Sperm DNA fragmentation negatively correlates with velocity and fertilization rates but might not affect pregnancy rates

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1 Sperm DNA fragmentation negatively correlates with velocity and fertilization rates but might not affect pregnancy rates Chun-Chia Huang, M.Sc., a,b,c David Pei-Cheng Lin, Ph.D., b Hui-Mei Tsao, M.Sc., a Tzu-Chun Cheng, Ph.D., a,b Chung-Hsien Liu, M.D., Ph.D., d and Maw-Sheng Lee, M.D., Ph.D. a,e a Division of Infertility Clinic, Lee Womens Hospital; b Institute of Biochemistry, Chung Shan Medical University; c Department of Medical Technology, Chungtai Institute of Health Science and Technology; d Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital; and e Department of Medicine, China Medical University, Taichung, Taiwan Objective: To evaluate sperm DNA fragmentation in correlation with sperm parameters and IVF/intracytoplasmic sperm injection (ICSI) outcomes. Design: Retrospective review. Setting: A tertiary infertility referral clinic. Patient(s): We collected 303 semen samples from patients undergoing IVF with or without ICSI. Intervention(s): Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay, measurement of fertilization rates, good embryo rates, and pregnancy rates for the IVF/ICSI program. Main Outcome Measure(s): The percentage of sperm with DNA fragmentation, correlated with semen analysis parameters and IVF/ICSI outcomes. Result(s): Sperm DNA fragmentation rates were significantly higher in patients with abnormal sperm parameters than in those with normal sperm parameters. When sperm DNA fragmentation was 10%, fertilization rates were affected. Sperm DNA fragmentation rates were negatively correlated with sperm velocity parameters but did not affect pregnancy outcomes. Conclusion(s): The results indicated that sperm DNA fragmentation affects fertilization rates and sperm motility but might not affect pregnancy rates. (Fertil Steril 2005;84: by American Society for Reproductive Medicine.) Key Words: Sperm DNA fragmentation, velocity, morphology, fertilization rate, embryo quality The male factor remains a common cause of infertility, with sperm defects found in 30% 50% of clinical infertility cases. Poor sperm quality is represented by abnormal sperm parameters, including low sperm concentration, poor sperm motility, and abnormal sperm morphology (1). The relationship between the percentage of morphologically abnormal spermatozoa and infertility has been widely described in men and animals. Nevertheless, morphology alone might not be sufficient to predict fertilization, and it has been suggested that sperm morphology should be associated with a careful investigation of motility parameters (2). Sperm motility has also been shown to correlate positively with IVF rates (3). Nevertheless, in some studies, sperm motility assessed microscopically was not always well correlated with either in vivo or IVF outcomes, except when the spermatozoa were absolutely immotile (4). Received April 19, 2004; revised and accepted August 16, Supported by the Chinese Infertility Foundation, Taichung, Taiwan, R.O.C. Reprint requests: Maw-Sheng Lee, M.D., Ph.D., Lee Womens Hospital, No. 263 Pei-Tung Road, Taichung 406, Taiwan (FAX: ; msleephd@giga.net.tw). With the advance of intracytoplasmic sperm injection (ICSI) for the treatment of male infertility, defects in sperm parameters have become less relevant. When a single sperm is chosen and injected directly into the cytoplasm of a mature oocyte, the normal physiologic selection processes involving sperm concentration, motility, and morphology are bypassed. With ICSI, artificial selection takes place. The selected single sperm relies heavily on the integrity of its genome, in cooperation with the oocyte genome, to initiate chromatin decondensation and subsequently to form pronuclei for a successful fertilization. In particular, ICSI is undertaken with severe defects in sperm parameters, raising debate as to the potential transmission of genomic defects to the next generation. Thus, it will be helpful to use markers other than traditional sperm parameters for ICSI. Sperm DNA fragmentation rate has been used as a marker to distinguish fertile from infertile men (5) and to predict IUI outcome (6). Increased sperm DNA fragmentation rates were found in couples with unexplained recurrent pregnancy loss (7), as well as in men with idiopathic male factor infertility (8). Sperm DNA fragmentation might affect the outcome of assisted reproduction in multiple ways, including effects on 130 Fertility and Sterility Vol. 84, No. 1, July /05/$30.00 Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 FIGURE 1 An example