Increased reactive oxygen species formation in semen of patients with spinal cord injury*t

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1 FERTILITY AND STERILITY Copyright c 1995 American Society for Reproductive Medicine Vol. 63, No.3, March 1995 Printed on acid-free paper in U. S. A. Increased reactive oxygen species formation in semen of patients with spinal cord injury*t Eve de Lamirande, Ph.D.:j: Bernard E. Leduc, M.D. II Akira Iwasaki, M.D.~ Magdy Hassouna, M.D., Ph.D.:j: Claude Gagnon, Ph.D.:j: Royal Victoria Hospital, McGill University, and Institut de Readaptation de Montreal Universite de Montreal, Montreal, Quebec, Canada Objectives: To evaluate reactive oxygen species production of semen samples and Percollwashed spermatozoa from men with spinal cord injuries and to determine if there is a relationship between this reactive oxygen species production and sperm motility. Participants: Semen samples from healthy volunteers and infertile patients were collected by masturbation. Interventions: Semen samples from men with a spinal cord injury were obtained by electroejaculation or by masturbation after treatment with physostigmine. Main Outcome Measurements: Motility was measured using the CellSoft computer-assisted analysis system (Cryo Resources Ltd., Montgomery, NY). Luminol-amplified chemiluminescence was used to measure reactive oxygen species production. Results: Semen samples and Percoll-washed spermatozoa from men with a spinal cord injury produced reactive oxygen species at much higher frequency and levels than equivalent preparations from infertile men or healthy volunteers. There was an inverse relationship between the percentage of motility and reactive oxygen species production in Percoll-washed spermatozoa from men with a spinal cord injury. Conclusion: Semen samples and Percoll-washed spermatozoa from men with spinal cord injury produce high levels of reactive oxygen species that may be related to the low sperm motility and infertility observed in these men. Fertil Steril 1995;63: Key Words: Male infertility, chemiluminescence, sperm motility, spermatozoa, Percoll separation The incidence of reactive oxygen species detection in semen samples from an un selected patient Received May 19, 1994; revised and accepted September 21, * Supported by a grant from the Medical Research Council of Canada, Ottawa, Ontario, Canada to C.G. t Presented in part at the 1992 Annual Meeting of the American Urological Association, Washington, D.C., May 10 to 14, :j: Urology Research Laboratory, Royal Victoria Hospital, Montreal, Canada. Reprint requests: Eve de Lamirande, Ph.D., Urology Research Laboratory, Room H6.47, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec H3A lal, Canada. II Institut de Readaptation de Montreal, Faculty of Medicine, Universite de Montreal. 11 Present address: Urology Department, Yokohama City University, Yokohama Red Cross Hospital, Yokohama, Japan. population consulting for infertility ranges from 25% to 40%, depending on the threshold used for reactive oxygen species measurements (1, 2). These reactive oxygen species are associated with a reduction in sperm motility (1, 3, 4) and egg-sperm fusion (5). Patients with spinal cord injury are subfertile (6). Efforts to correct this situation have focused primarily on the problem of sperm transport and means to overcome the barrier of anejaculation observed in 80% to 90% of these men (6). However, despite corrective measures to recover semen specimens from these patients, > 90% of them remain infertile (6). Even though the quantity of spermatozoa in semen is often more than adequate, spermograms from these men demonstrate asthenozoospermia according to World Health Organization Vol. 63, No.3, March 1995 de Lamirande et al. Reactive oxygen species in spermatozoa 637

