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Necrospermia and chronic spinal cord injury
FERTILITY AND STERILITY威 VOL. 74, NO. 2, AUGUST 2000 Copyright ©2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
MALE FACTOR
Necrospermia and chronic spinal cord injury Con Mallidis, Ph.D.,a,b,c Terence C. Lim, M.D.,d Steven T. Hill, M.D.,d Deborah J. Skinner, R.N.,d Douglas J. Brown, M.D.,d W. Ian H. Johnston, M.D.,b and H. W. Gordon Baker, M.D., Ph.D.a,b University of Melbourne and Reproductive Biology Unit, Royal Women’s Hospital and Melbourne IVF, Carlton, Victoria, Australia; University of Southern California School of Medicine, Los Angeles, California; and Victorian Spinal Cord Service, Austin and Repatriation Medical Centre, Melbourne, Victoria, Australia
Received November 1, 1999; accepted February 22, 2000. Reprint requests: H. W. Gordon Baker, M.D., Ph.D., Professorial Unit, Royal Women’s Hospital, Grattan Street, Carlton, Victoria 3053, Australia (FAX: 039347-1761; E-mail: g.baker @obgyn-rwh.unimelb.edu .au). a Department of Obstetrics and Gynecology, University of Melbourne. b Reproductive Biology Unit, Royal Women’s Hospital and Melbourne IVF. c University of Southern California School of Medicine. d Victorian Spinal Cord Service, Austin and Repatriation Medical Centre. 0015-0282/00/$20.00 PII S0015-0282(00)00650-6
Objective: To determine whether improvement in quality of semen over 4 consecutive days of electroejaculation in men with chronic spinal cord injury (SCI) was consistent with epididymal necrospermia. Design: Prospective study of a random sample of men with SCI. Setting: A southeastern Australian SCI management center in collaboration with the specialist andrology service of a university-based department of obstetrics and gynecology in a tertiary referral hospital. Patient(s): Nine men with chronic spinal cord injury. Intervention(s): Semen samples were obtained by using electroejaculation, and testicular biopsy samples were obtained by using fine-needle tissue aspiration. Main Outcome Measure(s): Semen analysis was performed according to World Health Organization criteria. Testicular biopsy and electron microscopy were done by using standard techniques. Result(s): During up to 4 days of consecutive-day electroejaculation, sperm motility and viability in semen obtained from men with chronic SCI increased by an average of 23% on days 2 and 3. The severity of the degenerative changes and the numbers of spermatozoa affected on day 1 became less marked by day 4. The changes were not present in late spermatids obtained from testicular biopsies. Conclusion(s): The asthenospermia of chronic SCI is similar to epididymal necrospermia and can be improved by consecutive-day electroejaculation. (Fertil Steril威 2000;74:221–7. ©2000 by American Society for Reproductive Medicine.) Key Words: Spinal cord injury, necrospermia, electroejaculation, epididymis, infertility
One of the major consequences of spinal cord injury (SCI) in male patients is infertility. Although reflex erections are common, ejaculation occurs rarely. It is estimated that less than 5% of men with chronic SCI can procreate without medical intervention (1). Studies in the past 40 years have shown that ejaculation can be stimulated by applying a vibrator to the ventral surface of the glans penis (vibration ejaculation) (2, 3) or an electrical current to the region of the prostate and seminal vesicles through a probe in the rectum (electroejaculation) (4, 5). These techniques have been widely used clinically; however, until the advent of intracytoplasmic sperm microinjection (ICSI), pregnancy rates were low despite improvements in equipment and the use of modern assisted reproduction techniques (6 –9).
The quality of semen obtained by assisted ejaculation from men with chronic SCI is almost always poor. It is characterized by small volumes, variable sperm concentrations, and, in particular, poor sperm motility (5, 6). This pattern of semen abnormality has been attributed to many factors, including stasis of accessory sex organ fluid, testicular hyperthermia, excessive reactive oxygen species generation, recurrent urinary tract infections, abnormal spermatogenesis, chronic medication, type of bladder management, changes in the hypothalamic-pituitary-testicular axis, sperm autoimmunity, general poor health, preexisting infertility, retrograde ejaculation, and urine contamination during assisted ejaculation (10 – 14). Although each of these factors could contribute to the overall deterioration in semen 221
quality, the mechanisms responsible for the semen defects in chronic SCI are poorly defined.
technique was explained to the patient and a consent form was completed.
