Use of hydrogen peroxide to assess the sperm susceptibility to oxidative stress in subjects presenting a normal semen profile

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1 international journal of andrology, 27:82 87 (24) Use of hydrogen peroxide to assess the sperm susceptibility to oxidative stress in subjects presenting a normal semen profile M. M. MISRO,* L. CHOUDHURY, K. UPRETI,* D. GAUTAM,* S. P. CHAKI,* A. S. MAHAJAN and R. BABBAR *Department of Reproductive Biomedicine, National Institute of Health and family Welfare, New Mehrauli Road, Munirka, New Delhi 1167, and Department of Physiology, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi 112, India Summary Human sperm susceptibility to oxidative stress is vital as it affects various characteristics of sperm function. In the present study, we report a simple, sensitive and quick method of assessing the capacity of the sperms to withstand increased oxidative stress. The basis for the test was derived from the fact that human sperms suspended in Ham s F-1 medium tend to lose the forward progressive motility when co-incubated with H 2 O 2 (6 lm). Replacement of the medium with seminal plasma (1 : 1) was able to reduce the loss of sperm motility (4%). Retention of sperm motility in semen ( 3%) following 1 min of H 2 O 2 (6 lm) exposure was taken as the criteria for delineating the quality of sperm as poor, moderate, good and excellent types. The protocol was tested in 87 subjects presenting a normal semen profile. On the basis of this test, 44% of the semen samples were classified as poor and the rest as moderate, good or excellent. Lipid peroxidation was found higher in the sperms from the ÔpoorÕ category. Activities of superoxide dismutase and catalase were also significantly elevated in the seminal plasma of these subjects as compared with combined categories of good or excellent. The test described here can be used routinely in laboratory investigations to assess sperm susceptibility to oxidative stress in subjects presenting a normal semen profile. Keywords: hydrogen peroxide, oxidative stress, sperm motility, sperm sensitivity Introduction Oxidative stress in semen has now been recognized as a link between peroxidative damage to human sperm and the incidence of male infertility (Aitken & Clarkson, 1987; Iwasaki & Gagnon, 1992; Sharma & Agarwal, 1996 and Griveau & Le Lannou, 1997). Oxidative damage has also been implicated as the possible cause of idiopathic infertility involving disruption of spermatogenesis (Lenzi et al., 1993). Correspondence: M. M. Misro, Department of Reproductive Biomedicine, National Institute of Health and family Welfare, New Mehrauli Road, Munirka, New Delhi 1167, India. mm_misro@yahoo.com High levels of free radicals were reported in the seminal fluid of 87% infertile and 55% fertile normozoospermic subjects (Mazzilli et al., 1994). This is suggestive of the fact that a high incidence of reactive oxygen species (ROS) formation might be associated with low fertilizing potential of sperms in both fertile and infertile individuals. Attempts to quantitate oxidative stress in semen as a predictive measure to male infertility have been made through measurements of ROS in the sperm, total antioxidant capacity (TAC) in the seminal plasma and ROS-TAC score for successful pregnancy outcome (Sharma et al., 1999). Using the same protocol, the presence of higher oxidative Ó 24 Blackwell Publishing Ltd.

