Hum. Reprod. Advance Access originally published online on March 5, 2008
Human Reproduction 2008 23(5):1023-1028; doi:10.1093/humrep/den060
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Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa
ART Research Center, Mizmedi Hospital, 701-4, Naebalsan-dong, Kangseo-gu, Seoul, Korea
1 Correspondence address. Tel: +82-22007-1840; Fax: +82-22007-1852; E-mail: ivf129{at}mizmedi.net
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Abstract |
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Top
Abstract
IntroductionBACKGROUND: Oxidative stress induced by reactive oxygen species (ROS) is associated with an impaired fertilization ability of spermatozoa. We investigated the effects of adding antioxidants to a sperm preparation medium on the functional parameters of the spermatozoa.
METHODS: Spermatozoa were washed with Ham's F-10 media containing the antioxidants, ethylenediaminetetraacetic acid (EDTA) and catalase, at various concentrations, and then the ROS levels in sperm suspensions, and the forward motility, acrosome reaction, DNA integrity and lipid peroxidation of the spermatozoa were assessed.
RESULTS: The ROS levels were significantly lower in sperm suspensions washed with the antioxidants (196
312 rlu; relative light units) than in control sperm (604 rlu, P < 0.05). The addition of 10 µM EDTA to the sperm preparation medium significantly improved the motility of the spermatozoa compared with the control group, the groups containing EDTA at other concentrations and the groups containing catalase. Catalase significantly increased the acrosome reaction rate of the spermatozoa. Both EDTA and catalase significantly decreased the DNA fragmentation rate of the spermatozoa. However, the antioxidants did not reduce lipid peroxidation.
CONCLUSIONS: Supplementing sperm preparation medium with EDTA or catalase significantly improved the overall functional parameters of the spermatozoa by reducing the ROS levels.
Key words: oxidative stress/spermatozoa/antioxidant/motility/DNA fragmentation
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Introduction |
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Certain levels of free radicals generated in the respiratory chain are necessary for the normal functions of sperm cells, including capacitation, hyperactivation, acrosome reaction, oocyte fusion and fertilization (Griveau and Le Lannou, 1997
; de Lamirande et al., 1998; Kim and Parthasarathy, 1998; Rivlin et al., 2004). However, the generation of excess reactive oxygen species (ROS) is associated with cell damage, including morphological defects (Aziz et al., 2004), DNA fragmentation and lipid peroxidation (Fraczek and Kurpisz, 2005), decreases in acrosome reaction and fusiogenic ability (Lemkecher et al., 2005), impaired fertilization ability of spermatozoa (Aitken and Baker, 2006) and lower pregnancy rates after IVF (Zorn et al., 2003; Hammadeh et al., 2006). These reports are consistent with the demonstration that oral antioxidant treatment appears to improve sperm DNA integrity and ICSI-IVF outcomes in those patients with sperm DNA damage or other severe male infertility (Greco et al., 2005; Menezo et al., 2007; Tremellen et al., 2007).
While seminal plasma is a rich source of antioxidants (Kim and Parthasarathy, 1998; Potts et al., 2000; Gallardo, 2007), semen also contains leukocytes that are major producers of ROS (Sharma et al., 2001). Moreover, it is well known that long-term exposure to seminal plasma is detrimental to the motility and survival of spermatozoa, with one of the major underlying factors being the presence of a seminal plasma motility inhibitor derived from semenogelin I and II (Murakami et al., 1998; Yoshida et al., 2003). This makes it necessary to remove seminal plasma by washing in order to maintain the motility of spermatozoa, but this procedure may also remove the endogenous antioxidants in seminal plasma.
Ironically, washing sperm by centrifugation can also generate ROS (Agarwal et al., 1994; Shekarriz et al., 1995), which has prompted some authors to add antioxidants to sperm preparations to avoid the oxidative stress induced by centrifugation. However, the addition of vitamin E to semen samples (Yenilmez et al., 2006) and the addition of glutathione or/and hypotaurine to a sperm preparation medium did not show beneficial effects on the sperm progressive motility or the baseline DNA integrity (Donnelly et al., 2000). Furthermore, the addition of superoxide dismutase (SOD) increases the DNA fragmentation of equine spermatozoa, rather than decreasing the oxidative stress (Baumber et al., 2005). Therefore, it is necessary to identify the optimal thresholds for ROS and antioxidant levels, and those antioxidants that efficiently protect and maintain functional sperm.