The quality of semen produced by assisted ejaculation from men with chronic SCI is similar to that seen with epididymal necrospermia, a rare condition affecting fewer than 1 in 200 men seen for infertility without SCI (15). Characteristics of this condition are sperm motility ⬍20% and low sperm viability (assessed by dye exclusion) ⬍30%; however, these characteristics tend to improve with frequent ejaculation. The more commonly encountered forms of asthenospermia associated with defective spermatogenesis or specific cilial defects usually have sperm viability ⬎50%.
Patients
With necrospermia, electron microscopic examination of ejaculated spermatozoa shows severe degenerative changes. Most flagella cross-sections lack clearly defined axonemal microtubules, having been replaced by granular material. The outer dense fibers are poorly defined. The mitochondria contain granular material, and the membranes are not clear. Most sperm have lost the plasma membrane and acrosome. However, sperm in the lumen of seminiferous tubules and corpus epididymis appear normal, with clear normal axonemes and acrosomes (15). In the first description of epididymal necrospermia, frequent ejaculation (two ejaculates per day for 4 or 5 days) was shown to improve sperm motility, viability and the appearance of the sperm on electron microscopy, in particular the proportion of tail cross-sections with distinguishable microtubules and heads with acrosomes. On the basis of these findings, it was suggested that the basic defect is in epididymal sperm storage (15). Subsequently, several patients with this condition have been able to produce pregnancies by practicing frequent ejaculation. More recently, it was shown that the method and the type of specimen collected were factors in predicting the degree of necrospermia in men with SCI (16, 17). Vibration ejaculation was found to result in lower percentages of immotile sperm that were dead compared with electroejaculation. In addition, the total percentage of dead sperm was lower with use of vibration ejaculation. Antegrade ejaculation specimens of semen showed a higher percentage of live immotile sperm and a lower total percentage of dead sperm compared with retrograde ejaculation samples. Some patients with chronic SCI who have undergone repeated vibration ejaculation or electroejaculation have shown improvement in semen quality, although rarely to the levels found in healthy men (18). Thus we aimed to determine whether men with chronic SCI had a condition similar to epididymal necrospermia.
To be included in the study, patients had to be 16 – 40 years of age, have SCI of at least 6 months’ duration, and have no known or suspected testicular dysfunction (e.g., past undescended testes or infertility). The aim was to perform electroejaculation on 4 consecutive days for semen analysis and cryopreservation. We also performed electron microscopy of the sperm obtained on day 1 and day 4 and of spermatids obtained from testicular biopsy samples when possible.
Electroejaculation The technique used for electroejaculation has been described in detail elsewhere (12). Briefly, the instrument used was the CGS Electrojector (Ratek Industries P/L, Melbourne, Victoria, Australia), a rechargeable, battery powered, electrical stimulator that provides a progressively increasing sine-wave current at 20 Hz to a maximum of 16 V and 500 mA. The stimulator is fitted with a blunt-end Delrin probe that houses three anteriorly placed stainless steel longitudinal bar electrodes. A thermocouple in the middle electrode monitors temperature at the electrode–mucosal interface. The temperature is read on an LED screen built into the stimulator casing. To prevent retrograde ejaculation, a nontoxic all-silicone Foley catheter was inserted into the bladder. Lubricating gels were avoided because they are known to be toxic to sperm. Once the catheter was placed, the balloon was inflated with 10 mL of normal saline. Urine was drained into a collection bag. An assistant applied constant, gentle traction to the catheter to tamponade the bladder neck (12). After proctoscopy to detect preexisting lesions, the rectal probe was inserted. The stimulator output was progressively increased toward the maximum output or until emission occurred. The temperature was kept below 40°C. Antegrade emission occurred through the urethra and around the catheter. The semen was collected in a warm, sterile, plastic jar. The proctoscope was reintroduced to check the condition of the rectal mucosa and the catheter was removed.
Semen Analysis
MATERIALS AND METHODS
On liquefaction, the ejaculate volume was measured in a graduated tube. The presence of gelatinous bodies and mucus streaks and the color and consistency of the sample were noted. Sperm concentration was measured in a hemocytometer chamber using an appropriate dilution. The percentage of dead sperm was determined by using the eosin Y wet method, which is based on the principle that dead sperm with damaged plasma membranes take up stain (20).