of TUNEL assay results. The green cells in B (arrows) were regarded as positive, compared with the DAPI-stained blue cells in A on the same observation field. The cells with relatively weak green fluorescence were regarded as negative. Original magnification, 600. the fertilization rates (9 14), sperm motility (8, 10, 14 23), sperm morphology (8, 10, 16, 23), embryo cleavage (8, 12), and pregnancy rates (24). It has also been reported that sperm concentration was related to sperm DNA fragmentation (8, 15, 22, 25). Despite previous studies, there is thus far no clear demarcation for sperm parameters, as measured by computerassisted sperm analysis (CASA), in relation to sperm DNA fragmentation. Because CASA is commonly used in clinical settings, making sense of CASA results in relation to sperm DNA fragmentation rates might help in the choosing of the right sperm for ICSI from different portions or batches of semen. At the same time, it might help in predicting IVF treatment outcome with or without ICSI. Here, we report on the correlation of sperm parameters from CASA with sperm DNA fragmentation profiles and IVF/ICSI outcomes. MATERIALS AND METHODS Patients A total of 303 patients undergoing IVF treatments with or without ICSI at the Division of Infertility Clinic, Lee Womens Hospital, Taichung, Taiwan were included in this study. Informed consent was received from patients from July to December All of the experimental procedures and sample procurements were approved by institutional bioethics review boards at both Lee Womens Hospital and Chung Shan Medical University. All of the patients underwent IVF treatments for the first time (217 IVF cycles and 86 ICSI cycles). Other assisted reproduction procedures involving cryopreserved sperm, donor sperm, or sperm derived from testicular biopsy were excluded from this study. Only sperm freshly ejaculated and prepared for IVF/ICSI procedures were included in this study. IVF/ICSI Treatment Procedure The controlled ovarian hyperstimulation protocol, oocyte retrieval, insemination, and ET were carried out as previously described (26). In brief, the controlled ovarian hyperstimulation protocol included gonadotropin (FSH [Metrodin; Serono, Rome, Italy], hmg [Pergonal; Serono]) administration and GnRH-agonist (leuprolide acetate, 1 mg/day; Abbott Laboratories, Chicago, IL) downregulation with the long protocol (26). Oocytes were retrieved through the vagina with sonographic guidance. The culture and insemination of oocytes were performed in human tubal fluid medium (Gibco BRL Life Technologies, Grand Island, NY) in an atmosphere of 5% CO 2 in air at 37 C. Sperm Preparation for Assisted Reproductive Techniques The 303 sperm samples were prepared with discontinuous PureSperm gradients in tubes (Nicadon, Gothenberg, Sweden). Each tube contained three layers of PureSperm at 90%, 70%, and 50% from bottom to top, respectively (1 ml for each layer). The 50% layer on the top was then covered with 1 ml of semen that had been collected and kept at room temperature for liquefaction. The preparation was then centrifuged at 300 g for 20 minutes. After centrifugation, the 90% layer was collected and washed with 5 ml of Ferticult Flushing (FertiPro, Beernen, Belgium) and then centrifuged at 600 g for 10 minutes. The pellet obtained after centrifugation was suspended in IVF medium (Scandinavian IVF, Gothenburg, Sweden) for IVF or in N-2-hydroxyethylpiperazine-N=-2-ethanesulfonic acid IVF medium for ICSI. Fertility and Sterility 131

3 TABLE 1 DNA fragmentation rates of the normal sperm used in IVF and the abnormal sperm used in ICSI. Normal sperm in IVF Abnormal sperm in ICSI No. of samples Sperm DNA fragmentation rate (%), a Fertilization rate (%), Good embryo rate (%), Pregnancy rate (%) a P.05 compared with normal sperm in IVF, Student s t-test. Sperm Evaluation All sperm samples were freshly prepared and evaluated on the day of IVF/ICSI. Each washed sample (5 L) was prepared with Papanicolaou stain, loaded on a 10- m Makler chamber (Sefi Medical Industries, Haifa, Israel), and processed through CASA machines (CHRISMAS Clinical 4.5; Image House, Copenhagen, Denmark), according to the manufacturer s instructions. To avoid variations between analyses, the setting for CASA was fixed according to the Kruger strict criteria (27). At least 100 spermatozoa were counted for each sample. Spermatozoa with borderline morphologies were counted as abnormal. Results were expressed as the percentage of normal spermatozoa observed in each chamber. Sperm samples with concentration /ml, motility 50%, and FIGURE 2 Raw scattered plots showing the fertilization rates and good embryo rates in correlation with sperm DNA fragmentation rates in the IVF/ICSI groups. 132 Huang et al. Sperm DNA fragmentation reduces velocity Vol. 84, No. 1, July 2005

