Formation of Reactive Oxygen Species by Spermatozoa From Asthenospermic Patients: Response to Treatment With Pentoxifylline

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Vol. 157, 214042146, June 1997 Printed in U S A .

TEEJOURNAL OF UROLOGY &pyright @ 1997 by AMERICAN

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FORMATION OF' REACTrVE OXYGEN SPECIES BY SPERMATOZOA FROM ASTHENOSPERMIC PATIENTS: RESPONSE TO TREATMENT WITH PENTOXIFYLLINE HIROSHI OKADA, NOBORU TATSUMI, MASANORI KANZAKI, MASATO FUJISAWA, SOICHI ARAKAWA AND SADAO KAMIDONO From the Department of Urology, Kobe University School of Medicine, Kobe, Japan

ABSTRACT

Purpose: We determined the incidence of reactive oxygen species formation by spermatozoa from asthenospermic patients, and the relationship between reactive oxygen species formation and sperm motion parameters. We also assessed the efficacy of in vitro and in vivo pentoxifylline treatment of asthenospermic patients whose spermatozoa generated high reactive oxygen species levels. Materials and Methods: Reactive oxygen species formation by spermatozoa from asthenospermic patients and fertile volunteers was measured by chemoluminescence. Reactive oxygen species formation by the sperm preparations was investigated without stimulation (steady state), or after stimulation with N-formyl-methionyl-leucyl-phenylalanine(f-MLP) or phorbol-12myristate-13-acetate. Spermatozoa from 15 asthenospermic patients whose spermatozoa produced high levels of reactive oxygen species at steady state were treated in vitro with pentoxifylline to determine its effect on reactive oxygen species generation and sperm motion parameters. These same 15 patients and 18 with asthenospermia whose spermatozoa did not produce reactive oxygen species a t steady state were treated with pentoxifylline a t 2 different dosages (300 and 1,200 mg. daily) to determine its effect on reactive oxygen species generation, sperm motion parameters and sperm fertilizing ability in vivo. Results: When reactive oxygen species formation was detected in the steady state that was not stimulated by f-MLP, the source of reactive oxygen species could be attributed t o the spermatozoa themselves. Spermatozoa from 15 of 7 1 asthenospermic patients generated reactive oxygen species at steady state. Pentoxifylline decreased reactive oxygen species generation by spermatozoa in these patients, and preserved the decrease of curvilinear velocity and beat cross frequency for 6 hours in vitro. For these patients orally administered pentoxifylline failed to decrease reactive oxygen species generation by spermatozoa, and had no effect on sperm motility, sperm motion parameters and sperm fertilizing ability a t low dosage (300 mg. daily). However, it increased motility and beat cross frequency a t high dosage (1,200 mg. daily) but it had no effect on sperm fertilizing ability. Conclusions: Stimulation of sperm preparations with f-MLP can identify the source of reactive oxygen species generated at steady state. Among asthenospermic patients there were some whose spermatozoa produced detectable steady state levels of reactive oxygen species. In this group pentoxifylline appeared to be effective for decreasing reactive oxygen species formation and preserving sperm motion parameters in vitro. Orally administered pentoxifylline had no effect at low dosage but it increased sperm motility and some sperm motion parameters without altering sperm fertilizing ability a t high dosage. KEYWORDS: infertility, male; spermatozoa; reactive oxygen species; pentoxifylline

Every cell that lives in a n aerobic environment and uses oxygen generates some level of reactive oxygen species. At the same time every cell has developed defense mechanisms (scavenger system) against reactive oxygen species. Several diseases have been attributed to an imbalance in the generation and scavenging of reactive oxygen Species. Several studies have examined the possible role of spermatozoa1 reactive oxygen species generation in defective sperm function and infertility. In most of these studies excessive reactive oxygen species generation with or without stimulation was associated with a defect in sperm function, particularly in patients with oligospermia. In contrast, lwasaki and G a p o n Accepted for publication November 22, 1996. Su ported in part by Grant-in-Aid #I07671725 for Scientific Researct C. 1995 to 1996, from the Japanese Ministry of Education, Science and Culture.