The research and ethics committees of the Austin & Repatriation Medical Centre and Royal Women’s Hospital approved the study. Before the procedure was begun, the
The percentage of motile sperm was determined by microscopic examination (⫻400) of 7 L of semen placed on a warm (37°C) glass slide and covered with a glass coverslip
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(22 mm ⫻ 22 mm). At least 200 sperm were counted and graded according to World Health Organization criteria. Nonmotile sperm were assigned a score of zero. Motile sperm were graded as 1 (no forward progression), 2 (forward progression), or 3 (rapid, linear forward progression). Computer-assisted semen analysis was not used because the low motility and dense background debris make the results unreliable. Sperm morphology was assessed from smears stained by using the Shorr procedure, and 200 sperm were graded according to the previously described criteria (21). The statistical significance of changes in semen analysis results was determined by using paired t-tests.
Fine-Needle Tissue Aspiration Biopsy Fine-needle tissue aspiration biopsy was used to obtain testicular tissue specimens from 5 patients. In 2 patients, they were obtained ⬎1 month before the daily electroejaculation studies; in 3 patients, they were obtained on the last day of the electroejaculation studies (22). The skin of the scrotum was prepared by applying an aqueous solution of 0.1% chlorhexidine. The positions of the epididymis and vas deferens were determined, and if the testis was retroverted, care was taken to avoid puncturing them. The testis was held in one hand, and a 20-gauge modified Turner needle (Portland Surgical Products, Portland, Victoria, Australia) was passed through the skin of the scrotum and into the substance of the testis to a depth of approximately 0.5 cm. The stylet was removed and a 20-mL plastic syringe with the plunger at the 5-mL mark was attached to the hub of the needle. The plunger was withdrawn to the 10-mL mark, and as pressure was maintained, the needle was advanced further into the testis. A quick thrusting movement was used to cut into the substance of the testis. The needle was pushed in three times in different directions to maximize the quantity of tissue recovered. The needle was then slowly removed from the testis and scrotal skin back pressure on the syringe was maintained. The skin was examined at the point of puncture because tubules sometimes came out of the needle and remained in the needle track. If possible, this tissue was recovered with forceps. The needle was detached, and its contents were expelled into a plastic Petri dish containing heparinized minimum essential medium. The syringe was also washed out with the medium to recover tissue aspirated into the barrel. Biopsy was then repeated on the other side. The tissue obtained was cut in half; one section was processed for histologic assessment and the other was prepared for electron microscopy. For standard histology, samples were embedded in wax and sections 3 m in thickness were cut and stained with a modified hematoxylin-eosin schedule (22).
Electron Microscopy Samples were processed by using the method described by Wilton et al. (15). To assess changes in sperm ultrastructure, 0.5-mL aliquots of the day 1 and day 3 or 4 specimens FERTILITY & STERILITY威
were processed for electron microscopy. A total of 15 semen samples (9 first and 6 final ejaculates) from nine patients were obtained. Each was centrifuged at 300 ⫻ g for 10 minutes and the seminal plasma was removed. The pellet was resuspended in warm normal saline and recentrifuged; the supernatant was then removed and the pellet was fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. Ejaculates obtained from four infertile patients were processed in tandem with ejaculates from patients with chronic SCI to act as controls for possible artifacts resulting from the processing procedure. Testicular tissue was obtained from 5 patients. Each sample was fixed by placing it into 5.5% glutaraldehyde in 0.1M sodium cacodylate buffer. Each specimen was postfixed in 2% osmium tetroxide, washed in sodium cacodylate buffer, and dehydrated through a graded series of alcohols before embedding in Spurr’s resin (62% nonenyl succinic anhydride, 23% vinylcyclohexene dioxide, 14% DER 736, and 1% dimethylaminoethanol). Embedding consisted of immersing the specimens into 1:1 resin:acetone, then 100% resin. The resin was polymerized at 70°C for 12 hours. Survey sections 1 m in thickness were cut by using glass knives on a Reichert Ultracut (Reichart Electrotechnik, Heimsheim, Germany) ultra-microtome. These sections were placed onto glass slides, stained with 1% toluidine blue, and examined by using a light microscope. Sections 60 nm in thickness were then cut using a diamond knife (Diatome U.S., Fort Washington, PA), placed onto copper grids, stained with uranyl acetate and lead citrate, and examined by using the Philips EM400 transmission electron microscope (Philips, Nijmegen, The Netherlands). Both semen and tissue samples were examined, and the numbers of head and tail cross-sections with acrosomal caps and a clearly distinguishable microtubule pattern were counted.