2 Assessment of sperm susceptibility to oxidative stress 83 stress even in normospermic men undergoing infertility evaluation has been confirmed very recently. (Pasqualotto et al., 21). Human spermatozoa are particularly susceptible to oxidative attack because of the presence of high concentration of unsaturated fatty acids in the plasma membrane (Jones et al., 1979). The sperm plasma membrane fluidity that enables membrane fusion at the time of fertilization is entirely derived from the properties of these unsaturated fatty acids. Thus, peroxidized sperm cells lack the membrane dynamics of fusogenicity and are deficient to fertilize the oocytes (Aitken et al., 1993). Depending on the antioxidant system prevalent within the sperm, it is expected that their sensitivity to cope with the oxidative stress would vary and thereby, it would be possible to distinguish populations of sperms on the basis of their sensitivity to oxidative stress. However, there is no method currently available to assess this aspect in sperms. In the present study, we have tested a quick, simple and sensitive method for assessing sperm susceptibility to oxidative stress. The test was found very successful in normozoospermic subjects with unexplained infertility, in order to identify those cases in which the sperms are more prone and sensitive to oxidative stress, or have a reduced capacity to withstand it. Materials and methods Subject selection and semen analysis Healthy male, normozoospermic subjects (n ¼ 87) attending a fertility clinic at National Institute of Health and Family Welfare (NIHFW), New Delhi were included in the study. The subjects had received no clinical or surgical intervention 3 months prior to the study. The mean age of the subjects was 3 ± 7.6 years. None of the subjects were habitual smokers and had no uni or bilateral varicocele. Further investigations related to infertility in these couples were confined to their female partners only. While nine subjects had more than 3 years of infertility, 31 had more than 2 years and the rest 47 couples were infertile for more than 1 year. Semen samples were collected after 2 4 days of sexual abstinence. After liquefaction for 3 min at 37 C, a routine semen analysis (semen volume, sperm count, motility, morphology and presence of leucocytes etc) was carried out as per WHO guidelines (WHO, 1999). Sperm count and motility of the subjects ranged from 2 to 12 million/ml and 5 75%, respectively. Sperm morphology was within the normal range as per WHO criteria. Ejaculates containing leucocytes (>1 1 6 /ml) were excluded from the study. Effect of H 2 O 2 on sperm motility in Ham s F-1 vs. replacement with seminal plasma Seminal plasma was replaced with Ham s F-1 medium, with or without H 2 O 2 (6 lm) and the sperm concentration was adjusted to /ml per well in a culture plate. After 1 min, a drop of the sperm preparation was examined at 4 under a phase contrast microscope to record the total rapid linear progressive and sluggish linear progressive motility of the sperm in the preparation (WHO, 1999). In a second set of experiments, the semen sample following liquefaction was divided into aliquots of.5 ml each. While one aliquot served as a control, seminal plasma from other aliquots were removed by centrifugation and sperm pellet was replaced with equal volume of seminal plasma diluted with Ham s F-1 medium at different proportions (1 : 1 or 1 : 2) with or without H 2 O 2 (6 lm). Sperm viability and motility were examined in all the fractions (World Health Organization, 1999). Test to assess sperm susceptibility to oxidative stress A simple protocol was developed to assess sperm susceptibility to oxidative stress. Accordingly, H 2 O 2 at the concentration of 6 lm per semen sample (1 ml) was utilized. Sperm motility was examined as usual following 1 min of H 2 O 2 treatment directly to the semen sample. Following the test incubation, the percentage of motility retained ( 3%) was used as the criterion to grade sperms/semen as poor, moderate, good and excellent types. For subsequent biochemical estimations Ôgood and excellentõ together was considered as ÔgoodÕ for comparison with the ÔpoorÕ category. Assay for lipid peroxidation (Lpx) A modified procedure of lipid peroxidation assay (Beuge & Aust, 1978) for spermatozoa was followed. In brief,.1 ml of sperm suspension ( /ml) was added to.1 ml of Tris (15 mm) buffer,.1 ml of (1.5 mm) ascorbic acid,.1 ml of FeSO 4 (2 mm) and.6 ml of dh 2 O. The sample was