reported that there was no correlation between reactive oxygen species formation and sperm concentration but a significant negative correlation was found between the level of reactive oxygen species formation and the percentage of motile spematozoa as well as linearity of movement,fi We investigated the production of reactive oxygen species by spematozoa from asthenospermic patients and fertile volunteers. we used special to differentiate the reactive oxygen species generated by the spermatozoa themselves. The reactive oxygen species levels in sperm preparations were measured first without stimulation (steady state) and then the same preparations were assayed after stimula(f-MLP). tion with N-formyl-methionyl-leucyl-phenylalanine PentOXifylline reportedly is effective in preserving sperm motility in vitro7 and in improving semen parameters in

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Vivo.s.9 However, to our knowledge no previous report has discussed the effects of pentoxifylline on sperm motion parameters with regard to reactive oxygen species formation using patients whose spermatozoa generate reactive oxygen species. Therefore, we investigated the effect of pentoxifylline on reactive oxygen species production by spermatozoa that generated reactive oxygen species at steady state and on sperm motion parameters in vitro. We also investigated the effects of its oral administration on reactive oxygen species production by spermatozoa, sperm motion parameters and sperm fertilizing ability in patients with asthenospermia, whose spermatozoa generated high levels of reactive oxygen species at steady state.

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was performed as described by Yanagimachi et aL16 except .hat the cryopreserved zona-free hamster eggs were pur:hased commercially. In brief, cryopreserved zona-free hamiter eggs were pre-incubated in microdroplets of BWW meiium under mineral oil for 3 hours at 37C in 5% carbon iioxide in air, and motile spermatozoa (1 to 10 X 106/ml.) Nere introduced into the microdroplets and co-incubated for t hours under mineral oil at 37C in 5% carbon dioxide in air. rhe penetration rate was determined under an inverted phase contrast microscope. For each assay at least 20 eggs were used. The normal control value a t our institution is 1more than 14%. Sperm preparation. ARer semen analysis the semen samples were fractionated on a discontinuous 2-step Percoll gradient according to the method described by Aitken and MATERIALS AND METHODS Reagents. 5-Amino-2,3,-dihydro-1,4 phthalazinedone (lu- Buckingham.l7 Briefly, 3 ml. of a 100% isotonic Percoll soluminol), f-MLP, phorbol-12-myristate-13-acetate(PMA), so- tion were overlaid with 3 ml. 70% Percoll in a sterile 15 ml. dium lactate, sodium pyruvate, bovine serum albumin, conical centrihge tube. Semen (2 ml.) was layered on top of each gradient and centrifuged at 600 X gravity for 20 minPercoll and 199 medium were purchased commercially. A utes. The sperm samples were collected from the base of the therapeutic preparation of pentoxifylline was obtained, and 100% isotonic Percoll layer, resuspended in 9 ml. BWW meantileukocytic common antigen (CD45) monoclonal antibody dium and centrifuged at 600 x gravity for 5 minutes. The (M0855) and a universal kit for the alkaline phosphatase: supernatant was discarded, the pellet was resuspended in anti-alkaline phosphatase technique were used. Biggers- BWW medium at a concentration of 2 X lo7 sperm per ml. Whitten-Wittingham (BWW) medium was prepared accordand this final sperm preparation was divided into 3,500 pl. ing to the procedure described by Biggers et al.l0An isotonic Percoll solution was made by adding 300 mg. bovine serum fractions to be used for the reactive oxygen species measurements at steady state (1) and for subsequent reactive albumin, 3 mg. sodium pyruvate and 0.37ml. sodium lactate oxygen species measurements after stimulation with f-MLP ~ medium 199 and mixing it syrup to 10 ml. 1 0 concentrated or PMA (2).A 5 pl. aliquot of each final sperm preparation with 90 ml. Percoll. This preparation was named 100% iso- was used for Papanicolaou staining to determine the mortonic Percoll solution, and a 70% isotonic Percoll solution was phological features of the spermatozoa according to World created by diluting