Leukocytes in semen from men with spinal cord injuries

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1 FERTILITY AND STERILITY VOL. 72, NO. 1, JULY 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Leukocytes in semen from men with spinal cord injuries Ian A. Aird, M.R.C.O.G.,* Gill S. Vince, Ph.D., Michelle D. Bates, B.Sc., Peter M. Johnson, Ph.D., and Iwan D. Lewis-Jones, M.D.* University of Liverpool, Liverpool, United Kingdom Objective: To assess the leukocyte populations in semen samples from men with spinal cord injuries (SCIs) and their relation to sperm motility. Design: Cross-sectional study. Setting: A joint spinal cord injury and fertility clinic at an academic tertiary referral center for fertility treatment and a university-based department of immunology. Patient(s): Nine men with chronic SCIs and seven healthy sperm donors as controls. Intervention(s): Semen samples were obtained by electroejaculation from men with SCIs and by masturbation from donors. Main Outcome Measure(s): Leukocyte populations determined by immunohistochemical techniques, bacteriologic assessment of urine, and sperm density and motility. Result(s): The most cellular specimens were antegrade specimens obtained from men with SCIs and coexisting urinary tract infections. The highest proportion of leukocytes occurred in retrograde samples from men with SCIs and urinary tract infections. The most predominant leukocytes in all specimens were granulocytes. Infection increased the number of T cells and the degree of cell activation. There was no significant correlation between leukocyte populations and total motile sperm counts. Conclusion(s): Increased numbers of leukocytes in semen samples from men with SCIs are the result of urinary tract infections. The reduced sperm motility seen in men with SCIs does not correlate with the numbers of leukocytes; therefore, other factors also contribute to the semen abnormalities in these patients. (Fertil Steril 1999;72: by American Society for Reproductive Medicine.) Key Words: Leukocytes, semen, spinal cord injury, sperm function, urinary tract infection Received August 19, 1998; revised and accepted February 8, Reprint requests and present address: Ian A. Aird, M.R.C.O.G., Queen Elizabeth Hospital, Sheriff Hill Gateshead, Tyne & Wear, NE9 6SX, United Kingdom (FAX: ). * Department of Obstetrics and Gynaecology. Reproductive Immunology Group, Department of Immunology /99/$20.00 PII S (99) More than 10,000 cases of spinal cord injury (SCI) occur annually in the United States (1). It has been estimated that 50% of these injuries occur in young men aged years (2). Loss of sexual function is a major cause of distress and anxiety to young men with SCIs; impaired sexual ability has been reported as more important than loss of the use of limbs or having a satisfactory social life (3). One of the major problems facing men with SCIs is adaptation to their altered sexual ability. Despite advances in rehabilitation medicine, infertility remains a significant complication; 5% of patients can procreate without medical intervention (2). Fertility problems in men with SCIs arise in two areas: erectile or ejaculatory disturbances secondary to neuromuscular dysfunction and poor semen quality (4). Various techniques have been used to overcome the ejaculatory dysfunction, the most frequent being vibrostimulation and electroejaculation. Using a combination of these two techniques, semen samples can be expected to be obtained in up to 80% of patients (5, 6). The cause of the abnormal sperm function remains uncertain. The most notable abnormality is a reduction in sperm motility, with some studies showing that 70% of men with SCIs have progressive sperm motility of 20% (7, 8). Possible factors contributing to poor semen quality include stasis of prostatic fluid, testicular hyperthermia, sperm contact with urine, changes in the hypothalamic-pituitarytesticular axis, sperm autoantibodies, recurrent urinary tract infections (UTIs), and the type of bladder management used (4). The last two potential causes of poor semen quality may 97