4 TABLE 2 Comparison between normal sperm used in IVF and abnormal sperm used in ICSI in OAT, AT, OT, and T subgroups. Normal sperm used in IVF Abnormal sperm used in ICSI OAT AT OT T No. of samples Sperm DNA fragmentation ab ab ab a rate (%), Fertilization rate (%), Good embryo rate (%), Pregnancy rate (%) Note: OAT oligoasthenoteratozoospermia; AT asthenoteratozoospermia; OT oligoteratozoospermia; T teratozoospermia. a P.05 compared with normal sperm used in IVF, Student s t-test. b P.05 compared with the teratozoospermia sperm used in ICSI, Student s t-test. normal morphology 14% were regarded as normal sperm and used for IVF. Others not reaching those criteria were regarded as abnormal sperm and used for ICSI. Fertilization Rates and Embryo Grading At hours after insemination or microinjection (i.e., at the two-pronuclei stage), the oocytes were assessed to determine whether fertilization had occurred. At 70 hours after oocyte retrieval, the embryos normally reached the eight-cell stage and were graded according to their morphology. Embryo grading criteria were as follows: grade 8A (graded A at the eight-cell stage): no fragmentation with equal-sized cells; grade 8B: 20% fragmentation with equal-sized cells; grade 8C: no fragmentation with unequal-sized cells; grade 8C : 20% fragmentation with unequal-sized cells; grade 8D: 50% fragmentation. Embryos graded 8A, 8B, and 8C were denoted as good embryos. Embryo transfers were performed with three to four good embryos at 72 hours after oocyte retrieval. Only the good embryos were chosen for ET. After transfer, the remaining blastocysts exhibiting good morphology were cryopreserved. TUNEL Assay for Sperm DNA Fragmentation Sperm DNA fragmentation was evaluated with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay (Boehringer Mannheim, Mannheim, Germany). A small portion of each semen sample that had been prepared for IVF/ICSI was used. The samples were washed twice in phosphate-buffered saline (PBS) and centrifuged for 5 minutes at 200 g to collect the sperm cells. The sperm cells were then suspended in PBS, and 25 L of each preparation was dropped onto slides, air-dried, then incubated in a solution of 0.1% Triton X-100 (Sigma, St. Louis, MO) and 0.1% sodium citrate (Sigma) for 2 minutes on ice. After incubation, the slides were washed twice and airdried. A 30- L TUNEL mixture, containing terminal deoxynucleotidyl transferase (TdT) and fluorescein-deoxyuridine triphosphate, was added to each slide. The slides were covered with mm coverslips and then incubated for 60 minutes at 37 C in a moist dark chamber. After incubation, the preparations were washed three times with PBS and then analyzed with a fluorescence microscope (Labopho-2; Nikon, Tokyo, Japan). At least 500 cells were counted on each slide to obtain the results. Only evident fluorescent signals were regarded as positive. When there was doubt, the same slides were stained with DAPI (4=, 6=-diamidino-2-phnylindole; Sigma) to confirm the presence of positive signals within the sperm head. Each TUNEL assay was performed with accompanying positive and negative controls. Positive control sperm samples were fixed and incubated with 1 g/ml DNase I (Sigma) for 10 minutes at room temperature to induce DNA fragmentation, which was then followed by the same procedures as described for the experiments. The negative controls were prepared accordingly, but without TdT. Statistical Analysis Student s t-test was used to assess statistical differences, with P.05 considered significant. The Spearman rank correlation was performed with raw scattered plots to show the Fertility and Sterility 133

5 TABLE 3 Comparison of various levels of sperm DNA fragmentation rates in 303 samples used in IVF or ICSI. Sperm DNA fragmentation rates (%) <4 4 <10 IVF ICSI Total IVF ICSI Total No. of samples Sperm DNA fragmentation rate (%), Fertilization rate (%), b Good embryo rate (%), Pregnancy rate (%) a P.05 compared with the total group of sperm samples with DNA fragmentation rates 4%, Student s t-test. b P.05 compared with the IVF group of sperm samples with DNA fragmentation rates 4%, Student s t-test. c P.05 compared with the ICSI group of sperm samples with DNA fragmentation rates 4%, Student s t-test. correlation of the sperm fragmentation rate with the fertilization rate and good embryo rate. RESULTS Sperm DNA Fragmentation Rates in IVF and ICSI Groups Sperm DNA fragmentation was detected with the TUNEL assay, in which a positive signal was judged by the presence of evident green fluorescence within the sperm