the 100% solution with BWW medium Health Organization recornmendations.1l before use. Measurement of reactive oxygen species production. ReacPatients, semen collection and assessment of sperm motion tive oxygen species production was measured in a Lumiphoparameters. We evaluated 71 idiopathic asthenospermic pa- tometer TD4000t at a chamber temperature of 37C for a total sperm mo- of 20 minutes in the integration mode. Luminol, which will tients (sperm density greater than 20 X 106/ml., tility less than 40%, abnormally formed sperm less than 50%) react with a variety of reactive oxygen species (hydrogen with more than a 2-year history of sterility who were attend- peroxide, *02-,*OH,lo2), was used as a luminescence probe. ing the male infertility clinic of the department of urology at A 500 pl. aliquot of the final sperm suspension was incubated our university hospital. All of these infertile patients, who with 1 p1. of a luminol stock solution (100 mM. solution in had never fathered a child, were otherwise healthy. Sperma- dimethyl sulfoxide). The luminescence was recorded subsetozoa from 10 healthy fertile donors who had fathered a child quently with a Lumiphotometer. The basal signal obtained in within 2 years of the study were used as controls. None had this manner was considered to represent the steady state a varicocele testis or abnormal serum luteinizing hormone, situation. Four sets of samples without a sperm suspension follicle-stimulating hormone, testosterone, prolactin or estra- were used as negative controls and the value of the mean diol levels. + 2 standard deviations of these negative controls was desSemen samples were produced by masturbation and col- ignated as the cutoff value for the detectable reactive oxygen lected into sterile containers that were transported immedi- species levels throughout the assay. ately to the laboratory. Semen analyses were performed after When the sperm preparation generated detectable steady complete liquefaction. A conventional semen profile was ob- state levels of reactive oxygen species the levels after stimutained for each sample using the procedures described by the lation were measured. In brief, a 500 pl. aliquot of the final World Health Organization.1' A 5 pl. aliquot was placed on a sperm suspension was incubated with 1 pl. of the luminol Makler chamber12 to determine the sperm concentration. stock solution in the presence of 50 pM. f-MLP or 100 nM. Aliquots (5 pl.) of samples were placed in a p-Cell* 20 pm. PMA. The luminescence was recorded for 20 minutes as deep disposable counting chamber and analyzed in a com- described. puter assisted semen analyzer to measure the sperm motion Determination of the source of reactive oxygen species deparameters, such as sperm motility, mean curvilinear veloc- tected in the sperm preparation and number of coexisting Ity, mean path velocity, linearity, lateral head displacement leukocytes. Ten sperm preparations obtained from 71 astheand beat cross frequency as described by Mortimer et d.13 nospermic patients produced detectable levels of reactive The settings used for the analyses were the same as those oxygen species at steady state. These sperm preparations used by Burkman'4 and Lewis15 et al, that is frame acquisi- were subsequently incubated with f-MLP or PMA and reaction rate 30 Hz., minimum contrast 7,minimum size 6, OW tive oxygen species generation was measured. The leukocytes Bize gate 0.4, high size gate 1.6,low intensity gate 0.4, high in the sperm preparations were differentiated from other mtensity gate 1.6 and magnification factor 2.04. During the cells, such as immature spermatids, by immunohistmhemiassay, the chamber was maintained at 37C and at least 200 cal staining using anti-CD45 monoclonal antibody as the motile sperm were examined. The assays were performed in primary antibody, followed by the alkaline phosphatase:antitriplicate for each sample. Values were expressed as the alkaline phosphatase technique using a commercially availmeans. able kit (K0670) according to the recommendations of the Zona-free hamster egg sperm penetration test (Hamster manufacturer. The concentration of contaminating leukotest). To evaluate sperm fertilizing ability the hamster test t Lab0 Science. Inc. Tokyo, Japan. * Fertility Technologies, Notick, Massachusetts.

