Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma*

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1 FERTILITY AND STERILITY Copyright <O 1993 The American Fertility Society Vol. 59, No. 6, June 1993 Printed on acid-free paper in U.S.A. Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma* Eve de Lamirande, Ph.D. t Claude Gagnon, Ph.D. Urology Research Laboratory, Royal Victoria Hospital, and Faculty of Medicine, McGill University, Montreal, Quebec, Canada Objectives: To characterize the very vigorous type of motility observed in the semen of some infertile men and to compare the superoxide anion scavenging capacity of the seminal plasma from these men and that from normal men. Subjects: Patients consulting for infertility related to sperm motility problems and men presenting as sperm donors. Methods: Motility patterns and measurements of sperm motility parameters were evaluated by computer-assisted digital image analysis system. The superoxide anion scavenging capacity of seminal plasma was measured by inhibition of nitroblue tetrazolium reduction due to the superoxide anion generated by the combination xanthine plus xanthine oxidase. Results: Spermatozoa from 9 of 68 semen samples with normal sperm concentration, morphology, and percentage of motility showed the typical motility patterns observed during hyperactivation (HA) and a significant level of HA (16% ± 3%) as compared with those in semen (2.3% ±.3%) from fertile volunteers. The superoxide anion scavenging capacity of the seminal plasma from men with sperm HA was 37% lower than that of control seminal plasma. Conclusions: Sperm HA can occur in whole semen and is associated with a low superoxide anion scavenging capacity in seminal plasma. It could be one of the causes for idiopathic infertility. Fertil Steril 1993;59: Key Words: Male infertility, idiopathic infertility, superoxide anion, superoxide dismutase, reactive oxygen species, spermatozoa, sperm motility Oligozoospermia ( <2 X 1 6 spermatozoajml) and asthenozoospermia ( <4% motile spermatozoa) are major causes of male infertility (1). However, in some cases, the number and motility of spermatozoa appear normal according to accepted standards (for example, the criteria defined by the World Health Organization [WHO]) (1), and yet no conception occurs despite female partners being normal. We Received November 9, 1992; revised and accepted February 17, * Supported by a grant from the Medical Research Council of Canada (Ottawa, Ontario, Canada) and by a chercheur boursier from the Fonds de Ia Recherche en Sante du Quebec (Quebec, Canada) to C.G. t Reprint requests: Eve de Lamirande, Ph.D., Urology Research Laboratory, H6.47, Royal Victoria Hospital, 687 ave des Pins Ouest, Montreal, Quebec, Canada H3A 1Al. have recently observed in a group of infertile patients that sperm motility in whole semen was very vigorous and characterized by very high velocity and lateral displacement of the head and low linearity. This type of movement presented similarities with that observed during hyperactivation (HA) when sperm motility dramatically changes from the linear, relatively slow, and low amplitude type of movement observed in seminal plasma to a high velocity, low progression, whiplash-like type of movement (2-5). Very low percentages of sperm HA are found in semen of fertile men (2% to 3%) (3). The first aim of our study was to further characterize this type of vigorous motility found in the semen of these men. Human spermatozoa can produce reactive oxygen species such as the superoxide anion (" 2 -) and hydrogen peroxide (H 2 2) (6, 7). Although hydrogen peroxide is known to be toxic for human spermatozoa Vol. 59, No.6, June 1993 de Lamirande and Gagnon Sperm hyperactivation in whole semen 1291

2 (6, 8), we have recently shown that the superoxide anion, generated by the combination xanthine plus xanthine oxidase (in the presence of catalase), induced HA and capacitation in human spermatozoa (9, 1). Furthermore, superoxide dismutase (SOD, the enzyme responsible for the degradation of the superoxide anion to hydrogen peroxide) not only blocked the HA and capacitation induced by the superoxide anion but also by fetal cord serum, a biological inducer of HA and capacitation (9). Seminal plasma has superoxide anion scavenging capacity because of the presence of SOD itself (11) and also the presence of proteins (such as albumin) that act as sacrificial scavengers (12) and of small molecules, such as the glutathione (12), vitamin E and C (13, 14), taurine and hypotaurine (15). The second aim of