incubated for 15 min at 37 C and centrifuged at 3 rpm for 15 min. Trichloroacetic acid (1 ml) was added to the supernatant to precipitate proteins. The contents were once again centrifuged, pellet discarded and 2 ml of thiobarbitceric acid (TBA) reagent was added to the supernatant to form thiobarbitaric acid reactive substances (TBARS). The solution was kept in a boiling water bath for 15 min to develop colour. Samples were read against a blank at 532 nm using a spectrophotometer and the activity was expressed as nmol TBARS/1 1 6 /ml spermatozoa. Assay for SOD activity The SOD activity in sperm suspension and in seminal plasma was measured through a slightly modified protocol as described by Das et al. (2). Briefly,.1 ml of seminal plasma or sperm suspension (1 1 6 /ml) was added to a cocktail mixture of reagents [phosphate buffer (ph 7.4), L-methionine (2 mm), hydroxylamine hydrochloride (1 mm), ethylenediaminetetraacetic acid (EDTA, 5 lm), Triton X-1 (1%), riboflavin (1 lm), Greiss reagent consisting of.1% napthethyldiamine (NED) and 1% sulphanilamide] and incubated at 37 C for 5 min. Riboflavin (8 ll) was added to each sample, which were further

3 84 M. M. Misro et al. incubated in a specially designed fluorescent illuminated light box. Following 1 min of incubation, Greiss reagent was added to each tube to develop colour. Samples were read against a blank at 543 nm using a spectrophotometer and the activity was expressed as U SOD/mg protein. Assay for catalase activity Catalase activity in sperm suspension and seminal plasma was measured as described by Aebi (1974) with a slight modification. Decomposition of 6 mm of H 2 O 2 was systematically followed by monitoring the decrease in absorbance at 24 nm. The change in absorbance is directly proportional to the measure of catalase activity. Values were expressed as Pkat/mg protein. Statistical analysis Data were analysed by analysis of variance (ANOVA). Multiple two-tailed t-test was utilized to find out whether the differences of mean value of parameters between groups are significant. Differences were considered as significant if the probability of their occurrence was <.5. Results H 2 O 2 (6 lm) in Ham s F-1 drastically reduced sperm motility in vitro (Fig. 1). Only about 4% of sperms were sluggishly motile after 1 min. However, gradual replacement of the medium with different dilutions of seminal plasma inhibited this quick loss of motility under similar test conditions. As a result, a higher percentage of the sperms were able to retain active motility (Fig. 2). Significantly high sperm viability (p <.1) was also observed in the fractions diluted (1 : 1) with seminal plasma as compared with those without it. On the basis of retention of sperm motility, semen samples (as indicated in parentheses) were categorized as poor (4), moderate (12), good (28) and excellent (7) as shown in Table 1. Various indicators of oxidative stress in sperm and seminal plasma fraction were evaluated from Motility (%) Control H 2 O 2 (6 µm) min 1 min Incubation Figure 1. H 2 O 2 (6 lm) mediated effect on motility of human sperms suspended in medium Ham s F-1 (n ¼ 15). Sperm motility was significantly (*p <.1) reduced. * Motility (%) a b c Figure 2. Effect of seminal plasma replacement on the retention of sperm motility following H 2 O 2 (6 lm) treatment. On an average, approximately 3 4% of the sperm motility could be retained following seminal plasma replacement. a, only medium; b, H 2 O 2 + medium; c, H 2 O 2 + medium/ seminal plasma (2 : 1) and d, H 2 O 2 + medium/seminal plasma (1 : 1, *p <.1 compared with b) (n ¼ 15). ÔpoorÕ and ÔgoodÕ category. Though the retention of sperm motility in the moderate group was 5 1%, the quality of motility was very slow and sluggish. Semen samples from this group were therefore excluded for subsequent biochemical analysis of oxidative stress. Sperms from the ÔpoorÕ category showed significantly (p <.1) increased lipid peroxidation levels as compared with the other group (Fig. 3). In the ÔpoorÕ group, there was also a significant rise in the activities of superoxide dismutase (SOD, p <.5) and catalase (p <.1) in the seminal plasma (Figs 4 & 5). Catalase activity in sperm fractions was found significantly (p <.5) elevated too in the ÔpoorÕ category (Fig. 5). Discussion Oxidative stress is not being routinely investigated though it plays an important role in male infertility (Lopes et al., 1998; Aitken, 1999; Saleh & Agarwal, 22). The source of generation of