The present study investigated the effects of adding ethylenediaminetetraacetic acid (EDTA) and catalase to a sperm preparation medium. Sperm motility, acrosome reaction, DNA integrity and lipid peroxidation were assessed, along with the optimal level of antioxidants required to minimize oxidative stress during sperm preparation.
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Materials and Methods |
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This study was approved by our institutional review board (Mizmedi hospital clinical research ethics committee), and informed consent was obtained from all patients who participated in this study.
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Semen sample preparation and antioxidants |
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Forty-two normozoospermic patients participated in this study. Semen inclusion criteria were as follows: concentration, >60 x 106/ml; motility, >50%; forward motility, >20%; total motile sperm count, >40 x 106 and strict morphology score, >10%. After semen analysis, the remnant semen samples were used in this study under the patient's agreement. We used EDTA and catalase as antioxidants in this study. EDTA prevents the conversion of the superoxide anion into hydrogen peroxide by inhibiting divalent-ion-dependent SOD. Catalase catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. Various concentrations of EDTA (100, 10, 1 µM/ml, Sigma, St Louis, USA) or catalase (100, 10, 1 U/ml, Sigma) were added to Ham's F-10 medium (Gibco, New York, USA) with 0.1% BSA. After liquefaction, semen samples were washed twice with the media containing antioxidants at 1000 rpm for 10 min.
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Sperm motility analysis |
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After washing the sperm with the Ham's F-10 medium or the media containing the antioxidants, the sperm suspensions were incubated for 1 h at 37°C and then forward motility of the sperm was measured using computer-aided semen analyzer (Medical-Supply, Seoul, Korea).
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Measurement of ROS |
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After washing of the sperm, levels of ROS in sperm suspensions were measured by chemiluminescence assay using lucigenin (bis-N-methylacridnium nitrate; Sigma). Lucigenin was prepared as 25 mM stock solution in dimethyl sulfoxide (DMSO, Sigma). A 4 µl of lucigenin stock solution was added to 400 µl of sperm suspension. A negative control was prepared by adding 4 µl of lucigenin to 400 µl of sperm suspension washed with antioxidant-free Ham's F-10 medium. Chemiluminescence was measured 15 min after addition of lucigenin using a luminometer (Bio-tek, Vermont, USA) at 37°C. Results were expressed as relative light units (rlu) per 2 x 107 sperm/ml.
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Acrosome reaction analysis: staining with FITC-ConA |
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Twenty-four hours after washing the sperm, the spermatozoa were fixed with 10% formaldehyde for 60 min at room temperature. After washing with phosphate-buffered saline (PBS), 100 µl of FITC-ConA solution (100 µg/ml, Sigma) was mixed with sperm pellet and staining was performed for 25 min at room temperature. After washing with PBS, the spermatozoa were mounted on a glass slide with 90% glycerol and observed at x400 under a fluorescence microscope (Nikon, Tokyo, Japan).
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Sperm DNA integrity analysis: comet assay |
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Twenty-four hours after washing the sperm, a single-cell electrophoresis, comet assay was performed to investigate the effect of antioxidants on sperm DNA integrity using a comet assay kit (Trevigen, Gaithersburg, USA). Five microlitre of semen sample was mixed with 45 µl of low melting agarose and layered onto a specially designed comet slide. The slide was covered with cover glass and transferred into 4°C refrigerator and cooled down for 10 min. The slide was submerged into a pre-cooled (4°C) lysing solution (containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, 10 mM DL-dithyothreitol, Sigma) for 2 h and then placed on a horizontal electrophoretic unit (BioRad, Hercules, USA). The slide was left in electrophoresis buffer (500 mM NaCl, 100 mM Tris, 1 mM EDTA, 0.2% DMSO, Sigma) for 20 min, and then electrophoresed for 60 min at 10 V and 250 mA. After electrophoresis, the slide was washed with PBS and immersed in neutralizing solution (50% Ethanol, 20 mM Tris, 1 mg/ml Spermine, Sigma) for 5 min. This step was repeated three times with fresh solution. After washing with PBS, the slide was stained with SYBR staining solution containing 10 mM Tris, 1 mM EDTA, 0.01% SYBR Green (Trevigen). After washing with distilled water, the slide was covered with vectashield mounting solution (Vector Labs, Burlingame, USA). Images at magnification x400 were captured using a digital camera imaging system (Nikon digital sight DS-U1) attached to a fluorescence-inverted microscope.