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to the known number of spermatozoa.ll Assignment of patient groups. We assigned the asthenospermic patients to groups based on levels of reactive oxygen species at steady state and the source of reactive oxygen species: group 1-15 whose sperm preparations generated detectable reactive oxygen species levels a t steady state that could be attributed to the spermatozoa themselves, group 2-21 whose sperm preparations generated detectable reactive oxygen species levels at steady state that could be attributed to spermatozoa and/or coexisting leukocytes, and group 3-18 of 35 with asthenospermia whose sperm preparations failed to generate detectable reactive oxygen species levels at steady state. Treatment of spermatozoa from group 1 patients with pentoxifylline in uitro. Spermatozoa from group 1 were preincubated for 30 minutes a t 37C with 1 or 10 mM. pentoxifylline and then washed with BWW medium. The steady state production of reactive oxygen species before and after treatment was measured. Treatment of spermatozoa from group I patients with pentoxi/jdline in vitro and its effects on sperm motion parameters. Spermatozoa from group 1 were pre-incubated with 1 mM. pentoxifylline in the same manner as described. Sperm motion parameters were examined a t 0,1,2,3and 6 hours after pre-incubation. The percentage of each value relative to the pretreatment value was calculated. Oral administration of pentoxifjlline in groups I and 3. Groups 1 and 3 patients were administered 300 mg. pentoxifylline daily for 4 months, and then the dosage was increased to and maintained a t 1,200 mg. daily for another 4 months. For each patient semen was analyzed and sperm motion parameters were measured every 4 weeks. Steady state reactive oxygen species production was measured a t the same intervals. The hamster test was done before, and a t 4 and 8 months during the treatment period. The serum concentrations of follicle-stimulating hormone, luteinizina hormone and testosterone were measured before and afterireatment. cytes was calculated relative

Case

Statistical analysis. The Mann-Whitney U test was used to evaluate the differences in reactive oxygen species production a t steady state and sperm motion parameters with or without pre-incubation with pentoxifylline. Wilcoxon’s signed rank test was used to evaluate the differences among the values before and after oral administration of pentoxifylline. Contingency table analysis was done to evaluate the effect of orally administered pentoxifylline on the hamster test. A value of p <0.05 was considered statistically significant. RESULTS

Determination of the source of reactive oxygen species detected at steady state in sperm preparations. Assay procedures were established to determine the source of reactive oxygen species detected at steady state. A total of 10 sperm preparations (cases 1 to 10) obtained from asthenospermic patients generated detectable levels of reactive oxygen species a t steady state. When stimulated by f-MLP, 7 sperm preparations (cases 1 to 7) increased reactive oxygen species generation by 2.1 to 4.5-fold. The remaining 3 sperm preparations (cases 8 to 10) did not increase reactive oxygen species generation in response to f-MLP. All sperm preparations increased reactive oxygen species production after stimulation with PMA to various extents (fig. 1). The former 7 and latter 3 sperm preparations contained more than lo6 and fewer than lo4 leukocytes per ml., respectively, by immunohistochemical staining. Leukocytes suspended in BWW medium (104/ml.) did not generate reactive oxygen species at steady state (data not shown). Thus, these results suggested that the reactive oxygen species detected a t steady state, which were not increased by stimulation with f-MLP, were generated by the spermatozoa themselves. In subsequent experiments we adopted this f-MLP stimulation to differentiate sperm preparations whose reactive oxygen species production could be attributed to sDermatozoa themselves. Reactive oxygen species formaiion by spermatozoa from fer-

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FIG.1. Reactive oxygen species production by sperm preparations at steady state and after stimulation with f-MLP or PMA. Chemolu. minescence was generated by sperm preparations and expressed in log,,,(relative light units [rlulllo’)).