this stress however varies. Men with clinical confirmation of varicocele, those with significantly higher number (>1 1 6 /ml) of leucocyte contamination or with higher percentage of morphologically abnormal spermatozoa are reported to manifest increased ROS levels in the semen (Pasqualotto et al., 21). It is also believed that an imbalance between the production of ROS and its disposal through naturally occurring antioxidants might also lead to a rise in the ROS levels in the semen (Geva et al., 1998). A higher level of ROS would not only be detrimental to the unique ability of male germ cells to move forward but probably also affect their ability to fertilize the oocyte. Fertilizing ability of human spermatozoa is reported to be inversely proportional to the sperm ROS production (Sukcharoen et al., 1996). This ÔmaleÕ factor, which normally goes undiagnosed in routine semen analysis, may be responsible for preventing conception in an otherwise normal couple. * d

4 Assessment of sperm susceptibility to oxidative stress 85 Table 1. Human sperm sensitivity to oxidative stress as determined by the retention of sperm motility following H 2 O 2 (1.2 mm) treatment to semen samples (n ¼ 87) S. No. No. of subjects/ semen samples used Sperm count prior to H 2 O 2 treatment (millions) Sperm motility prior to H 2 O 2 treatment (%) Mean ± SEM Mean ± SEM Mean ± SEM Sperm motility after 1 min of H 2 O 2 treatment (%) Oxidative stress resistance capacity of sperms (Grade) ± ± 18.5 Non-motile/non-progressive (26) Poor or beating type motility (14) ± ± 8.8 Slow forward progressive motility Moderate 6.89 ± ± ± 9.5 Moderate forward progressive motility Good 14.2 ± ± ± 12.5 Good forward progressive motility Excellent 25 ± 4. 5 Numbers in parentheses ( ) show the number of semen samples classified in that category. nm TBARS/mg protein Good Poor Seminal plasma Sperms Figure 3. Characterization of lipid peroxidation in sperms and seminal plasma from the poor vs. good category of semen samples (n ¼ 75). Significant (*p <.1) increase in the levels of TBARS was observed from the sperms of former category. SOD (U/mg protein) ** Seminal plasma Good * Sperms Poor Figure 4. Superoxide dismutase (SOD) activity in sperms and seminal plasma from the poor and good category of semen samples (n ¼ 75). Significant rise in the activities of SOD (p <.5) was observed in seminal plasma from poor category. Catalase (P kat/mg protein) Seminal plasma * ** Good Poor Sperms Figure 5. Catalase activity in sperms and seminal plasma from the poor and good category of semen samples (n ¼ 75). Activity was significantly higher in seminal plasma (*p <.1) and sperms (**p <.5) from the poor category. The risk of oxidative stress affecting sperm function may come from factors present either in the semen or in the secretions of female genital tract. The relevance of exposing the sperms to an additional oxidative stress comes from the fact that sperms might have to withstand similar stress conditions in the female reproductive tract. Surprisingly low concentrations of H 2 O 2 in semen had little effect on sperm motility. However, the effect was more pronounced if sperms are suspended in artificial medium (Chaki & Misro, 22). The strength of H 2 O 2 and the duration of the test period were standardized following several pilot experiments using H 2 O 2 at various concentrations. The reproducibility of the test may be judged from the fact that in spite of the huge differences in the sperm count and motility of the semen samples used, the retention of sperm motility following the test was graded under the four categories of poor, moderate, good and excellent ones (Table 1). With the help of the test

5 86 M. M. Misro et al. developed, a screening is now possible to identify those semen samples in which sperms are more susceptible to oxidative stress effects. This detection of differential sensitivity of human sperms to oxidative stress as delineated in the present test is not possible through routine semen analysis. Seminal plasma is believed to be the richest source of antioxidants, which reduce the harmful effects of ROS on sperms. Our studies also confirmed that sperms were able to retain up to 4% motility when test medium with H 2 O 2 was replaced in part with seminal plasma (Fig. 2). Besides motility, H 2 O 2 exposure also affects other functional characteristics in human sperms (Chaki & Misro, 22). However, motility being a prime sperm function, H 2 O 2 effect on the former was further