head (Fig. 1). The sperm DNA fragmentation rate in the ICSI group was significantly higher than that in the IVF group, being 6.8% in the abnormal sperm for ICSI, in contrast to 1.9% in the normal sperm for IVF (Table 1). No significant differences in mean fertilization rates, good embryo rates, and pregnancy rates were found between the IVF and the ICSI groups. With raw scattered plots, however, the good embryo rate and fertilization rate distributions were different between the IVF and ICSI groups (Fig. 2). Although both groups seemed to exhibit a negative correlation between sperm DNA fragmentation rates and fertilization rates, the ICSI group seemed to be less affected. To better elucidate the sperm DNA fragmentation rates, we further divided the ICSI group into four subgroups: oligoasthenoteratozoospermia, oligoteratozoospermia, asthenoteratozoospermia, and teratozoospermia. All four subgroups had significantly higher sperm DNA fragmentation rates than the normal sperm group for IVF (Table 2). The sperm DNA fragmentation rates in the oligoasthenoteratozoospermia, oligoteratozoospermia, and asthenoteratozoospermia subgroups were also higher than that for the teratozoospermia subgroup (Table 2). When the sperm DNA fragmentation rates of the 303 samples were further divided into four subgroups of 4%, 4% 10%, 10% 15%, and 15%, significantly lower fertilization rates and good embryo rates were found in the 10% 15% and the 15% subgroups, and a lower good embryo rate was found in the 10% 15% subgroup, when compared with the 4% subgroup (Table 3). When each subgroup was further split into IVF and ICSI subdivisions, IVF fertilization rates in the 4% 10% and the 10% 15% subgroups were significantly lower than that in the 4% subgroup. The IVF good embryo rate in the 10% 15% subgroup was also significantly lower than that in the 4% subgroup. The ICSI fertilization rate in the 15% subgroup was significantly lower than that in the 4% subgroup (Table 3). The negative correlation between the sperm DNA fragmentation rates and fertilization rates or good embryo rates was further examined with Spearman rank correlation coefficients. The results showed that fertilization rates in both the IVF and ICSI subdivisions were negatively correlated with the sperm DNA fragmentation rates, with the correlation coefficients at and 0.222, respectively (Table 4). Sperm DNA Fragmentation with Morphology Correlation To further understand the effects of sperm DNA fragmentation, we analyzed different percentages of normal sperm morphology in relation to the sperm DNA fragmentation rate, fertilization rate, good embryo rate, and pregnancy rate (Table 5). The sperm DNA fragmentation rates were negatively correlated with the percentage of sperm with normal morphology, when the samples exhibiting normal morphology at 1% or less were compared with those exhibiting 14% normal morphology (Table 5). No correlation was 134 Huang et al. Sperm DNA fragmentation reduces velocity Vol. 84, No. 1, July 2005

6 TABLE 3 Continued Sperm DNA fragmentation rates (%) 10 <15 15 IVF ICSI Total IVF ICSI Total b a c a b a found between the percentages of sperm exhibiting normal morphology and good embryos rates, nor between normal morphology rates and pregnancy rates (Table 5). Sperm DNA Fragmentation with Sperm Motility Parameter Correlation To make sense of the routinely available data from CASA, we correlated the sperm motility parameters, including average path velocity (VAP) and straight line velocity (VSL), with sperm DNA fragmentation rates and reproductive performance (Tables 6 and 7). We found that the percentage of TABLE 4 Spearman rank correlation coefficients between sperm DNA fragmentation rates and fertilization rates or good embryos rates in the IVF and ICSI groups. Spearman rank correlation coefficient ( ) P value IVF fertilization rate a.000 IVF good embryo rate ICSI fertilization rate b.040 ICSI good embryo rate a P.01. b P.05. sperm DNA fragmentation had to be 3.0% to achieve the normal criteria of VAP ( 30 m/s) and VSL ( 20 m/s). The fertilization rate was significantly decreased in sperm with VAP 10 m/s, as compared with those with VAP 30 m/s. The sperm DNA fragmentation rates were negatively correlated with VAP when they were 30 m/s (Table 6), as compared with those with VAP 30 m/s. Similarly, the sperm DNA fragmentation rates were negatively correlated with VSL when they were 20 m/s (Table 7), when compared with those with VSL 20 m/s. However, as in the subgroups that depict rates of normal sperm morphology, no correlation was found between VAP/ VSL parameters and good embryos rates or between VAP/ VSL parameters and pregnancy rates. DISCUSSION Although ICSI