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Measurement of lipid peroxidation |
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Lipid peroxides, derived from polyunsaturated fatty acids, are unstable and decompose to form a complex series of compounds including malondialdehyde (MDA). In the present study, the levels of MDA in sperm suspensions were assessed using a commercial MDA-586 kit (OxisResearch, Portland, USA). After the treatment with, or without, antioxidants, semen samples (1 x 108/ml) were washed twice with PBS at 1000 rpm for 10 min. Five hundred microlitre of ice cold PBS and 5 µl of butylated hydroxytoluene were added to the sperm pellet and the cells were homogenized. After incubation at 48°C for 10 min, 10 µl of pubucol and 640 µl of N-methyl-2-phenylindole were added to 200 µl of semen sample and mixed thoroughly by vortex. After incubation at 45°C for 1 h, the samples were centrifuged at 10 000 rpm for 10 min to obtain the clear supernatants. MDA levels of semen samples were measured using a spectrometer (BioRad) at 586 nm.
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Statistical analysis |
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Values in figures are presented as mean ± standard deviation (SD). Differences between treatment groups were analyzed with
2 test and, when appropriate, two-tailed Student's t-test. For all statistical procedures performed, a P-value <0.05 was considered significant. |
Results |
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The levels of ROS in the sperm suspension were measured by a chemiluminescence assay using lucigenin after washing the sperm with the antioxidants (Fig. 1). The ROS levels in the sperm suspensions were significantly lower in the experimental groups [100 µM EDTA, 287 ± 91 (mean ± SD) rlu; 10 µM EDTA, 264 ± 89 rlu; 1 µM EDTA, 312 ± 209 rlu; 100 U of catalase, 196 ± 76 rlu; 10 U of catalase, 212 ± 93 rlu; 1 U of catalase, 218 ± 151 rlu] than the control group (604 ± 290 rlu, P < 0.05). Although the ROS levels of the catalase groups were lower than those of the EDTA groups, the differences were not statistically significant.
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The effect of adding antioxidants to the sperm-washing medium on the forward motility of spermatozoa was investigated (Fig. 2). We also assessed the curvilinear velocity, straight-line velocity, average path velocity, amplitude of lateral head displacement, beat cross frequency and hyperactivation, but the figure only shows forward motility data since the patterns of these parameters were similar to that of motility. The forward motility did not differ between the control group (22.6 ± 7.9%) and the catalase groups (24.8 ± 11.8 to 27.9 ± 11.5%), but it was significantly improved with 100 µM EDTA (32.0 ± 12.3%) and 10 µM EDTA (38.3 ± 12.0%, P < 0.05).
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The effect of antioxidants on the acrosome reaction of spermatozoa was assessed with FITC-ConA staining 24 h after antioxidant treatment (Fig. 3). The acrosome reaction rate did not differ between the control group (23.2 ± 9.7%) and the EDTA groups (21.7 ± 12.0 to 24.0 ± 12.7%), but it was significantly increased by the addition of a low level of catalase (10 U of catalase, 26.4 ± 14.1%; 1 U of catalase, 26.2 ± 15.0%).