2 explain the increased numbers of round cells observed in semen samples from men with SCIs (9). The significance of increased numbers of leukocytes in seminal fluid and abnormal sperm function remains controversial (10, 11). One contributing factor is the different methods used to enumerate leukocytes in seminal fluid. Immunocytochemistry is now the accepted standard for the detection of all leukocyte populations in semen (11). According to the World Health Organization (12), concentrations of leukocytes of /ml are considered increased and the ejaculate is termed leukocytospermic. A relation between increasing seminal granulocyte concentrations and poor semen parameters has been reported previously (13). This study was designed to identify the leukocyte populations in semen from men with SCIs and to compare these populations with those from prospective sperm donors. Our aims also were to assess whether leukocytospermia in general or changes in the profile of leukocyte subpopulations in particular are associated with sperm count and motility in patients with SCIs, and also to identify factors associated with SCIs that may affect the semen quality of such men. MATERIALS AND METHODS Men with SCIs referred to the combined infertility and SCI clinic at the Regional Spinal Injuries Unit, Southport and Formby District Hospital, from February 1996 to December 1996 were included in the study. This study was approved by the local ethics committee, and informed consent was obtained. At the initial clinic visit, a full history and examination was performed and the optimum method of sperm collection was decided. All patients (n 9) included in the study required electroejaculation treatments to procure semen specimens. Vibrostimulation either had failed (three patients) or was deemed unsuitable because the site of injury was below T-10 (i.e., a lower motor neuron lesion; six patients). Four patients underwent repeated electroejaculations on two further occasions, three patients had two electroejaculation procedures performed, and two patients had a single electroejaculation procedure performed. Recurrent electroejaculation procedures were carried out at an interval of 2 4 weeks. The characteristics of the subjects who took part in this study are shown in Table 1. Their mean age was 34.7 years (range, years) and their mean duration of injury was 13.6 years (range, 6 43 years). Seven prospective sperm donors were used as a control group. All prospective donors had undergone a detailed bacteriologic assessment of the genitourinary tract and showed no evidence of infection before sperm banking. The average age of the donors was 27.8 years (range, years). TABLE 1 Characteristics of nine men with spinal cord injuries. Patient no. Age (y) Duration of injury (y) Level of injury Degree of injury Bladder management T-10 Incomplete Normal C-4 Complete Indwelling catheter L-1 Incomplete Reflex void and sheath T-12 Complete Reflex void and sheath L-1 Incomplete Intermittent catheter T-8 Complete Bladder stimulator T-8 Complete Indwelling catheter T-12 Complete Reflex void and sheath C-5 Complete Bladder stimulator Semen Collection Patients with SCIs underwent electroejaculation to procure semen specimens. Before the electroejaculation procedure, the bladder was catheterized and a sample of urine was sent for bacteriologic assessment. Specimens that contained white blood cells per milliliter and organisms per liter were considered infected. Ten milliliters of Ham s F-10 culture medium (ICN Biomedicals, Thame, United Kingdom) then was instilled into the bladder. The electroejaculation procedures were performed with the use of Seager Electroejaculation equipment (Dalzell USA Medical Systems, Dungannon, Northern Ireland). The patient was placed in the lateral decubitus position and a 31-mm-diameter probe was inserted into the rectum. Stimulation was begun at 7 V, and each stimulation lasted 2 4 seconds. The maximum number of stimulations was 15, and the voltage ranged from 7 35 V. During stimulation, the perineum and bulbous urethra were massaged and the antegrade fraction of the ejaculate was collected in a sterile plastic container. After the procedure, the bladder was recatheterized and the retrograde fraction was recovered. Proctoscopy was performed both before and after the procedure to ensure that no thermal damage to the rectal mucosa had occurred. Patients with lesions above T-5 were premedicated with 10 mg of nifedipine sublingually to reduce the risks of autonomic dysreflexia. All but one patient had the procedure carried out under light general anesthesia. Donor samples were produced by masturbation after 4 days of sexual abstinence and collected in sterile plastic containers. Semen Analysis Sperm density and motility assessment was performed according to standard World Health Organization criteria (12). The volume of retrograde specimens occasionally was 15 ml. In such circumstances, the specimen was centri- 98 Aird et al. Seminal leukocytes after SCI Vol. 72, No. 1, July 1999