has long been regarded as a successful infertility treatment technique that addresses the male factor, particularly for those with conditions of oligoasthenoteratozoospermia, oligoteratozoospermia, asthenoteratozoospermia, and teratozoospermia (28), it remains controversial whether ICSI procedures increase the risk of transmitting impaired DNA to the next generation (8, 29 31). Although abnormal sperm motility and morphology represent increased levels of DNA damage (10), it is mainly patients with severe semen parameter defects who require treatment by ICSI (28). Hence, choosing the right spermatozoa poses a challenge for the ICSI therapist (10). Sperm motility and morphologic profile, although routinely obtained through semen analysis, might not be of paramount value in choosing a specific sperm for ICSI. Lopes et al. (28) demonstrated that one quarter of unfertil- Fertility and Sterility 135

7 TABLE 5 Comparison of various percentages of sperm with normal morphology in 303 samples used in IVF or ICSI. Sperm normal morphology (%) <14 14 No. of samples Sperm DNA fragmentation rate (%), a a Fertilization rate (%), a a Good embryo rate (%), Pregnancy rate (%) a P.05 compared with sperm normal morphology 14%, Student s t-test. ized ICSI oocytes were most likely injected with a spermatozoa containing fragmented DNA. A recent report published by Morris et al. (31) stated that sperm selection for ICSI in terms of DNA was random. Thus, there is clearly a great need for reference criteria to help in choosing the right sperm for ICSI. Apart from sperm motility and morphology, another potentially useful indicator is the sperm DNA fragmentation rate. Extensive literature has shown that sperm DNA fragmentation might affect the outcome of assisted reproduction through effects on fertilization (8, 10 14), sperm motility (8, 10, 14 23), sperm morphology (8, 10, 16, 23), sperm concentration (8, 15, 22, 24), embryo cleavage (8, 12), and pregnancy rate (25). Indeed, some investigators have used the sperm DNA fragmentation rate as a marker to differentiate fertile from infertile men (9) and to predict the outcome of IUI (5). However, sperm DNA fragmentation is not currently used as part of the reference criteria in most assisted reproduction laboratories because it is not routinely included in semen analyses. In addition, the extent to which sperm DNA fragmentation rates can be applied in clinical settings remains to be evaluated, because only a few specific sperm cells are chosen for ICSI. In this study, we examined sperm DNA fragmentation rates in correlation with sperm parameters from CASA, in an attempt to relate them to fertilization rate, embryo quality, and pregnancy outcome. Our aim was to assess the possibility of depicting the DNA fragmentation rates in terms of CASA results and to evaluate the prognostic and diagnostic value of CASA data as part of the reference criteria for ICSI treatment. Our data showed higher DNA fragmentation rates in abnormal sperm for ICSI, as compared with normal sperm for IVF (6.8% vs. 1.9%) (Table 1), strongly correlating sperm DNA fragmentation with poor sperm quality. Because we defined abnormal sperm samples as not reaching the standard concentration /ml, motility of 50%, and normal morphology 14%, we further correlated various defective sperm parameters with sperm DNA fragmentation by subdividing the abnormal sperm samples into four subgroups: oligoasthenoteratozoospermia, oligoteratozoospermia, asthenoteratozoospermia, and teratozoospermia. All of the four subgroups exhibited significantly higher sperm DNA fragmentation rates than the normal sperm group (Table 2), suggesting no preferential DNA impairment effect on the concentration or morphology. Interestingly, DNA strand breaks did not seem to affect the fertilization rate, embryo quality, and pregnancy rate in the abnormal sperm group with ICSI, when compared with the normal sperm group with IVF treatment (Table 1). Neither was there any significant difference in the fertilization rate, embryo quality, and pregnancy rate between any of the four subgroups of abnormal sperm with ICSI and the 136 Huang et al. Sperm DNA fragmentation reduces velocity Vol. 84, No. 1, July 2005