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To evaluate the protective effect of antioxidants on sperm DNA integrity, the DNA fragmentation rate of the spermatozoa was assessed by the comet assay 24 h after washing the sperm (Fig. 4). The DNA fragmentation rates of the spermatozoa were significantly lower in the antioxidant groups (100 µM EDTA, 23.0 ± 10.8%; 10 µM EDTA, 20.7 ± 10.0%; 1 µM EDTA, 20.8 ± 4.8%; 100 U of catalase, 23.1 ± 6.2%; 10 U of catalase, 20.0 ± 2.8%; 1 U of catalase, 20.1 ± 2.6%) than in the control group (27.2 ± 8.0%, P < 0.05). Although the rates of DNA fragmentation between the EDTA and catalase groups were not different, the protective effect of catalase on the sperm DNA fragmentation was more consistent and stable because the catalase group showed a small size of the SD compared with the EDTA group.
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The effect of antioxidants on lipid peroxidation of the spermatozoa was also investigated (Fig. 5). The level of MDA was estimated with the MDA-586 kit and a spectrometer at 586 nm. The MDA levels did not differ significantly between the control group (3.61 ± 0.94 µM) and the antioxidant groups (100 µM EDTA, 2.56 ± 1.80 µM; 10 µM EDTA, 3.3 ± 0.72 µM; 1 µM EDTA, 4.23 ± 1.25 µM; 100 U of catalase, 3.29 ± 1.57 µM; 10 U of catalase, 3.81 ± 1.49 µM; 1 U of catalase, 4.12 ± 1.19 µM per 1 x 108 sperm/ml). These data indicate that the antioxidants did not have beneficial effects on lipid peroxidation of the spermatozoa.
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Discussion |
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Ham's F-10 medium was used as a basic medium for sperm preparation in the present study. For oocyte or embryo culture, this medium is well known to be unsuitable because it contains hypoxanthine that induces meiotic arrest and a cell block. Hypoxanthine increases the cellular cAMP level by inhibiting phosphodiesterase (PDE), which results in meiotic arrest of oocytes. However, pentoxifylline, which is also an inhibitor of PDE, improves sperm motility by prompting the cAMP-dependent tyrosine phosphorylation of sperm proteins (Yunes et al., 2005
). Therefore, we considered that Ham's F-10 was a suitable medium for sperm preparation in the present study.
In the present study, the ROS level in the sperm suspension was significantly reduced by the addition of antioxidants to the sperm preparation medium. In contrast, washing the sperm sharply increased the ROS level in the sperm suspension (data not shown). This result is consistent with reports that washing the sperm by centrifugation is associated with the generation of ROS and physical damage to spermatozoa (Alvarez et al., 1993; Agarwal et al., 1994; Shekarriz et al., 1995), and that the antioxidant capacity is higher for sperm separated with a swim-up than for the sperm separated with a percoll gradients, which requires more vigorous centrifugation (Fraczek et al., 2004). Interestingly, the beneficial effect of antioxidants on reducing the ROS level was not observed when antioxidants were added to ejaculated whole semen that had not been washed by centrifugation. Moreover, we did not find an acceptable value of ROS levels even in the seminal plasma of the control (data not shown). The low level of ROS in seminal plasma was probably due to the antioxidant levels already being high in seminal plasma (Kim and Parthasarathy, 1998; Potts et al., 2000; Gallardo, 2007). This would play a role in protecting ejaculated spermatozoa from the occurrence of oxidative stress in the female reproductive tract (Zini et al., 2002).
Luminol- and lucigenin-dependent chemiluminescences are the most commonly used methods for measuring ROS in sperm samples (Sikka, 2004). We attempted to detect the ROS level in sperm suspensions using luminol or lucigenin, but only obtained reliable values when using lucigenin. Said et al. (2004) was also only able to detect the ROS level in human sperm suspensions when using lucigenin, and not when using luminol. This may be due to lucigenin not only crossing the plasma membrane of cells, but also penetrating the mitochondria to measure ROS generated within the cell (Van Dyke et al., 2003). However, Roca et al. (2005) found that the level of ROS in thawed boar sperm suspensions was significantly higher when using luminol than when using lucigenin, and further studies should attempt to clarify the reasons for this difference, which might be simply reflect species specificity.