3 TABLE 2 Monoclonal antibodies used in the study. Antibody to Specificity Clone Source Dilution CD3 T cells UCHT1 Serotec, Oxon, UK 1:100 CD4 Helper T cells (Th) MT310 Dako Ltd., Bucks, UK 1:50 CD8 Cytotoxic T cells (Tc) DK25 Dako Ltd. 1:50 CD14 Monocytes/macrophages 3C10 Cell supernatant Neat CD16 Granulocytes, NK cells 3G8 Cell supernatant 1:100 CD22 B cells 4KB128 Dako Ltd. 1:20 CD45 Pan-leukocyte (leukocyte common antigen) F Cell supernatant Neat CD69 Activated T cells, B cells, macrophages CH/4 Serotec 1:20 Class II MHC L243 Cell supernatant Neat Vimentin V9 Serotec 1:50 Cytokeratin CK18 Serotec 1:50 IgG Control Coulter, Luton, UK 1:100 Note: IgG immunoglobulin G; MHC major histocompatibility complex; NK natural killer; UK United Kingdom. fuged at 3,000 g for 10 minutes and resuspended in 2.5 ml of Ham s F-10 culture medium before analysis. Immunocytochemistry Semen samples were diluted with phosphate-buffered saline (ph 7.2) and washed twice by centrifugation at 600 g. The resulting cell pellet was resuspended in phosphatebuffered saline and counted in a hemocytometer. The percentage viability was estimated by Trypan blue exclusion, and the cell concentration was adjusted to /ml in phosphate-buffered saline containing 0.1% bovine serum albumin (Sigma, Poole, United Kingdom). Fifty microliters of the cell suspension was spotted onto multispot glass slides (four spots per slide; C. A. Hendley Ltd., Essex, United Kingdom). The slides were air-dried, fixed in acetone, and stored sealed at 20 C if not used immediately. For immunocytochemistry, the cells first were stained for 30 minutes with the mouse monoclonal antibodies detailed in Table 2. After two 5-minute washes in 0.05M tris(hydroxymethyl)aminomethane buffered saline (TBS; ph 7.6), bound antibodies were detected by the APAAP method (14) using rabbit anti-(mouse immunoglobulin G) immunoglobulin G (diluted 1:25; Dako Ltd., High Wycombe, United Kingdom) for 30 minutes, washed in TBS, and then incubated with a preformed complex of calf intestinal alkaline phosphatase and mouse monoclonal anti-(alkaline phosphatase) (APAAP, diluted 1:50; Dako Ltd.) for a further 30 minutes. Staining was developed with Napthol AS-MX phosphate and Fast Red (Sigma) with the inclusion of 1 mm of levamisole to block any endogenous alkaline phosphatase. Slides were counterstained with hemalum and mounted in Aquamount (BDH, Poole, United Kingdom). Mouse immunoglobulin G was used in place of the first antibody as a negative control. The number of positively stained cells for each leukocyte subpopulation was counted in 10 high-power fields. Germinal cells were assumed to be CD45-negative round cells. Thus, for the CD45-labeled slide in each sample, both the positive and negative cells were counted to obtain the percentage of CD45-positive cells and the percentage of immature germ cells. Statistical Analysis Statistical analysis was performed using the Arcus Biomedical computer software package (Arcus Biomedical, Medical Computing, Cambridge, United Kingdom). Nonparametric data are expressed as medians with ranges in parentheses, and the Mann-Whitney U test was used to compare nonuniformly distributed data. Spearman s rank correlation coefficient was used to assess the correlation between total motile sperm count and leukocyte count. P.05 was defined as statistical significance. RESULTS Twenty-one electroejaculation procedures were performed, resulting in 33 semen samples for analysis, of which 18 were antegrade specimens and 15 were retrograde specimens. Sixteen (76.2%) of the 21 electroejaculation procedures were successful in producing specimens (either antegrade or retrograde) that contained motile spermatozoa. Nine of the antegrade samples and 7 of the retrograde samples came from men with coexisting UTIs. The samples were separated into four groups for analysis of leukocyte concentrations based on the nature of the sample (antegrade or retrograde) and the presence or absence of a coexisting UTI. Table 3 shows the effect of SCI on the total motile sperm count, total cell count, and leukocyte content of the semen specimens. The total cell count is given rather than the cell concentration because the retrograde specimens were col- FERTILITY & STERILITY 99