our study was to investigate whether the occurrence of HA in semen was associated with a low SOD-like activity in seminal plasma. Semen Samples MATERIALS AND METHODS Semen samples were collected after 3 days of sexual abstinence from patients consulting for infertility (n = 115) related mostly to sperm motility problems (second referral) and men presenting as sperm donors (n = 34) without any prior selection. Sperm concentrations ranged from 1 to 84 X 1 6 spermatozoa/ml (mean± SEM, 87 ± 1 X 1 6 spermatozoa/ml; median, 48 X 1 6 spermatozoa/ml) and sperm motility ranged from % to 92% (mean ± SEM, 39% ± 2%; median, 35%). Spermograms indicated that 81 of these men suffered from oligozoospermia and/or asthenozoospermia ( <2 ± 1 6 spermatozoa/ml and/or <4% sperm motility). The present study concerns the 68 men (44 patients and 24 potential donors among whom were 3 fertile men) whose semen characteristics were considered normal according to the WHO. In this group of 68 men, the average sperm concentration was 15 ± 2 X 1 6 spermatozoa/ml (median, 88 X 1 6 spermatozoa/ml), motility was 64% ± 2% (median, 63%), and at least 4% of spermatozoa were morphologically normal. Motility Analysis Thirty minutes after collection, semen samples were placed in a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel) kept at 37 C on a heated plate (Microwarm plate 3; Kita Zato, Japan). Motility was analyzed using the CellSoft computer-assisted digital analysis system (Cryo Resources, Montgomery, NY) as previously described (16, 17). Criteria for HA were the following: curvilinear velocity (VCL, total distance traveled divided by the total time the cell was tracked) >8 J.Lm/sec, linearity (LIN, ratio of the straight line distance to the actual track distance times 1) <6.5, and amplitude of the lateral head displacement (ALH, deviation of the sperm head from the mean trajectory) >6.5 11m (17). Motility measurements were determined for at least 2 motile spermatozoa. The percentage of HA was then obtained using a program written to select from individual cell data files, only data from those cells that showed the specific characteristics of HA (17). Samples showing sperm HA.:::; 4% of motile spermatozoa were considered normal and included in the control group because fertile volunteers never had >4% sperm HA. Samples showing >4% sperm HA were included in the test Superoxide Dismutase-Like Activity Superoxide dismutase-like activity (total superoxide anion scavenging capacity) was measured by the inhibition of nitro blue tetrazolium reduction due to the superoxide anion generated by the combination xanthine plus xanthine oxidase (18, 19). The reaction mixture contained HEPES-balanced saline (13 Mm NaCl, 4 mm KCl,.5 mm MgC1 2, 14 mm fructose, and 25 mm N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid [HEPES] adjusted to ph 8), xanthine (.25 mm), nitroblue tetrazolium (2 mm), bovine serum albumin (1 mg/ml), and various volumes of seminal plasma. The reaction was initiated by the addition of xanthine oxidase (.5 U /ml) and monitored at 22 C by the increase of absorbance at 57 nm. Under these conditions, the reduction of nitroblue tetrazolium is linear for the first 4 minutes. Measurements of the SOD-like activity were taken at 2 minutes. One unit of SODlike activity was defined as the amount of seminal plasma capable of decreasing by 5% the reduction of nitroblue tetrazolium. Seminal plasma samples used for the determination of SOD-like activity were obtained by centrifugation of semen at 6 X g for 15 minutes and recentrifugation of the supernatant at 1, X g for 2 minutes. Clear seminal plasma was frozen ( -2 C) until used. Statistics Student's t-test (2 tails, unpaired values) was used to determine the difference between groups de Lamirande and Gagnon Sperm hyperactiuation in whole semen Fertility and Sterility

3 Control / / Hyperactivated lou'm Figure 1 Representative sperm motility patterns observed in the semen of control men and in the semen of men with sperm HA. Fertile volunteers had sperm motility patterns similar to those of control spermatozoa (upper panel) and very few of their spermatozoa had motility patterns characteristic of HA (lower panel). High numbers of spermatozoa with motility patterns characteristic of HA were found in the semen of 9 of 68 men with normal sperm concentration, morphology, and percentage of motility. Tracks were obtained using the CellSoft Research Module. Cells were tracked for second at a rate of 3 frames/sec, and the bar corresponds to 1 11m. T, trashing; S, starspin; C, circlinghigh