RESULTS Semen Analyses
Nine men 22–36 (mean [⫾SD], 28 ⫾ 4) years of age underwent electroejaculation beginning 8 to 103 (54 ⫾ 40) months after injury (Table 1). Six patients had consecutive daily electroejaculation for 4 days, two for 3 days, and one for 1 day. Patient 1 had no ejaculate on day 2 and a low volume of semen with no sperm present on day 3. Patient 4 had semen suitable for storage on day 1 and was excluded from the rest of the study because he did not have necrospermia. Patient 8 had no sperm on day 3. Results of semen analysis are shown in Figure 1. On average, with successive ejaculations, the semen volume and sperm concentration decreased but the initially low sperm motility increased significantly on days 2 and 3 by 23% (Figure 1). In three of the seven patients, sperm motility remained low (⬍10%). Viability also increased on day 2. Sperm morphology was less affected (data not shown). Pa223
TABLE 1 Clinical details of nine men with chronic spinal cord injury.
Patients
SCI level
Frankel gradea
No. of months since injury
1 2 3 4 5 6 7 8 9
C5 C6 T4 T6 T8 T8 T10 T11 L2
B A A A A A A A B
8 20 11 103 83 19 82 103 61
No. of consecutive days with electroejaculation
Testicular biopsy result
3 4 4 1 4 4 4 3 4
N ND N/MD ND N N ND N-SL N
Note: N ⫽ normal; ND ⫽ not done; N/MD ⫽ normal one side, moderate hypospermatogenesis on the other side; N-SL ⫽ normal to slight hypospermatogenesis; SCI ⫽ spinal cord injury. a The Frankel grade is an index of the severity of SCI ranging from A, complete motor and sensory, and B, complete motor, incomplete sensory, to E, minimal long tract signs. Mallidis. Necrospermia and chronic SCI. Fertil Steril 2000.
tient 7 had initial rates of motility (24%) and live sperm (32%) that were slightly higher than usually seen with necrospermia. In this patient, previous motilities of sperm obtained by electroejaculation varied from 4% to 40%. However, marked improvements in motility (51%–55%) and viability (58%– 60%) were seen with repeated ejaculation, which is characteristic of necrospermia. In contrast, patient 9 had an initial live sperm percentage of 49% that decreased with repeated daily ejaculation; this patient also had little improvement in motility (1% on day 1 to 8% on day 2). Viability in other samples from this man ranged from 8% to 36%. Overall, the major gains in sperm motility and viability were achieved by day 2, although in three patients, improvement in motility and viability continued into day 3 of the consecutive-day semen collection process.
Testicular Histology Tubule wall thickness, tubule diameter, epithelial lining, and cell complement and proliferation were normal in three patients. One patient had slight hypospermatogenesis. In patient 5, one testis had normal spermatogenesis and the other had moderately severe hypospermatogenesis with few spermatogenic cells remaining and thickened tubule walls.
Electron Microscopy All nine day 1 samples showed marked degeneration of spermatozoa (Fig. 2). Sperm heads had either completely or partially disrupted acrosomal caps (Fig. 2A) and the midpieces and tails had poorly defined indistinct or fuzzy membranes, mitochondria, outer dense fibers, and axonemes (Fig. 2A, insert). The latter were also partially obscured by dense material. Samples after repeated ejaculation showed im224
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proved ultrastructure with fewer degenerative features (Fig. 2B). Although most sperm still had partially disrupted or detached acrosomal caps, absent plasma membranes, and mitochondrial changes, the microtubules were clearly distinguishable in some midpieces and flagellae (Fig. 2B, insert). Regardless of the state of spermatogenesis, all five biopsy samples contained ultrastructurally normal late spermatids with intact acrosomes and midpieces (Fig. 2C).