REACTIVE OXYGEN SPECIES FOR?dATION BY SPERM IN A8TkIBNOSPEUWC PATIENTS tile volunteers and asthemperm& patients. Sperm preparetions from 10 fertile volunteers failed to produce detectable reactive oxygen species levels at steady state (fig. 2). In contrast, sperm preparations obtained from 36 of 71 asthenospermic patients generated detectable levels at steady state. In 15 of these 36 patients reactive oxygen species generation was not increased after stimulation with f - W (group 1). In the remaining 21 patients f-MLP stimulation had increased reactive oxygen species production (group 2). In uitro effect of pentoxifilline on remtive oqygen species production and motion parameters of spermtozw from group 1 patients. Reactive oxygen species production by spermatozoa from group 1 was decreased significantly by preincubation in vitro with the 2 concentrations of pentoelline examined (table 1). The effects of preincubation with pentoxifylline on sperm motion parameters were also evaluated. Without pre-incubation with pento+lline all sperm motion parameters decreased with time of incubation in BWW medium to various extents. fie-incubation with pentoxSylline (1 mM./ml.) prevented the decrease in the curvilinear veiocity and beat cross frepuency for 6 hours (fig. 3). However, pentoxifylline had no effect on sperm motility, linearity, mean path velocity and mean lateral head displacement. Effectsof pentoxifilline on reactive oxygen species production, sperm motion parameters and fertilizing ability of spermatozoa from asthenospermic patients in viuo. To assess the effects of pentoxifylline in vivo groups 1 and 3 patients were treated with 300 mg. pentomlline orally daily. Such treab ment had no signiscant effect on reactive oxygen species generation by the spermatozoa or on sperm motion parameters in group 1 in contrast to the in vitro results (table 2). The dosage then was increased from 300 to 1,200 mg. daily but again no change was observed in the steady state production of reactive oxygen species. Both dosages had no effect on the reactive oxygen species production and sperm motion parameters of the sperm preparations of group 3 (data not shown). However, in group 1 treatment at this dosage increased sperm motility and curvilinear velocity without altering reactive oxygen species production. Pentox@lline treatment at both dosages had no effect on sperm fertilizing ability

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either group as 888e88ed by the hamstar teet. Serum folliclestimulating hormone, luteinizing hormone and teatuetemne levels did not change before or after treatment with pentoxifylline (data not shown). DI8CUSSION

Several studies demonstrated that spermatozoa can produce reactive oxygen species, and the incidence and level of reactive oxygen species production were greater in spermatozoa from infertile patients than from fertile volunteers.6.17slS However, the methods used for sperm preparation and reactive oxygen species detection varied among the studies. In addition, the study results could be confounded by the fact that there are 2 possible source8 of reactive oxygen species in the ejaculated semen: 1) spermatoeoa and 2) umtaminating leukocytes. It is impossible to remove completely the contaminatingleukocytes from sperm preparations, even using Percoll and anti-CD45 coated magnetic beade.18 We used a new technique, which waa a M a i t i o n of the method described by Krausz et al,’S to determine the 8ource of reactive oxygen speciea &tected at steady state. We measured steady state reactive oxygen species production by the sperm preparations, that is without stimulation. We then measured reactive oxygen spaciea praduction by the sperm preparations after stimulation with f-MLP. Formyl peptides, such as f-MLP, are receptor mediated chemoattrac-

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REACTIVE OXYGEN SPECIES FORMATION BY SPERM IN ASTHENOSPERMIC PATIENTS sperm motility

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R G . 3. EKects of pre-incubation with pentoxifylline on spermatozoa from group 1. Horizontal axis is hours (hr) after pre-incubation. Vertical axis is percentage relative to that before treatment. Values are median of 15 s rm preparations. Asterisk indicates values sigruficantly different from those without re incubation (p 10.05, Mann-Whitney U test). V E ,mean curvilinear velocity. VAP, mean path velocity. LIN, linearity. ALH, lateral h e a l displacement. BCF, beat cross frequency.