4 TABLE 3 Total motile sperm counts and cell counts of healthy donors and men with spinal cord injuries. Patient group Total motile sperm count ( 10 6 ) Total cell count ( 10 6 ) Percentage of viable sperm Percentage of CD45- positive cells Donors (84.8 1,482) 3.8 (0.2 24) 70 (50 80) 22.9 ( ) Men with SCIs Nature of sample Antegrade SCI 6.6 (0 16) 6.9 ( ) 74 (20 85) ( ) Retrograde SCI 0 ( ) 2.9 ( ) 75 (50 87) 67.8 ( ) UTI Present 0.53 ( ) 7.44 ( ) 60 (20 82) 69.6 ( ) Absent 0.76 ( ) 3.12 ( ) 75 (40 87) 46.6 ( ) Level of injury Above T ( ) 6.86 ( ) 75 (20 87) 63.6 ( ) Below T-10 0 (0 262) 3.91 ( ) 72.5 ( ) 56.5 ( ) Extent of injury Complete 5.04 ( ) 4.6 ( ) 75 (20 87) 54.8 ( ) Incomplete 0 (0 6.57) 4.24 ( ) 68 ( ) 67.1 ( ) Duration of injury 10 y 5.9 ( ) 4.6 ( ) 77 (40 87) 48.5 ( ) 10 y 0 (0 262) 4.9 ( ) 60 (20 87) 67.1 ( ) Bladder management Catheter 4.88 ( ) 3.9 ( ) 75 (20 87) 48.5 ( ) No catheter 0 (0 262) 7.2 ( ) 75 ( ) 67.1 ( ) Note: Values are medians with ranges in parentheses. See text for results of statistical comparisons. SCIs spinal cord injuries; UTI urinary tract infection. lected into culture medium and volumes could be 30 ml, far greater than normal ejaculates. The cell concentration in the donor samples had a mean value of /ml (range, /ml); however, most cells were immature germ cells because the CD45- positive cell concentration in the donor samples had a mean value of /ml (range, /ml). The semen samples obtained from the donors contained significantly more motile sperm than those obtained from the men with SCIs (P.0001). Analysis of the samples from the men with SCIs showed that antegrade samples contained more motile sperm than retrograde samples (P.01). Significantly greater total numbers of motile sperm were observed in samples from men in whom the spinal cord lesion was above T-10 (i.e., an upper motor neuron lesion; P.02). The duration of injury had a significant effect on the total motile sperm count for each specimen; men with a duration of injury of 10 years had greater numbers of motile sperm than men with a longer duration of injury (P.04). The extent of the SCI (complete or incomplete) did not have a significant effect on semen quality. The type of bladder management used had a significant effect on the total motile sperm count; patients who used catheter drainage of the bladder (either indwelling or intermittent) had significantly more motile sperm than men who used reflex voiding or bladder stimulators (P.04). Antegrade specimens from men with SCIs were more cellular than retrograde specimens and donor samples, but this was not statistically significant (P.06 and P.14, respectively). In men with SCIs, the presence of infection significantly increased the total numbers of cells in the semen compared with the absence of infection (P.049). The most cellular specimens were antegrade samples obtained from men with SCIs and coexisting UTIs; these contained significantly more cells than infected retrograde specimens or donor samples (total cell count versus for retrograde specimens, P.04, and for donors, P.04). Retrograde samples contained the highest proportion of leukocytes (CD45-positive cells). This was significantly different from donor samples (P.03). Infected retrograde specimens showed the highest proportion of leukocytes of all samples analyzed (80.7%). It is of interest that these specimens also contained the lowest numbers of motile sperm (median total motile sperm count, 0; range, The incidence of infected samples was higher in men who used catheters, but this was not statistically significant (71.4% versus 38.1%; P.11, 2 test). The proportion of leukocytes in semen samples from men with SCIs was not significantly affected by the level, extent, or duration of injury or by the method of bladder management used. Cell viability was not significantly different between healthy donors and men with SCIs. It also was unaffected by the nature of the sample, the presence of a UTI, the 100 Aird et al. Seminal leukocytes after SCI Vol. 72, No. 1, July 1999

5 TABLE 4 Leukocyte populations in the different study groups. Study group Leukocyte population Donor (n 7) Antegrade, infected (n 9) Antegrade, uninfected (n 9) Retrograde, infected (n 7) Retrograde, uninfected (n 8) CD3 0.4 a 12.3 b 0 c 1.6 d 0.1 (0 1.2) (0 735) (0 0.79) (0 420) (0 80) CD b 0 c 2 d 0 (0 1.8) (0 375) (0 6.5) (0 290) (0 10) CD8 0 e 17 b (0 0.8) (0 218) (0 2.8) (0 132) (0 52) CD b,f (0 1,350) (1.3 1,470) (0 65) ( ) ( ) CD a c 2, (0 2,610) (0 3,760) (0 700) (5 10,000) (0 4,240) CD22 0 a 0 g (0) (0 14) (0 0.2) (0 33) (0 0.7) CD ,120 b,f 61 c 3,400 d 233 (4.4 2,315) (430 10,000) (10 810) (138 10,000) (16.4 4,600) CD (0 9.6) (0 1.8) (0 12) (0 3.6) (0 0.2) MHC class II 37 e 454 b,f d 43 (3 666) (26 1,530) ( ) (15.7 2,700) ( ) Vimentin 1.8 a,e,h ( ) (0 2,140) (2 290) (0.2 4,220) (3.8 46) Cytokeratin 14.2 a ( ) ( ) (9 500) (5 830) (39 