DISCUSSION Our findings show that a condition consistent with epididymal necrospermia occurs in men with chronic SCI. Sperm motility and viability are low but improve with frequent ejaculation. The ultrastructural characteristics of degenerate spermatozoa in the ejaculate without such characteristics in the late spermatids in the testis are similar to those described in epididymal necrospermia (15). With daily ejaculation, motility and morphology of sperm from men with chronic SCI improved but did not return to normal. This observation is in keeping with the findings in able-bodied men with necrospermia (15). The rapid decrease in sperm output with daily ejaculation also suggests that epididymal storage is impaired in men with chronic SCI. We postulate that necrospermia occurs in SCI because of defective temperature regulation and reproductive tract stasis. Brindley (23) showed that intrascrotal temperatures are elevated in men with chronic SCI. Bedford (24) claimed that elevated scrotal temperature has the greatest effect on cauda epididymal function. It has long been known that the scrotal contents are maintained below core body temperature and that increasing the temperature to or slightly above body temperature produces semen abnormalities. Placing the testes or epididymides in the abdomen of rats, rabbits, and hamsters produces major disturbances in epididymal function, and Bedford (25) noted parallel effects in humans. In animals exposed to scrotal heating, the size of the tail of the epididymis is decreased and the rate of transit of sperm through the epididymis is increased (25). In the rat, sperm in the tail of the epididymis are reduced in number, motility, and viability; frequent ejaculation improves these abnormalities. Of note, although the overall fertility of the cauda cryptepididymal sperm is reduced, there is a decrease in capacitation time, with earlier hyperactivation, acrosome reaction and fertilization in vitro. The epididymal changes are reversible if the epididymis is returned to the scrotum within a few weeks. These results have led to speculation that the generally poor semen quality, rapid epididymal transit, small sperm reserves, and short capacitation times of humans compared with those of domestic and laboratory animals may result from interference with testicular cooling caused by clothing and the common habit of sitting with their legs crossed (24, 25). Vol. 74, No. 2, August 2000
FIGURE 1 Changes in semen with daily electroejaculation in nine men with chronic spinal cord injury. Sperm motility increased significantly on days 2 and 3 (mean [⫾SE] difference from day 1 to day 2: 23% ⫾ 6.9%, paired t ⫽ 3.32, P⫽.016; mean difference from day 1 to day 3: 23.3% ⫾ 6.7%, paired t ⫽ 3.50, P⫽.017). Sperm viability also increased on day 2 (24.9% ⫾ 6.8%, paired t ⫽ 3.63, P⫽.011).
Mallidis. Necrospermia and chronic SCI. Fertil Steril 2000.
Men with chronic SCI sit in wheelchairs and have scrotal temperatures about 1°C higher than those of clothed, sitting, able-bodied men (23). Men with SCI who have higher scrotal temperatures are less likely to have motile sperm in electroejaculation specimens (23). This finding was not confirmed by Brackett et al. (26); however, these investigators measured scrotal temperatures in men with SCI who were seated on an examination table with their trousers removed and after a 15-minute adaptation period, not while the men were sitting in their wheelchairs. Instead of or in addition to defective temperature regulation with chronic SCI, the contents of the vas may fail to clear completely because of neurologic dysfunction. Hence, the vas FERTILITY & STERILITY威
and perhaps the seminal vesicles may become packed with sperm, secretions, and cells involved in removal of the aging sperm. This effect would be somewhat akin to the finding of degenerating sperm in the epididymal tubules most distal from the testis in congenital agenesis of the vas. The semen containing the degenerating sperm and its products would be collected with the first assisted ejaculation procedure. Subsequent frequent ejaculation would clear the vasa, caudae epididymides, and seminal vesicles of accumulated degenerating sperm, allowing younger sperm to later appear in the ejaculate. This possibility is supported by reports that highly motile sperm can be obtained by aspirating or flushing the proximal end of the vas in men with chronic SCI (27, 28). 225
FIGURE 2 Transmission electron micrographs of spermatozoa from a man with chronic spinal cord injury who underwent daily electroejaculation for 4 days (overall magnification, ⫻20,000; magnification of inserts, ⫻40,000). (A), On day 1, the acrosome is missing and the midpiece structures (inset) are indistinct. (B), On day 4, the acrosome is better preserved and the midpiece (inset) has distinct microtubules and outer dense fibers, but the mitochondria and plasma membrane are still abnormal. (C), In the testis, the acrosomes and midpiece (inset) of late spermatids are normal.
Mallidis. Necrospermia and chronic SCI. Fertil Steril 2000.