explored and tested in the present study for assessing sperm susceptibility to oxidative stress. The results of the test vindicated the hypothesis that sperm sensitivity to oxidative stress varies with the individual semen sample. Sperms from approximately 44% of the semen samples which otherwise present a normal profile showed little resistance to the elevated oxidative stress under test conditions. The delineation of ÔpoorÕ category from ÔgoodÕ was seen further validated when increased lipid peroxidation levels were observed in the sperm fractions categorized as ÔpoorÕ (Fig. 3). A reactive oxygen species and total antioxidant capacity (ROS-TAC) score was demonstrated as a better indicator to distinguish fertile from infertile men than ROS alone (Pasqualotto et al., 21). However, the equations and calculations of this score appear too cumbersome. These investigations need a sophisticated laboratory infrastructure, the protocol requires considerable expertise and hence, is difficult to follow on a routine basis. However, the test protocol developed in the present study is less time consuming, easy to execute and can be carried out along with the routine semen analysis. However, though ROS- TAC score can be successfully applied to assess semen samples from different types of male infertility, the present test has the limitations of screening those ÔidiopathicÕ cases presenting a normal semen analysis but probably with a higher susceptibility to oxidative stress. It is reported that the usual ROS scavengers such as catalase and SOD are effective only against ROS produced by an external source. However, these enzymes are relatively ineffective when ROS production is endogenous (Sharma et al., 1999). Increased lipid peroxidation is suggestive of the rising levels of ROS in sperms of ÔpoorÕ category (Fig. 3). Only one-third of the ROS produced by spermatozoa is released extracellularly (Plante et al., 1994). However, it is only this externally available fraction ready for decomposition by the enzymatic or non-enzymatic components present in the seminal plasma. Therefore, the consequence of the remaining intracellular, un-decomposed ROS might be sufficient to affect the sensitivity of the sperms in terms of their ability to withstand further oxidative stress. Seminal plasma from ÔpoorÕ category showed a significant increase in SOD and catalase activities (Figs 4 & 5). Our results are in conformity with the earlier observation (Zini et al., 1993) that catalase activity of sperms and seminal plasma was significantly greater in those samples that produce ROS as compared with those that did not. This extracellular catalase, however, would not have much impact on the sperm antioxidant status. Exogenous supplementation with non-enzymatic antioxidants such as vitamin E was therefore recommended in cases of asthenozoospermia to overcome the effects associated with the intracellular peroxidative damage (Suleiman et al., 1996). The clinical significance with respect to identification of such infertile cases as ÔpoorÕ type can only be firmly established with benefits of treatment in the above lines. The in vitro protocol tested in the present study is simple to execute and appears to be a novel approach. As a pilot work, the present study indicates that infertile men presenting a normal semen profile need to be assessed carefully on the basis of their sperm sensitivity to oxidative stress as providing therapeutic management to some of them probably require a more rigorous nonenzymatic antioxidant therapy than others. However, this in vitro ÔtestÕ is now being developed in a kit form to make it more user-friendly and is under extensive evaluation in different groups of infertile and fertile subjects for further confirmation and consolidation of the present findings. Aebi, H. (1974) Catalase. In: Methods in Enzymatic Analysis, Vol. 2. (ed. H. U. Bergmeyer), pp Academic Press, New York. Aitken, R. J. (1999) The Amoroso lecture, the human spermatozoa-a cell in crisis. Journal of Reproduction and Fertility, 115, 1 7. Aitken, R. J. & Clarkson, J. S. (1987) Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. Journal of Reproduction and Fertility, 81, Aitken, R. J., Harkiss, D. & Buckingham, D. (1993) Relationship between iron-catalysed lipid peroxidation potential and human sperm function. Journal of Reproduction and Fertility, 98, Beuge, J. A. & Aust, S. D. (1978) Microsomal lipid peroxidation. 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