8 TABLE 6 Comparison of various levels of average path velocity in 303 samples used in IVF or ICSI. Sperm average path velocity ( m/s) 10 >10 15 >15 20 >20 25 >25 30 >30 No. of samples Sperm DNA fragmentation rate (%), a a a a a Fertilization rate (%), a a Good embryo rate (%), Pregnancy rate (%) a P.05 compared with sperm average path velocity 30 m/s, Student s t-test. normal sperm with IVF (Table 2). Notably, however, we observed a more than twofold higher pregnancy rate in normal sperm as compared with that in the oligoteratozoospermia subgroup (55.3% vs. 25%), though no statistical difference was found. This raised the possibility that the data herein might not have enough statistical power to assert a correlation or a noncorrelation, at least between the normal sperm group and the oligoteratozoospermia subgroup. Regarding the fertilization rate, our data were contrary to those previously published by Sun et al. (10), Lopes et al. (9, 28), and Host et al. (32). These investigators asserted a negative DNA fragmentation correlation with the fertilization rate. On the other hand, our data were in agreement with reports published by Morris et al. (31), Tesarik et al. (33), and Henkel et al. (25), who described no sperm DNA fragmentation or paternal factor correlation with the fertilization rate. In terms of embryo quality and pregnancy rates, there also are formerly reported data showing a contradictory outcome. Shoukir et al. (34) and Benchaib et al. (13) presented data indicating no correlation between sperm DNA fragmentation and embryo quality. Oehninger et al. (35) suggested no significant severe male infertility effect, oligoasthenoteratozoospermia for example, on implantation and pregnancy outcome. In contrast, Sun et al. (10) proposed a significant negative association between the percentage of sperm with DNA fragmentation and the embryo cleavage rate, and Henkel et al. (25) found a significantly reduced pregnancy rate in IVF patients inseminated with TUNEL-positive spermatozoa. Thus, whether increased sperm DNA fragmentation rate might be a detrimental factor in embryo quality and pregnancy outcome remains unsettled. The previous contradictory reports, however, reflect the complexity of sperm TABLE 7 Comparison of various levels of straight line velocity in 303 samples used in IVF or ICSI. Sperm straight line velocity ( m/s) 5 >5 10 >10 15 >15 20 >20 No. of samples Sperm DNA fragmentation a a a a rate (%), Fertilization rate (%), Good embryo rate (%), Pregnancy rate (%) a P.05 compared with sperm average path velocity 30 m/s, Student s t-test. Fertility and Sterility 137

9 genomic integrity as a determining factor in IVF/ICSI treatments. Taken further, oocytes might also contain fragmented or damaged DNA, as suggested by studies in mice (38) and in humans (29, 36, 37). It has been suggested that oocytes contain a limited capacity to repair sperm DNA fragmentation (38), which might have greatly affected the outcome of formerly reported data, owing to different criteria in recruiting oocytes in the studies. Furthermore, sperm DNA integrity does not represent all of the paternal effects controlling early embryonic activities after IVF/ICSI treatment. Tesarik et al. (33) found that paternal effects actually exist during the first cell cycle of human preimplantation development after ICSI, whereas the major embryonic gene activation occurs between the four-cell and the eight-cell stages in human embryos (39, 40). They also demonstrated that early paternal effects could compromise assisted reproduction outcomes in the absence of increased sperm DNA fragmentation (40), suggesting that sperm DNA integrity only affects morphologic abnormalities beyond the zygote and early cleavage stages. This might partly explain the lack of correlation between sperm DNA fragmentation and fertilization rates and between sperm DNA fragmentation and embryo quality (expressed as the good embryo rate) in the present study and in previous reports. However, contrary to the impaired implantation rates observed by Tesarik et al. (40) in their experimental groups, we did not see comparable results because our pregnancy rate in the normal sperm group for IVF did not differ significantly from that in the abnormal sperm group for ICSI, even though their DNA fragmentation rates were significantly different (1.9% 3.1% vs. 6.8% 6.1%) (Table 1). Because we transferred only good embryos on day 3 after IVF or ICSI, one possibility is that the detrimental sperm DNA fragmentation effects were excluded during embryo selection. Another possibility might be that the level of DNA fragmentation, averaging between 1.9% 3.1% and 6.8% 6.1% in our 303 sperm samples, did not affect the pregnancy outcome. A third possibility is that we did not distinguish live from dead sperm when performing the TUNEL assay, because only live sperm were selected for ICSI. This could have distorted the correlation picture. To distinguish the possibilities mentioned above and further clarify the level of sperm DNA fragmentation at which fertilization, embryo quality, and pregnancy outcome might be affected, we examined raw scattered plots in the IVF and the