curvature; H, helical; B, biphasic. Motility Patterns RESULTS Spermograms performed on the 149 semen samples studied revealed that 68 of the samples were normal according to the WHO criteria. We observed in 9 (6 infertile men and 3 men presenting as sperm donors) of these 68 cases that sperm motility in whole semen was very vigorous and differed greatly from that observed in semen samples from fertile donors. The motility patterns of spermatozoa from fertile donors and from these 9 men were therefore compared. Spermatozoa in semen from fertile donors had relatively straight trajectories and low speed, and the ALH was narrow (Fig. 1). However, in the subgroup of 9 men with very vigorous sperm motility, there was a significant proportion of spermatozoa displaying the typical motility patterns of hyperactivated spermatozoa (3, 17) (Fig. 1). Trashing, helical, starspin, circling-high curvature, and biphasic motility patterns were always observed in these samples. Motility Measurements Motility parameters were determined for semen samples from the 9 men with sperm HA and from 12 control men (3 fertile men and 9 sperm donors with normal spermograms according to the WHO criteria) (Table 1). Curvilinear velocity and ALH were significantly higher, and linearity was lower in semen showing motility patterns typical of HA as compared with control semen values. These differences in the motility parameters are consistent with the presence of HA (3, 17). The percentages of sperm HA were 2.6% ±.3% and 16% ± 3% in control and test (sperm HA) groups, respectively (P <.1) (Table 1; Fig. 2). The sperm HA present in semen did not appear to be a transient phenomenon. Three of the 9 men with sperm HA in semen came back for a second test and had the same type of sperm motility and similar percentages of sperm HA (data not shown). Superoxide Dismutase-Like Activity Superoxide dismutase was shown to block the HA induced by the superoxide anion and by fetal cord serum (9). The levels of SOD-like activity (the superoxide anion scavenging capacity) of seminal plasma from 7 of the 9 men with sperm HA and from 7 of the 12 sperm donors investigated for motility patterns were therefore determined (Fig. 2). There was a 37% lower SOD-like activity in the seminal plasma from semen with sperm HA as compared with that found in control seminal plasma (P <.2). Two ofthe 7 men with sperm HA in semen came back for a second test, and SOD-like activities were again found to be lower than that of control. DISCUSSION Our data indicate that sperm HA can occur in whole semen and that this HA may be related to a Table 1 Motility Parameters in Semen Samples From Control Men and Men With Sperm HA * Group Motility VCL LIN ALH % p,m/sec HA Control 73 ± 4 58 ± ± ± ±.3 HA 59 ± 4 72 ± 3t 5.:3 ±.2:j: :3.6 ±.2:j: 16 ± :>:j: * Motility parameters and percentages of HA were determined as described in Materials and Methods. Semen samples from 12 control men (:3 fertile men and 9 sperm donors with normal spermograms according to the WHO criteria and 9 men with sperm HA in the semen were analyzed). t Statistically different, P <.1, as compared with control :j: Statistically different, P <.1, as compared with control % Vol. 59, No.6, June 1993 de Lamirande and Gagnon Sperm hyperactivation in whole semen 1293

4 , : ' 4 c:: 3 > 2 t; I!.r Cll a. 1 >- ::c -lb::. Control HA 12...J E 2. 8 > ti as c (/) 4 Control Figure 2 Sperm HA and seminal plasma SOD-like activity in control semen and in semen with sperm HA. Sperm HA (top panel) and seminal plasma SOD-like activity (bottom panel) were determined as described in Materials and Methods. Semen samples tested for HA are described in the footnote of Table 1. For SOD-like activity, seminal plasma from 7 control men (3 fertile men and 4 men with sperm motility characteristics similar to those of the fertile men) and from 7 men with sperm HA in semen were analyzed. Values for sperm HA and SOD-like activity differ between the two groups, (P <.1 andp <.2, respectively). low level of SOD-like activity in seminal plasma. In 9 of 68 (13%) semen samples, a significant number of spermatozoa (>4% of the motile spermatozoa) had motility patterns and parameters similar to those found during HA (Fig. 1, 2; Table 1). The percentage of HA found in these samples ranged from 7% to 33%. Sperm HA occurs at low levels in semen from fertile men (Table 1; Fig. 2) (Burkman [3]) and the absence of sperm capacitation in semen was previously related to the presence, in seminal plasma, of antifertility and/or decapacitation factors (2). The present data