2 (WHO) standards (7) and abnormal sperm function as evaluated by sperm penetration assay (8). The aims of the present study were to determine whether whole semen samples from patients with spinal cord injury produce reactive oxygen species and whether spermatozoa from the same semen samples, washed through a Percoll gradient, also generate reactive oxygen species. The data indicate that 97% of the semen samples produce reactive oxygen species and that, even after washing, spermatozoa from these patients still produce reactive oxygen species, the level of which is correlated inversely with the percentage of motile cells. Materials MATERIALS AND METHODS Percoll was purchased from Pharmacia Fine Chemicals (Dorval, Quebec, Canada) and luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) from Boehringer Mannheim (Dorval, Quebec, Canada). All other chemicals used were at least of reagent grade. Normal Subjects and Patient Selection The volunteers (n = 20) who participated in this study were healthy men between 23 and 43 years of age and producing more than 40 X 10 6 spermatozoa/ml (range, 46 to 250 X 10 6 /ml) characterized by normal motility (> 50% forward progression) and morphology as defined by the WHO (7). The group of infertile patients was composed of an unselected population of 166 patients presenting to the Infertility Clinic ofthe Royal Victoria Hospital, Montreal, Quebec, Canada. Semen specimen from volunteers and infertile patients were produced by masturbation after 3 days of sexual abstinence. A group of 21 paraplegic patients with traumatic spinal cord injury associated with ejaculatory dysfunction participated in the study. The mean age of the participants was 28.1 years (range, 20 to 40 years), and the mean time since injury was 5.8 years (range, 0.5 to 14 years). The level of the lesion was cervical in 9 cases (5 complete and 4 incomplete) and thoracic or lumbar in 12 cases (10 complete and 2 incomplete). All of these men failed to ejaculate with vibratory stimulation. Patients were informed of the protocols and signed a consent form. Nineteen of the patients with spinal cord injury were injected with 40 mg of butylbromide at time o. A half hour later, 2 mg of physostigmine were administered SC, and semen was collected by masturbation 15 minutes later (9). In two other patients semen was obtained by electroejaculation. Nifedipine (5 to 10 mg sub lingually ) was first administered to prevent autonomic dysreftexia that may accompany ejaculation. After verification of the status of the rectal mucosa, the electroejaculation probe (model II; G and S Instrument Co., Duncanville, TX) was lubrified and introduced into the anal canal. The prostatovesicle area was stimulated at 5 to 10 volts for 30 to 40 stimuli (1 to 2 second per stimulus). Penile tumescence was observed before ejaculation, and semen was collected by manually milking the urethra. Patient's blood pressure, pulse, and rectal temperature were monitored during the procedure, and integrity of rectal mucosa was confirmed after the procedure. No significant difference was observed in the results obtained with these two semen collection techniques, and results were therefore pooled. Sperm Preparation and Motility Measurements All semen samples were handled by the same method and within 30 to 60 minutes of collection time. Semen samples were layered on discontinuous Percoll gradients buffered with HEPESbalanced saline (130 mm NaCI, 4 mm KCI, 0.5 mm MgCI 2, 14 mm fructose, and 25 mm N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid [HEPES] adjusted to ph 8.0) and centrifuged as previously described (10). Spermatozoa at the 65% to 95% Percoll interface and in the 95% layer were collected and pooled. Sperm motility measurements were obtained using the CellSoft computer-assisted image analysis system (Cryo Resources Ltd., Montgomery, NY) both before and after washing on PercolI gradient (10). Reactive Oxygen Species Determination Formation of reactive oxygen species was measured using a computer-driven LKB Wallac 1251 Luminometer (LKB-Wallac, Turku, Finland). Luminescence was recorded at 27.7 C after the addition of luminol (0.2 mm final concentration) to the original semen (0.2 ml) diluted with HEPES-balanced saline (0.3 ml) or Perc oil-washed spermatozoa (0.05 ml) diluted lo-fold with HEPES-balanced saline. The reactive oxygen species formation detected by luminescence was recorded in the integration mode for 10 seconds with constant stirring of the analyzed sample. Readings were taken every 5 minutes for 30 minutes with a 638 de Lamirande et al. Reactive oxygen species in spermatozoa Fertility and Sterility