TABLE2. Effects of pentoxifylline on reactive oxygen species production. swrm motion parameters and hamster test in vivo 5% Before Treatment

4 Mos. at 300 MaDay

4 Mos. at 1,200 Mg./Day

Reactive oxygen species production and sperm parameters (mean % 2 SO)* 100 101 2 3.5 104 2 3.3 Reactive oxygen species 100 96 -C_ 5.6 151 2 9.4t Motility 100 103 2 4.5 122 2 5.5t Mean curvilinear velocity Mean path velocity 100 98 2 7.9 101 2 8.8 100 104 t 5.7 100 2 4.4 Linearity 100 95 2 13.2 102 2 10.2 Lateral head displacement Beat cross frequency 100 97 2 5.5 96 2 8.9 Hamster test in uivo (No. pts.)$ Group l(15 pts.): 1 2 3 Normal Abnormal 14 13 12 Group 3 (18pts.): Normal 6 7 5 Abnormal 12 11 13 * Group 1 patients were given pentoxifylline and reactive oxygen production and sperm motion parameten were evaluated. Values are reported as relative percentage of each parameter. t Significantly increased compared to the baseline value before treatment ( p 10.05,Wilcoxon signed rank test). $ Groups 1 and 3 patients were administered pentoxifylline and the results of the hamster test before and after treatment were evaluated. Pentoxifylline treatment in vivo did not alter the results of the hamster test (contingency table analysis).

tants.20 Sperm lack a n f-MLP receptor on the surfaces19 and, therefore, cannot be stimulated to produce reactive oxygen species by incubation with f-MLP. In contrast, leukocytes possess an abundance of f-MLP receptors and the production of reactive oxygen species by leukocytes can be stimulated by f-MLP. If reactive oxygen species production is detected in a sperm preparation a t steady state and can be stimulated further by f-MLP, it is likely that the reactive oxygen species were generated not only by the spermatozoa but also by the contaminating leukocytes. We also confirmed that sperm preparations that produced detectable steady state levels of reactive oxygen species and could be stimulated further by

f-MLP contained more than lo6 leukocytes per ml. by immunohistochemical staining. In a preliminary experiment we also found that a leukocyte concentration of 104/ml.in BWW medium could not generate detectable levels of reactive oxygen species at steady state in our assay system used. Thus, in the sperm preparations with detectable steady state levels of reactive oxygen species that did not increase aRer f-MLP stimulation the source of the reactive oxygen species had to be the spermatozoa themselves. Using this strategy we assigned 31 asthenospermic patients, whose sperm preparations generated reactive oxygen species at steady state, to groups 1 and 2. PMA activates protein kinase C directly and stimulates reactive oxygen species production by all cells. Therefore, stimulation with PMA cannot be used to differentiate the source of the reactive oxygen species. T o determine if there were contaminating leukocytes in the sperm preparation precisely immunohistochemical staining was used. However, the study took at least 4 hours to complete. On the other hand, our method needed only a n additional 20 minutes after measuring steady state reactive oxygen species levels. In this series sperm preparations from 36 of 71 asthenospermic patients generated reactive oxygen species at steady state and 21 of them could be stimulated further by incubation with f-MLP (group 2). The remaining 15 patients whose sperm preparations were not stimulated to generate reactive oxygen species by f-MLP were designated as group 1. Theoretically, the reactive oxygen species detected at steady state in the sperm preparations of group 2 could be attributed to spermatozoa and/or contaminating leukocytes, while those detected at steady state in the sperm preparations of group 1 could be attributed solely to spermatozoa. The incidence of reactive oxygen species formation by spermatozoa from asthenospermic patients has not yet been established. To our knowledge our report is the first to suggest that at least 21.1%(15 of 71) of asthenospermic patients have spermatozoa that produce detectable steady state levels of reactive oxygen species. The plasma membrane of human