352) Note: Values represent numbers of cells that stained positively per 10 high-power fields and are expressed as medians with ranges in parentheses. The letters denote significant differences between groups (P.05) as follows: a donor versus retrograde; b antegrade, infected versus antegrade, uninfected; c antegrade, uninfected versus retrograde, infected; d retrograde, infected versus retrograde, uninfected; e donor versus antegrade, infected; f antegrade, infected versus retrograde, uninfected; g antegrade, infected versus retrograde, infected; and h donor versus antegrade, uninfected. MHC major histocompatibility complex. level, extent, or duration of injury, or the method of bladder management used. Seven men with SCIs underwent repeated electroejaculation procedures. Three of these men had a UTI at the time of the initial electroejaculation procedure and, despite treatment with the appropriate antibiotics, a UTI was still present at the time of the repeated procedures. Analysis of the samples showed no differences in total cell counts or total motile sperm counts between any of the specimens collected. In men who had a UTI at the time of the initial procedure but who were treated successfully with antibiotics (n 2), there was a substantial decrease in both the total numbers of cells and the numbers of CD45-positive cells. However, this was not associated with an increase in the total motile sperm count. Two men underwent recurrent electroejaculation procedures and had no evidence of a UTI at any time. In these samples, there were no obvious changes in either the cell populations or the numbers of motile sperm. The relation between the total motile sperm count and the numbers of cells in the ejaculate was assessed for total cell number (all round cells), CD45-positive cells, and each of the individual leukocyte populations. No statistically significant negative correlations were noted for any of these analyses. Table 4 shows the different subpopulations of leukocytes in the different groups according to the nature of the sample and the presence or absence of infection. In all groups, granulocytes (CD16) were the most predominant leukocyte population, followed by macrophages (CD14). Increased numbers of T cells were seen in both antegrade and retrograde samples in the presence of a UTI; infection also increased the numbers of cells that expressed major histocompatibility complex class II, which is considered a measure of cell activation. Seminal B cells were found only in one man with an SCI who also had the largest number of T cells. It is of interest that this was the only patient who had a weakly positive mixed agglutination reaction test result for antisperm antibodies. Further testing revealed a weakly positive immunobead test result. However, sperm motility is poor in these patients, and testing is difficult when sperm motility is 10%. FERTILITY & STERILITY 101

6 DISCUSSION This study initially confirmed the findings of other investigators that men with SCIs have abnormal sperm function (4). The major abnormality appears to be a reduction in sperm motility, with retrograde specimens being more adversely affected than antegrade specimens. Men with upper motor neuron lesions and injuries of 10 years duration have significantly more motile sperm per ejaculate than men with longer-standing or lower motor neuron injuries; this is in contrast to other reports that suggest no effect of the level or duration of injury on sperm function (2). Although the use of a urinary catheter was associated with an increase in the number of samples that showed evidence of infection, significantly greater numbers of motile sperm were harvested from men in whom bladder management involved the use of urinary catheters. This may suggest that, in terms of preserving sperm function, protection of sperm from contact with urine is of greater significance than prevention of UTI. Studies involving in vitro exposure of sperm to electrical currents have suggested that the electroejaculation procedure itself may affect sperm motility (15); however, any effect is likely to be small given the low electrical currents used. Samples from men with SCIs that were produced by vibrostimulation or masturbation showed a similar incidence of low sperm motility. It also has been suggested that sperm quality may be improved by performing recurrent ejaculation procedures on men with SCIs (16). The findings of this study do not support this suggestion. This may be because the number of patients involved in this study was low or because not