EDTA at 10 µM significantly increased the sperm motility in the present study. Interestingly, this concentration of EDTA is already widely used in human embryo culture media. However, when we added EDTA to the sperm preparation medium at a higher level (1 mM), the motility of the sperm decreased sharply to near zero (data not shown), which indicates that excessive chelation of divalent ion such as calcium is detrimental to sperm motility. However, unlike EDTA, the addition of catalase did not show the beneficial effect of increasing the sperm motility. This is consistent with Calamera et al. (2001) finding no positive effect of catalase on human sperm motility. Therefore, adding EDTA during sperm preparation to inhibit the production of ROS appears to be more effective at improving the sperm motility than adding catalase to scavenge the ROS produced.
The acrosome reaction is started with the fusion of the outer acrosome membrane and the plasma membrane of the sperm cell, and hence the fusiogenic ability of the membranes is critical to its success. Lipid peroxidation of the cell membrane is one type of cell damage induced by ROS. Therefore, the fusiogenic ability of the sperm cell membrane can be damaged by ROS, which results in inhibition of the acrosome reaction (Ichikawa et al., 1999; Lemkecher et al., 2005). In the present study, we found that the acrosome reaction rate of spermatozoa was significantly increased by the addition of catalase at low concentrations (10 and 1 U), but not at a high concentration (100 U). This result indicates that there is an optimal concentration for the beneficial effects of ROS on sperm capacitation including the acrosome reaction, with ROS at significantly higher or lower concentrations inhibiting this process (Rivlin et al., 2004).
It is well known that the integrity of sperm DNA is also affected by ROS (Fraczek and Kurpisz, 2005; Menezo et al., 2007), which has been associated with poor semen quality, low fertilization rate, impaired implantation, increased abortion rate and even an elevated incidence of disease in the offspring (Lewis and Aitken, 2005). Oral antioxidant treatment appears to improve sperm DNA integrity (Greco et al., 2005; Menezo et al., 2007; Tremellen et al., 2007). However, the addition of glutathione and hypotaurine (Donnelly et al., 2000) or SOD (Baumber et al., 2005) to a sperm preparation medium did not reduce the DNA fragmentation of the spermatozoa. In particular, the addition of SOD appears to be inappropriate since rather than acting as an antioxidant, it produces ROS such as hydrogen peroxide by scavenging the superoxide anion. In the present study, we found that adding EDTA and catalase as antioxidants reduced the fragmentation of sperm DNA. EDTA prevents the conversion of the superoxide anion into hydrogen peroxide by inhibiting divalent-ion-dependent SOD. Catalase directly catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. Therefore, using EDTA or catalase appears to protect DNA integrity from the powerful attack by hydrogen peroxide. Although we did not show the data from a combined treatment of EDTA and catalase in this study, we are accumulating the data about a possible synergistic effect of the combined treatment in a sequential study.
In the present study, we measured the MDA levels in semen samples in the absence of stimulating lipid peroxidation, unlike other authors who measured MDA levels following promotion with Fe2+/ascorbate (Williams et al., 2005; Aitken et al., 2007). Those studies were probably prompted by it being considered necessary to promote a lipid peroxidation chain reaction in order to generate a detectable level of MDA in human semen samples (Gomez et al., 1998). Therefore, our levels of MDA might have been too low to detect a beneficial effect of antioxidants on lipid peroxidation. Another possibility is that the lipid peroxidation was entirely dependent on MDA values in our study. This would lead to misinterpretation of our results, because MDA is only one of the several degradation products generated during the lipid peroxidation process of sperm membranes (Gomez et al., 1998). Therefore, further study on the effect of antioxidants on lipid peroxidation of spermatozoa is necessary.
In conclusion, preparing sperm with media containing antioxidants at the appropriate concentrations improves the overall functional parameters of the spermatozoa by reducing the ROS level, and hence may improve the outcome of ART programmes by protecting the spermatozoa from oxidative stress.
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Funding |
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This work was supported financially by our hospital (Mizmedi Hospital). There was not any commercial grant related to this study.
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Submitted on November 23, 2007; resubmitted on February 4, 2008; accepted on February 7, 2008.
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