3 peak of luminescence observed between 5 and 15 minutes. After subtracting the appropriate blanks, the peak luminescence was expressed in mv /s per 10 9 spermatozoa. Formation of reactive oxygen species was considered detectable when the luminescence was equal to or above 0.05 m V Is. This value corresponds to two times the maximal noise (0.025 mv Is) spontaneously generated by the luminometer under our experimental conditions (1). Statistical Analysis The x 2 method was used to compare frequency of events between different groups. Student's t-test was used to evaluate the differences between reactive oxygen species production of sperm samples. Regression curves were calculated by the least squares method, and the coefficient of correlation (r) was calculated. RESULTS The presence of reactive oxygen species in whole semen was detected in 97% of patients with spinal cord injury, and in 81 % of these semen samples, reactive oxygen species levels were> 10 mv /s per 10 9 spermatozoa. The average level of reactive oxygen species detected in this population of samples was 884 ± 305 m V /s per 10 9 spermatozoa (mean ± SEM) (Table 1). In contrast, only 15% and 10% of normal men and only 40% and 25% of an unselected population of patients consulting for infertility (1) produced reactive oxygen species at detectable levels or levels> 10 m V /s per 10 9 spermatozoa, respectively, in semen. In normal men and in infertile patients, the levels of reactive oxygen species measured in semen were, respectively, 40- and 14- fold lower than those detected in semen from patients with spinal cord injury (Table 1). Within the group of men with spinal cord injury, no difference in the level of reactive oxygen species or frequency of reactive oxygen species detection was observed for the level of lesion or duration of injury (data not shown). When semen specimens from patients with spinal cord injury were cell;trifuged on a Percoll gradient to obtain a preparation enriched in morphologically normal spermatozoa of sufficient density to penetrate the 65% Percoll layers, 90% of sperm specimens still produced reactive oxygen species (Table 2). By comparison, only 10% and 22% of spermatozoa from normal and infertile men, respectively, still produced reactive oxygen species after washing on Percoll (Table 2). The mean (±SEM) reactive oxygen species level in washed spermatozoa from men with spinal cord injury, 640 ± 303 mv /s per 10 9 spermatozoa, was sixfold and 140-fold higher than those of Percollwashed spermatozoa from infertile patients and normal volunteers, respectively (Table 2). A majority of patients with spinal cord injury had sperm motility between 0% and 20% in whole semen (23 of 32 semen samples) and after Percoll gradient centrifugation (20 of 32 sperm samples) (Table 3). The levels of reactive oxygen species detected in these two suspensions of poorly motile spermatozoa were not statistically different. However, there was a significant 17-fold difference between the levels of reactive oxygen species measured in semen samples and in Percoll-washed spermatozoa when semen samples with > 20% sperm motility were evaluated (Table 3). The majority of semen samples with 0% to 20% sperm motility (13/23) and with sperm motility Table 1 Reactive Oxygen Species in Semen Samples From Normal Volunteers, Infertile Men, and Men With Spinal Cord Injury Subjects Incidence of reactive oxygen species detection* Incidence of high reactive oxygen species production* Reactive oxygen species production* Normal Infertile Spinal cord-injured 3/20 (15)t 66/166 (40) 31/32 (97) 2/20 (10)t 42/166 (25) 26/32 (81) 22 ± 16 [0 to 318]t:l: 63 ± 19 [0 to 3,160] 884 ± 305 [0 to 6,772] * The level of reactive oxygen species produced by semen sampies (from 20 volunteers, 166 infertile men, and 21 spinal cordinjured men) were measured as described in Materials and Methods, are expressed in mv/s per los spermatozoa and are mean ± SEM. A sample had detectable reactive oxygen species production when the chemiluminescence recorded was at least twice that of the background levels and a high reactive oxygen species production when it was above 10 m V /s per 10 9 spermatozoa. t Results obtained with normal subjects are lower than those obtained with infertile men (P < 0.05) or with spinal cord-injured men (P < ). Results obtained with infertile men are lower than those obtained with spinal cord-injured men (P < ). Values in parentheses are percentages. :I: Values are means ± SEM with ranges in brackets. From Iwasaki and Gagnon, 1992 (1). Vol. 63, No.3, March 1995 de Lamirande et al. Reactive oxygen species in spermatozoa 639