REACTIVE OXYGEN SPECIES FORMATION BY SPERM IN ASTHENOSPERMIC PATIENTS

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sperm is highly susceptible to lipid peroxidation21 because of nia. To develop new treatment strategies it is important to earn more about the nature of these deficiencies. Our results its high content of fatty acids. Therefore, the massive reactive oxygen species production by spermatozoa can cause nay provide clues to a therapeutic approach for the treatoxidative changes in the plasma membranes, including the ment of idiopathic male factor infertility. For confirmation of loss of integrity and fluidity.22 It is possible that defective these results a prospective and double blind clinical trial, sperm function in this same group of asthenospermic pa- with fecundity as the crucial outcome measure, is necessary. tients can be partly attributed to excessive reactive oxygen species generation. None of the sperm preparations from CONCLUSIONS fertile volunteers produced reactive oxygen species, consistent with previous results by Iwasaki and Gagnon.6 After measuring steady state Levels stimulation of sperm When we discuss the deleterious effect of reactive oxygen preparations by f-MLP can identify the source of reactive species on sperm we should consider the effects of its scav- oxygen species. Among asthenospermic patients there was a enger in the seminal plasma. However, in the process of in group whose spermatozoa produced detectable steady state vitro fertilization seminal plasma is removed and reactive reactive oxygen species levels. In this group pentoxifylline oxygen species generated by spermatozoa can affect the out- was effective in decreasing reactive oxygen species formation come of the procedure directly. Pentoxifylline has been re- and preserving sperm motion parameters in vitro. Orally ported to decrease reactive oxygen species production23.24 administered pentoxifylline had no effect at low dosage. How. ~ ~it increased sperm motility and some sperm motion and to increase sperm motion parameters in v i t r ~ . ~ever, Tesarik et a1 demonstrated that in unselected asthenosper- parameters without altering sperm fertilizing ability at high mic patients pentoxifylline increased the curvilinear velocity, dosage. path velocity and beat cross frequency but did not increase Hoechst Japan Ltd. provided the pentoxifylline. the percentage of motile spermatozoa.25 However, to our knowledge no data have been reported concerning the effects of pentoxifylline on sperm motion parameters in select astheREFERENCES nospermic patients whose spermatozoa produced detectable 1. Aitken, R. J., Buckingham. D., West, K, Wu, F. C., Zikopoulos, steady state levels of reactive oxygen species. Therefore, we K and Richardson, D. W.: Differential contribution of leucoinvestigated the effect of pentoxifylline on sperm motion cytes and spermatozoa to the generation of reactive oxygen parameters and spermatozoa1 reactive oxygen species prospecies in the ejaculates of oligozoospermic patients and fertile duction in asthenospermic patients in vitro and in vivo. Our donors. J. Reprod. Fertil., 94:451, 1992. study confirmed that pre-incubation of spermatozoa from 2. Aitken, R. J. and Clarkson, J. S.: Cellular basis of defective asthenospermic patients with this agent suppressed reactive sperm function and i t s association with the genesis of reactive oxygen species generation in group 1 and increased 2 of the oxygen species by human spermatozoa. J . Reprod. Fertil., 81: sperm motion parameters. It is noteworthy that pre459, 1987. incubation of sperm with pentoxifylline prevented the de3. DAgata, R., Vicari, E., Moncada, M. L., Sidoti, G., Calogero, A. E., Fornito, M. C., Minacapilli, G., Mongioi, A. and Polosa, crease in the curvilinear velocity and beat cross frequency for P.: Generation of reactive oxygen species in subgroup of inferup to 6 hours but had no effect on the other sperm motion tile men. Int. J . Androl., 1 3 344, 1990. parameters