enough ejaculation procedures were carried out to show a significant improvement. The previously reported increased cellularity of specimens from men with SCIs (9) appears to be due to the increased incidence of UTIs, because semen samples from men with SCIs in the absence of UTIs showed a similar number of cells as donor samples. The presence of a UTI increases the total number of cells in the samples, but this does not correlate with an adverse effect on sperm motility. No significant adverse effect on sperm function was noted when comparing total numbers of cells, numbers of CD45- positive cells, or individual leukocyte populations with sperm motility. Recurrent ejaculation does not appear to affect cell populations in semen samples. However, adequate treatment of UTIs did reduce the numbers of cells in semen samples from men with SCIs, although this did not lead to an improvement in sperm function. The lack of a significant correlation between total motile sperm counts and leukocyte populations in patients with SCIs may be due in part to the small number of patients in this study. However, because of the relative rarity of SCI in the population studied, it is difficult to recruit large enough numbers of subjects to increase the statistical impact of the study. Some of this difficulty could be overcome with a multicenter study design. The lack of a correlation also may suggest that increased numbers of white blood cells in semen from men with SCIs are not responsible for the abnormalities noted in sperm function or, more likely, that the observed abnormalities are multifactorial in nature. Larger studies involving healthy men have shown a relation between increasing granulocyte concentrations and reduced sperm motility (13). Increased numbers of seminal leukocytes also have been implicated in abnormal hamster ovum penetration test results (17) and reduced sperm concentrations and motility (18). Leukocyte concentrations of /ml also have been associated with reduced success rates after IVF-ET procedures (19). Other studies, however, have failed to show an association between leukocytospermia and semen quality (20). The level of leukocytospermia as defined by the World Health Organization ( leukocytes per milliliter) may not be clinically useful, and higher levels of leukocytes are required to produce significant effects. Because granulocytes appear to be the predominant leukocyte found in semen, they may be the leukocyte population most likely to cause sperm damage. However, in the present study, we found no significant relation between granulocyte counts and total motile sperm counts. Although no direct relation has been shown between the numbers of leukocytes found in the semen of men with SCIs and reduced sperm motility, increased numbers of leukocytes may be a contributing factor to the sperm abnormality because activated leukocytes are known to produce large amounts of reactive oxygen species (20). The results of this study have shown that, in the presence of infection, the degree of cell activation (as measured by major histocompatibility complex class II expression) is increased. It is known that reactive oxygen species can lead to peroxidative damage of the sperm membrane by the saturation of fatty acids and that this may cause loss of motility and sperm viability (21). Previous studies have reported an increase in reactive oxygen species in semen from men with SCIs that has been correlated with poor sperm motility (7, 22). A significant positive correlation also has been shown between leukocyte concentrations and levels of reactive oxygen species in semen from men with SCIs (22). Conventional sperm stains (e.g., Giemsa) fail to differentiate immature germ cells from leukocytes. The introduction of cytochemical techniques (e.g., the peroxidase method) brought an improvement in the detection of leukocytes in semen because these stains reliably identify granulocytes (11). However, assessments using this method do not identify lymphocytes or monocytes. With the advent of monoclonal antibodies, it is now possible to stain for all the leukocyte populations in semen samples. Hence, the prevalence of leukocytospermia, as defined by the World Health 102 Aird et al. Seminal leukocytes after SCI Vol. 72, No. 1, July 1999