4 Table 2 Reactive Oxygen Species in Percoll-Washed Spermatozoa From Normal Volunteers, Infertile Men, and Men With Spinal Cord Injury Subjects Incidence of reactive oxygen species detection* Incidence of high reactive oxygen species production Reactive oxygen species production Normal Infertile Spinal cord-injured 2/20 (10lt 14/65 (22) 29/32 (90) 1/20 (5) 13/65 (20) 24/32 (75) 4.6 ± 3.6 [0 to 72]:1: 110 ± 40 [0 to 1,580] 640 ± 303 [0 to 8,521] * Parameters are defined in Table 1. Semen samples are from 20 volunteers, 166 infertile men, and 21 spinal cord-injured men. t Results obtained with normal subjects are lower than those obtained with infertile men (P < 0.03) or with spinal cord-injured men (P < ). Results obtained with infertile men are lower than those with spinal cord-injured men (P < ). Values in parentheses are percentages. :I: Values are means ± SEM with ranges in brackets. From Zini et ai., 1992 (2). > 20% (8/9) yielded sperm populations that produced less reactive oxygen species after Percoll gradient than in the original semen specimens. However, in 9 of 23 samples with poor sperm motility, there was a rise in reactive oxygen species production after the Percoll wash, whereas no increase in reactive oxygen species was noted after Percoll wash (0 of9 samples, P < 0.05) when the same comparison was made but with semen specimens with sperm motility> 20%. There was a negative relationship (r = 0.39, P = 0.026) between the level of reactive oxygen species and the percentage of motility in Percoll-washed spermatozoa from patients with spinal cord injury (Fig. 1). However, no significant relationship was observed between sperm motility and reactive oxygen species levels when semen samples, instead of washed sperm suspensions, were analyzed (data not shown). DISCUSSION The spontaneous formation of reactive oxygen species by cells present in semen has been associated with reduced sperm motility (1, 3, 4), abnormal sperm morphology (11), decreased sperm-egg interaction (5), reduced fertility in vivo (12), and the presence of leukocytes (13). A greater number of men from an unselected population consulting for infertility produce semen specimens that generate reactive oxygen species far in excess of what is observed in semen from normal fertile men (1). In the present study, the frequency at which reactive oxygen species were detected, as well as the levels of reactive oxygen species measured in semen samples and in Percoll-washed spermatozoa from patients with spinal cord injury, were much higher than those found in semen of normal or infertile men (Tables 1 and 2). Reactive oxygen species in semen may have a variety of cellular origins. They may be generated by damaged spermatozoa and by leukocytes sedimenting at the 40% to 65% interface of the Percoll gradient (1). The latter cells are known to produce significant amounts of reactive oxygen species even in their unstimulated state (14) and to discharge massive amounts of superoxide anion after stimulation (14, 15). The cell populations accumulating at the 65% to 95% Percoll interface and within the 95% Percolllayer are also other sources of reactive oxygen species (1). These cell populations are composed of morphologically but not necessarily func- Table 3 Relationship Between Sperm Motility in Semen and Reactive Oxygen Species Formation in Patients With Spinal Cord Injury Sperm specimen No. of samples Motility Reactive oxygen species production* Semen Semen Percoll-washed spermatozoa Percoll-washed spermatozoa o to 20 >20 o to 20 > ± 335 (2 to 6772lt 759 ± 624 (0 to 5741) 998 ± 472 (0 to 8521) 44 ± 17 (0 to 154):1: * Reactive oxygen species production is expressed in m V /s per 10 9 spermatozoa. t Values are means ± SEM with ranges in parentheses. :I: Statistically different (P < ) from all the values obtained for reactive oxygen species production. 640 de Lamirande et al. Reactive oxygen species in spermatozoa Fertility and Sterility