examined (sperm motility, path velocity, linear4. de Lamirande, E. and Gagnon, C.: Reactive oxygen species and ity and amplitude of lateral head displacement). These findhuman spermatozoa. I. Effects on the motility of intact sperings, combined with the results of previous reports, suggest matozoa and on sperm axonemas. J . Androl., 13: 368, 1992. that pentoxifylline treatment may be useful for in vitro 5. Alvarez, J. G.,Touchstone, J . C., Storey. B. T. and Blasco, L.: sperm augmentation in asthenospermic patients by decreasSpontaneous lipid peroxidation and production of hydrogen ing the reactive oxygen species production by spermatozoa. peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicPentoxifylline is widely used in Japan at a dose of 300 mg. ity. J. Androl., B: 338, 1987. daily for treatment of patients with cerebrovascular disor6. Iwasaki, A. and Gagnon, C.: Formation of reactive oxygen speders. However, treatment with pentoxifylline at this dosage cies in spermatozoa of infertile patients. Fertil. Steril., 57: 409, for 4 months did not affect reactive oxygen species genera1992. tion, sperm motion parameters or sperm fertilizing ability in 7. Pang, S.C., Chan, P. J. and Lu, A.: Effect of pentoxifylline on groups 1 and 3. Therefore a low oral dose of pentoxifylline sperm motility and hyperactivation in normozoospermic and does not produce a beneficial effect in patients with asthenonormokinetic semen. Fertil. Steril., 60.336, 1993. spermia. Tournaye et a1 treated infertile patients with pen8. Marrama, P., Baraghini, G. F., Carani, C., Celani, M. F., Giovenco, P., Granh, F. and Montanini, V.: Further studies on toxifylline and documented that a low dosage had poor efthe effect of pentoxifylline on sperm count and sperm motility feck26 Thus, the dosage of pentoxifylline was increased to in patients with idiopathic oligo-asthenozoospermia.Androlo1,200 mg. daily in the same patient population. At this dosgia, 1 7 612,1985. age pentoxifylline improved sperm motility, as previously 9. Yovich, J. M., Edirisinghe, W. R., Cummins, J. M. and Yovich, demonstrated in some reports,26 and increased the curvilinJ. L.: Influence of pentoxifylline in severe male factor infertilear velocity only in group 1 but not in group 3. Combined ity. Fertil. Steril., 5 3 715, 1990. with the results of previous reports, our findings support the 10. Biggers, J. D.,Whitten, W. K and Whittingham, D. G.: The in vitro use of pentoxifylline in select asthenospermic paculture of mouse embryos in vitro. In: Methods in Mammalian tients whose spermatozoa generate detectable steady state Embryology. Edited by J. C. Daniel. Jr. San Francisco: Freelevels of reactive oxygen species. However, therapeutlc dosman, pp. 86-116,1971. age of pentoxifylline did not alter reactive oxygen spec.~es 11. W H O Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction, 3rd ed. New York: production. A possible explanation is that the concentratlon Cambridge University Press, 1992. of the drug in the reproductive tract does not approach that A.: The improved ten-micrometer chamber for rapid used in vitro, its mode of action may be different in vivo and 12. Makler, sperm count and motility evaluation. Fertil. Steril.. 33: 337, in vitro, or the scavenger in the seminal plasma modified the 1980. effects of reactive oxygen species in vivo. Moreover, pentoxi- 13. Mortimer, D., Serres, C., Mortimer, S. T. and Jouannet, P.: f y h e did not improve the sperm fertilizing ability as evalInfluence of image sampling frequency on the perceived moveuated by the hamster test, the reason for which is not clear ment characteristics of progressively motile human spermatozoa. Gamete Res., 2 0 313, 1988. from our study. Currently, there is no effective treatment for patients with 14. Burkman, L. J.: Discrimination between nonhyperactivated and classical hyperactivated motility patterns in human spermadeficient sperm functions, such as idiopathic asthenosper-

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