ICSI groups. We found that both groups seemed to exhibit a negative correlation between sperm DNA fragmentation and fertilization rates. The ICSI group seemed to be less affected, which suggests that artificial sperm selection during the ICSI procedure might have helped in evading the negative effect to some extent. This prompted us to further divide the 303 sperm samples into four groups according to their DNA fragmentation rates. A decreasing trend of fertilization rates and good embryo rates was found along with the increase in DNA fragmentation (Table 3). Further examination with Spearman rank correlation coefficients showed that sperm DNA fragmentation rates were negatively correlated with fertilization rates in both the IVF and the ICSI groups but not with good embryo rates (Table 4). There seemed to be a demarcation at the 10% rate of sperm DNA fragmentation defining significantly lower fertilization rates, when samples with a 10% rate were compared with those with a 4% rate (Table 3). A comparable demarcation at 10% was also reported in a recent study by Benchaib et al. (13). It therefore seems that sperm DNA fragmentation might indeed impair fertilization, but only for those with higher rates. It is possible that if the sperm DNA fragmentation rate is low, the oocytes might be able to repair and probably reverse the condition, as suggested by Ahmadi and Ng (37). When the limited DNA repair capacity is overloaded with higher levels of DNA fragmentations, at 10% for example, the compensatory mechanism is surpassed, resulting in lower fertilization rates. The DNA fragmentation effect on sperm morphology seemed to be evident in the samples containing only 1% normal-morphology sperm (Table 5). Similarly, Sills et al. (23) compared sperm chromatin fluorescence characteristics and standard semen analysis parameters and found significant inverse correlations between normal sperm morphology and sperm chromatin fluorescence characteristics that were mainly represented by the DNA fragmentation index and high DNA stainability. It is noteworthy that although the sperm DNA fragmentation rate was relatively low in samples of 14% good morphology, there remained a 2.3% 7.4% DNA fragmentation rate in the sperm used for IVF/ ICSI treatment in our study (Table 5). No correlation was found between the percentages of sperm exhibiting normal morphology and good embryos rates, nor between normal morphology rates and pregnancy rates (Table 5). The present data reflect the inadequacy of using morphology as the main criterion in selecting a specific sperm for ICSI. A significant negative correlation was observed between DNA fragmentation rates and sperm motility in recent reports (8, 10, 14 23). Nevertheless, previous studies did not give details of sperm motility in terms of VSL or VAP; both are routinely available from CASA analyses. Our results in this study clearly demonstrated a negative association between VAP and sperm with fragmented DNA, when the samples with a VAP 30 m/s were compared with those with a VAP 30 m/s (Table 6). A comparable negative association between VSL and sperm with fragmented DNA was also found when the samples with a VSL 20 m/s were compared with those with a VSL 20 m/s (Table 7). When the VAP was 30 m/s or the VSL was 20 m/s, the sperm DNA fragmentation rates were expected to be no more than 3.0%, a status similar to that of the normal sperm group used for the IVF treatments (Tables 1, 6, and 7). We suggest that the status of VAP 30 m/s and VSL 20 m/s might serve as part of the reference criteria for specific sperm selection from portions of samples used for ICSI. 138 Huang et al. Sperm DNA fragmentation reduces velocity Vol. 84, No. 1, July 2005

10 In conclusion, our data supported a strong correlation between sperm DNA fragmentation and poor sperm quality, although no preferential effect on sperm concentration or morphology seemed to be present. In addition, to depict the inadequacy of sperm selection based mainly on morphology, our data offered a clear demarcation of sperm DNA fragmentation that might affect fertilization. We have also reported the minimal ICSI sperm VSL and VAP that might offer a reproductive performance similar to non-icsi IVF. These demarcations might help in the interpretation of CASA results and in sperm selection for ICSI. Acknowledgments: The authors thank Chiu-Ping Chen, Ming-Chou Hung, and Hsiu-Hui Chen for their laboratory support, and Han-Hsin Chang for critical comments on the manuscript. REFERENCES 1. World Health Organization. WHO laboratory manual for the examination of human semen sper-cervical mucus interaction. 4th ed. 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