5 - 80 t = ~ 60 = 0 40 Ei! 20 C) ~ Ul y = 31-7 l[ r = Log (1 + ROS) Figure 1 Relationship between the percentage of motility and reactive oxygen species production in Percoll-washed spermatozoa from men with spinal cord injury. Spermatozoa were washed on Percoll gradients, and motility and chemiluminescence measurements were performed as described in Materials and Methods. Production of reactive oxygen species is expressed as log (1 + reactive oxygen species) instead of log reactive oxygen species to avoid negative values. P = tionally or biochemically normal spermatozoa. For example, the great majority of patients with spinal cord injury had < 20% motile spermatozoa within the dense 65% to 95% Percoll layers (Table 3), whereas this sperm fraction is made up of 75% to 95% of progressively motile spermatozoa when a semen specimen from a normal man is separated on a similar Percoll gradient (10). The levels of reactive oxygen species detected in original semen and Percoll-washed spermatozoa may provide some indications of the cellular source for reactive oxygen species. Because human seminal plasma contains a variety of reactive oxygen species scavengers including superoxide dismutase (80D) (16), catalase (17), and 80D- and catalaselike (2) scavengers, the amount of reactive oxygen species detected in the presence of seminal plasma generally reflects an underestimated value of reactive oxygen species production. Thus, a level of reactive oxygen species detected in Percoll-washed spermatozoa lower than that of whole semen (as it was observed in 21 of 32 cases in men with spinal cord injury) indicates that leukocytes and morphologically abnormal spermatozoa accumulating at 40% to 65% Percoll interface are likely important sources of reactive oxygen species produced in semen. Conversely, a reactive oxygen species level in Percoll-washed spermatozoa higher than that detected in whole semen (as it was observed in 9 of 32 cases in men with spinal cord injury; Table 3) probably reflects the predominance of morphologically normal but biochemically or functionally abnormal spermatozoa as a significant contributor to the reactive oxygen species produced in semen. There was a significant inverse relationship between the percentage of motile spermatozoa and the levels of reactive oxygen species in Percollwashed spermatozoa from patients with spinal cord injury (Fig. 1). A similar relationship was found between reactive oxygen species levels and sperm motility in semen samples from infertile patients (1). Even though in the present study no significant correlation was established between sperm motility and level of reactive oxygen species in semen samples, the level of reactive oxygen species observed in samples with sperm motility> 20% was much lower than that detected when sperm motility was < 20% (Table 3). The absence of linear relationship possibly may be explained by a higher concentration of polymorphonuclear leukocytes (PMN) in semen of patients with spinal cord injury as compared with that of infertile patients (6). Even in their basal state, these PMN would cause an increase in reactive oxygen species detection without causing an immediate loss of sperm motility. Except for epididymitis where spermatozoa are in contact with PMN for long periods of time, spermatozoa are usually in contact with PMN only after ejaculation when they are protected partially from extracellular reactive oxygen species by the presence of scavengers in seminal plasma (14). In patients with spinal cord injury, the presence of round cells and morphologically damaged spermatozoa in semen is a common feature (6). These patients are prone to infection and infiltration of white blood cells (6), PMN being the predominant vanguard cell species encountered (18). Testicular hyperthermia because of a constant sitting position and intrinsic damage to testicle (6) possibly may be responsible for the increased number of morphologically abnormal spermatozoa and immature germ cells (spermatids and spermatocytes) in semen of men with spinal cord injury. Furthermore, elevated scrotal temperature also is known to decrease spermatogenesis (6). Thus, for these patients, spermatozoa, when produced, may be either morphologically abnormal or normal (but functionally abnormal) depending on where hyperthermia exerts its greatest damage. Whether hyperthermia can be linked to the increased reactive oxygen species production by semen components from patients with spinal cord injury remains to be established. Vol. 63, No.3, March 1995 de Lamirande et al. Reactive oxygen species in spermatozoa 641