7 Organization, is often higher than when techniques such as the peroxidase method were used. One of the seven healthy donors in this study had a leukocyte concentration of /ml, in keeping with other studies that have found that the prevalence of leukocytospermia, when immunocytochemistry is used, is 10% 20% (11). The predominant leukocytes in semen from both healthy sperm donors and men with SCIs are granulocytes, followed by T cells. B cells are found only rarely. Samples from men with SCIs contain a larger proportion of CD45-positive cells than donor samples. Most cells in samples from donors were CD45-negative (77.1%) and assumed to be immature germ cells. Samples obtained from men with SCIs and coexisting UTIs contain more leukocytes; the number of granulocytes is increased markedly, but there also are significant increases in the number of T cells present. T cells can produce the cytokine interferon-, which has been reported to inhibit sperm motility (23). Men with SCIs usually are anejaculatory, and it has been suggested that the longer spermatozoa are stored in the epididymis, the more macrophages and granulocytes are attracted by aging semen, and thus the more leukocytes will be shed in the next ejaculation (11). However, we have shown herein that, in the absence of infection, men with SCIs do not have increased numbers of seminal leukocytes compared with healthy donors who are ejaculating regularly. Semen samples from men with SCIs with and without UTIs may give insight into the etiology of poor sperm motility. The suggestion that increased cellularity is a cause of the abnormal sperm function seen in these men is not supported by the results of this study; in uninfected men with SCIs, there is no increase in leukocyte populations yet there is still a deterioration in sperm quality. However, UTIs may be a contributing factor to the positive correlation between leukocyte concentrations and reactive oxygen species seen in men with SCIs (22). The cause of abnormal sperm function in men with SCIs is likely to be multifactorial; altered neurologic control to the testis and spermatogenesis may be another contributing factor that remains to be investigated. References 1. Bennett CJ, Seager SW, Vasher EA, McGuire EJ. Sexual dysfunction and electroejaculation in men with spinal cord injury: review. J Urol 1988;139: Buch JP, Zorn BH. Evaluation and treatment of infertility in spinal cord injured men through rectal probe electroejaculation. J Urol 1993;149: Ver Voort SM. Ejaculatory stimulation in spinal cord injured men. Urology 1987;19: Linsenmeyer TA, Perkash I. Infertility in men with spinal cord injury: review. Arch Phys Med Rehabil 1991;72: Bekerman H, Becher J, Lankhorst GJ. The effectiveness of vibratory stimulation in anejaculatory men with spinal cord injury. Paraplegia 1993;31: Denil J, Ohl DA, McGuire EJ, Jonas U. Treatment of anejaculation with electro ejaculation. Acta Urol Belg 1992;60: De Lamirande E, Leduc BE, Iwasaki A, Hassouna M, Gagnon C. Increased reactive oxygen species formation in semen of patients with spinal cord injury. Fertil Steril 1995;63: Chung PH, Yeko TR, Mayer JC, Sanford EJ, Maroulis GB. Assisted fertility using electroejaculation in men with spinal cord injury a review of literature. Fertil Steril 1995;64: Sedor JF, Hirsch IH. Evaluation of sperm morphology of electroejaculates of spinal cord injured men by strict criteria. Fertil Steril 1995;63: Tomlinson MJ, Barratt CL, Cooke ID. Prospective study of leukocytes and leukocyte subpopulations in semen suggests they are not a cause of male infertility. Fertil Steril 1993;60: Wolff H. The biologic significance of white blood cells in semen. Fertil Steril 1995;63: World Health Organization. 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Fertil Steril 1982;37: Wolff H, Politch JA, Martinez A, Haimovici F, Anderson DJ. Leukocytospermia is associated with poor sperm quality. Fertil Steril 1990; 53: De Geyter C, De Geyter M, Behre HM, Schneider HPG, Nieschlag E. Peroxidase positive round cells and microorganisms in human semen together with antibiotic treatment adversely influence the outcome of in-vitro fertilization and embryo-transfer. Int J Androl 1994;17: Aitken RJ, West K, Buckingham D. Leukocytic infiltration in the human ejaculate and its association with semen quality, oxidative stress, and sperm function. J Androl 1994;15: Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 1989; 40: Padron OF, Brackett NL, Sharma RK, Lynne CM, Thomas AJ, Agarwal A. Seminal reactive oxygen species and sperm motility and morphology in men with spinal cord injury. Fertil Steril 1997;67: Hill JA, Haimovici F, Politch JA, Anderson DJ. Effects of soluble products of activated lymphocytes and macrophages (lymphokines and monokines) on human sperm motion parameters. Fertil Steril 1987;47: FERTILITY & STERILITY 103