indicate that the presence of sperm HA in semen is associated with levels of SODlike activity in the seminal plasma (Fig_ 2) that were 37% lower than those found in control seminal plasma. These results are in agreement with the involvement of the superoxide anion in human sperm HA (9, 1). The superoxide anion generated by the combination xanthine plus xanthine oxidase (in the presence of catalase) induced higher levels of HA and capacitation than fetal cord serum, a biological inducer of these processes. Furthermore, SOD blocked the HA and capacitation induced by the soperoxide anion and by fetal cord serum. The fact HA that SOD reversed the HA once induced suggested that a constant generation of superoxide anion by spermatozoa is needed to maintain HA and that the superoxide anion generated by spermatozoa is, at least in part, released outside the cells (9, 1). The low levels of seminal plasma SOD-like activity associated with the presence of HA in the semen do not exclude that deficiencies in other factors, such as the antifertility and/or decapacitation factors, (2) or an overstimulation of the superoxide generating enzyme present in spermatozoa, could participate in the induction of sperm HA in the semen of these nine men. As it is often observed in biological systems, several control mechanisms participate in the regulation of a given phenomenon. Although sperm HA is a necessary step for spermatozoa to acquire a full fertilizing capacity (2, 3), the presence of HA in semen is nevertheless abnormal and could be a cause of infertility. Hyperactivation confers a mechanical advantage on spermatozoa in the oviduct where they may encounter viscous oviductal fluid and a viscoelastic cumulus matrix (21), but it remains to be investigated whether this high amplitude type of motility would favor the passage of spermatozoa through cervical mucus. A positive correlation was found between the degree of lateral head displacement and the success of sperm-cervical mucus interaction (22), but donor spermatozoa was used for this study and the sperm lateral head displacement was therefore lower than what is observed during HA. It was recently shown that mouse spermatozoa pretreated with calcium (therefore hyperactivated and capacitated) fertilized significantly fewer eggs after artificial insemination than did spermatozoa preincubated in calcium deficient medium (progressive motility, no HA) (23). The authors concluded that sperm transport to the oviduct was impaired by the calcium pretreatment probably because of loss of progressive motility (24). Similarly, lower numbers of hamster spermatozoa were found in oviducts of mice inseminated with capacitated spermatozoa as compared with that found with uncapacitated spermatozoa (25), and the number of spermatozoa found in the oviducts appeared to be inversely proportional to the percentage of hyperactivated spermatozoa in the sample used for insemination (25). It is possible that the same type of phenomenon happens in humans, and that, when HA occurs in semen, there is an impaired transport of spermatozoa through the female genital tract. It remains also possible that the HA observed in semen is leading to premature capacitation and ac de Lamirande and Gagnon Sperm hyperactivation in whole semen Fertility and Sterility

5 rosome reaction. Some previous studies have shown a close correlation between these events (3, 1). These prematurely hyperactivated spermatozoa possibly undergo the acrosome reaction too early and/or die before they reach the fertilization site. The present data emphasize the need for a more careful examination of sperm motility characteristics in semen samples classified normal. Sperm samples showing hyperactivated motility can easily be missed by an untrained eye and may be considered as samples with very good motility, especially when a visual evaluation is performed at room temperature (HA has to be evaluated at 37 C). The presence of sperm HA in semen (found in 13% of the semen samples classified normal according to WHO criteria) could be a cause for male idiopathic infertility. Further studies are needed to understand the role of the SOD-like activity of seminal plasma in this phenomenon. Acknowledgments. The authors thank Mrs. Lina Ordonselli and Mrs. Andree F. 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A quantitative comparison of the passage of capacitated and uncapacitated hamster spermatozoa through the uterotubal junction. Bioi Reprod 1992;46: Vol. 59, No.6, June 1993 de Lamirande and Gagnon Sperm hyperactivation in whole semen 1295