6 At the present time, there is no treatment known to allow a decrease of reactive oxygen species production and of the related damages in semen. However, it is known that hydrogen peroxide, formed from the dismutation of the superoxide anion is the primary reactive oxygen species responsible for toxic effects on sperm motility via a decreased intracellular adenosine triphosphate content (19). Two cellular systems, a possible nicotinamide adenine dinucleotide phosphate oxidase at the level of sperm membrane (3, 5) and the sperm mitochondrial diaphorase (a nicotinamide adenine dinucleotide oxydoreductase) (20) were proposed as generating systems for the superoxide anion in spermatozoa. In the first case, most of the hydrogen peroxide would be released extracellularly, and addition of catalase to semen samples and all solutions needed for the Percoll wash would perhaps help to maintain sperm motility and prevent reactive oxygen species-induced damages. In the second case, most of hydrogen peroxide would be formed inside the cells, and there would be a need for a cell permeant hydrogen peroxide scavenger because catalase cannot enter cells. In conclusion, semen samples and Percollwashed spermatozoa from men with spinal cord injury produce reactive oxygen species at a higher frequency and higher levels than equivalent samples from normal or infertile men. These facts as well as inverse correlation observed between reactive oxygen species production and sperm motility may explain, at least partly, the infertility observed in these men. Acknowledgment. The authors thank Mrs. Lina Ordonselli for her secretarial assistance in the preparation of this manuscript and all the volunteers who participated in this study. REFERENCES 1. Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil Steril 1992;57: Zini A, de Lamirande E, Gagnon C. Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa. Int J AndroI1993;16: Aitken JR, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 1987;81: Alvarez J, Touchstone J, Blasco L, Storey B. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as a major enzyme protectant against oxygen toxicity. J Androl 1987;8: Aitken JR, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation and human sperm function. BioI Reprod 1989;40: Linsenmeyer T A, Perkash 1. Infertility in men with spinal cord injury. Arch Phys Med Rehabil1991;72: World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. Cambridge: The Press Syndicate of the University of Cambridge, Hill J, Murphy B, Johnson A, Lipshultz L1. Sperm penetration bioassay to evaluate semen obtained from electroejaculated patients. J Urol 1987;138:162A. 9. Leduc BE, Roy D, Poulin O. The use of physostigmine in men with spinal cord injury with ejaculatory dysfunction. Can J Rehabil 1992;4: de Lamirande E, Gagnon C. Quantitative assessment ofthe serum induced stimulation of human sperm motility. Int J Androl 1991;13: Rao B, South JC, Martin M, David G. Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility. Gamete Res 1989;24: Aitken RJ, Irvine DS, Wu FC. Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol 1991;164: Aitken JR, West KM. Analysis of the relationship between reactive oxygen species production and leukocyte infiltration in fractions of human semen separated on Percoll gradients. Int J Androl 1990;13: Kovalski NN, de Lamirande E, Gagnon C. Reactive oxygen species generated by human neutrophils inhibit sperm motility: protective effect of seminal plasma and scavengers. Fertil Steril1992;58: Morel F, Doussiere J, Vignais PV. The superoxide generating oxidase of phagocytic cells. Physiological, molecular and pathological aspects. Eur J Biochem 1991;201: Nissen HP, Kreysel HW. Superoxide dismutase in human semen. Klin Wochenschr 1983;61; Jeulin C, SoufirJC, Weber P, Laval-MartinD, Calvayrac R. Catalase activity in human spermatozoa and seminal plasma. Gamete Res 1989;24: Ganong WF. Review of medical physiology. California: Lange Medical Publications, de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility. J Androl 1992;13: Gavella M, Lipovac V. NADH-dependent oxidoreductase (diaphorase) activity and isozyme pattern of sperm in infertile men. Arch Androl 1992;28: de Lamirande et ai. Reactive oxygen species in spermatozoa Fertility and Sterility