatozoa. Therefore, the cut-off values of the study groups were lower than those reported in the literature due to extensive evaluation of the effects of sperm DNA damage. One can assume that effects of sperm DNA damage on acrosome reaction could be seen even in cases with low sperm DFI values currently presumed as normal sperm DNA integrity values. However, high DFI values (17 36%) have been indicated previously in relation to the negative effects of sperm DNA damage on assisted outcomes (Henkel et al., 2003; Sergerie et al., 2005; Evenson, 2006). On the contrary, the cut-off value for assisted outcomes was >10% DFI in the present study due to observation of all live births under this critical value. This low DFI cut-off was another point of interest in this study. Two explanations for this conflict might be that: the threshold values of DFI could be different according to the assessment method of sperm DNA damage and there were lower DFI cutoffs with TUNEL assay (Evenson, 2006); and the methodology of making dry smears for the TUNEL assay has been claimed to produce poor fluorescence readings (Cuello Carrión and Ciocca, 1999). In this study, observation of correlations, especially in cases with low fertilization rates (<60%), indicated that a critical impact of DNA damage on acrosome reaction and fertilization should be expected even after ICSI. On the other hand, there were no correlations observed between variables among cases with fertilization rate >60%. Thus, this study indicated that there is no single factor among the level of DFI, the acrosome reaction or use of ICSI that accounts for maintenance and restoration of fertilizing capacity of DNA-damaged spermatozoa. Furthermore, in this study, the percentages of motility and normal morphology were found to be inconsistent with the fertilization rates. Nevertheless, the small sample size in the study should be considered as the main limiting factor, whereby the correlations of these traditional variables could be dismissed. More recently, it has been indicated that human spermatozoa, even with high DNA damage, might retain the ability to fertilize an oocyte (Emery and Carrell, 2006). Further, full-term 213

7 214 pregnancies have been achieved with ICSI despite high levels of sperm DNA damage (Gandini et al., 2004). A potential role of the oocyte in recovering some degree of critical errors and impact of DNA-damaged spermatozoa after fertilization has therefore been claimed (Tesarik et al., 2002). These workers also indicated that this potential might be age limited and the couples with young female partners could be at an advantage compared with couples with older female partners (Tesarik et al., 2002). The findings of this study are also consistent with this theoretical idea and couples with younger female partners were more likely to have higher fertilization rates (>60%). Regarding observed negative correlations, it could be postulated that acrosome reaction or sperm DNA damage assessments could be used either alone or in conjunction in prediction of prognosis in order to increase sensitivity and specificity. Logically, cost efficiency should be the decisive factor for preferred prognostic test in male infertility. A theoretical cost reduction of assisted reproduction treatment, especially targeting unexplained infertility and male infertility, might also be expected due to avoidance of unnecessary IVF and IUI attempts. Therefore, routine application of these tests, especially assessments of sperm DNA integrity, should be a part of the current management. However, there are still some missing points, especially the role of oocyte, in DNA damage of human spermatozoa and its fertilizing capacity. References Benchaib M, Braun V, Lornage J et al Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Human Reproduction 18, Carrell DT, Liu L, Peterson CM et al Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. 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