Although our findings indicate that necrospermia is common in men with chronic SCI, the problem is clearly more complex, with many other mechanisms also operating to impair sperm function. Large numbers of polymorphonuclear leukocytes are almost always present in the ejaculate of these men. The leukocytes may be present because of infections in the accessory sex glands associated with catheterization or urinary infections. Leukocytes may further compromise sperm function by various mechanisms (29). High reactive oxygen species generation by semen and progressive gradient centrifugation from men with chronic SCI have been reported (14). However, this cannot be the only mechanism of sperm damage, as it has been shown that leukocyte counts in semen from men with SCI did not correlate with reduced sperm motility (30). Patient 7 had variable sperm motility in samples obtained by electroejaculation. Although the initial motility and viability of sperm were higher than usual in this patient, repeated ejaculation produced marked improvement in semen quality. Able-bodied men with epididymal necrospermia may also have variable semen analysis results. Sperm motility may be reduced for other reasons. For example, patient 9 had low motility but near-normal viability that decreased with repeated ejaculation, suggesting that he did not have necrospermia, although electron microscopy of the initial 226
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semen sample contained degenerate sperm suggestive of the condition. Of note, this man had low (below L2) and incomplete paraplegia. In addition, spermatogenesis can be reduced in some men with chronic SCI. Although none of the patients in our study had severe hypospermatogenesis in both testes, we have tried the repeated ejaculation procedure in other men with both SCI and severe spermatogenic disorders without success. There are also patients in whom it is difficult to obtain semen by using assisted ejaculation despite normal results of testicular biopsy. Finally, some men with SCI, such as patient 4 in our study, have reasonably good semen quality. It is possible that this particular patient had spontaneous ejaculation or emission unbeknownst to him; he was thus excluded from the study. The deterioration in genital tract function leading to reduced semen quality appears rapidly after SCI. We have been able to collect normal semen for cryopreservation in the acute phase of SCI once patients have recovered from spinal shock. However, the quality of the semen deteriorated after about 10 days, despite application of electroejaculation three times per week (19). This knowledge can be used to optimize the clinical management of fertility in men with SCI. Where possible, Vol. 74, No. 2, August 2000
normal semen should be collected and cryopreserved in the first 2 weeks after the injury. This should enable pregnancies to be produced by artificial insemination and avoid the need for more expensive interventions, such as the use of assisted reproductive technology. For men with SCI who require assisted ejaculation, part of the fertility work-up could include an approach similar to that described in our paper— electroejaculation or vibration ejaculation performed on 3 to 4 consecutive days. We already know that semen quality can be improved with repeated assisted ejaculation (7, 18). Our findings indicate that the quality of semen, in particular sperm motility, is improved on the second and third days of repeated assisted ejaculation, which allows the possibility of semen cryopreservation. Once this pattern of improvement in semen quality is known, these findings can be reproduced at a later date and be timed for the provision of fresh samples for artificial insemination or IVF. The previously cryopreserved semen can be considered as back-up in case of failure to obtain the expected semen sample. Men with chronic SCI who can produce semen by safe vibration ejaculation might be advised to perform regular ejaculation 2 to 3 times per week, with the aim of maintaining semen quality so that home insemination can be performed. Evidence indicates that semen quality is better when vibration ejaculation rather than electroejaculation is used (16). In summary, we report a practical approach to improving the quality of semen in men with chronic SCI. Use of electroejaculation on consecutive days can improve the quality of semen. However, the improvement in quality was not always good enough for artificial insemination. Thus, ICSI with sperm aspirated from the genital tract is a major advance in the treatment of infertility in men with chronic SCI, in which other methods of obtaining sperm have failed. The results raise further questions about the causes of poor quality of semen after SCI. Semen quality in men with SCI is similar to that seen in able-bodied men with epididymal necrospermia. Perhaps SCI can be viewed as an experiment in nature that allows clinical research to better elucidate the mechanisms of necrospermia in able-bodied persons. References 1. Talbot HS. The sexual function in paraplegia. J Urol 1955;77:91–100. 2. Bors E, Comarr AE. Neurological disturbances of sexual function with special reference to 529 patients with spinal cord injury. Urol Surv 1960;10:191–222. 3. Brindley GS. Reflex ejaculation under vibratory stimulation in paraplegic men. Paraplegia 1981;19:299 –302. 4. Brindley GS. Electro-ejaculation: its technique, neurological implication and uses. J Neurol Neurosurg Psychiatry. 1981;44:9 –18.
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5. Brindley GS. The fertility of men with spinal injuries. Paraplegia 1984;22:337– 48. 6. Halstead LS, VerVoort SM, Seager SW. Rectal probe electrostimulation in the treatment of anejaculatory spinal cord injured men. Paraplegia 1987;25:120 –9. 7. Ohl DA, Bennett CJ, McCabe M, Menge AC, McGuire EJ. Predictors of success in electroejaculation of spinal cord injured men. J Urol 1989;142:1483– 6. 8. Bennett CJ, Seager SW, Vasher EA, McGuire EJ. Sexual dysfunction and electroejaculation in men with spinal cord injury: review. J Urol 1988;139:453–547. 9. Perkash I, Martin DE, Warner H Speck V. Electroejaculation in spinal cord injured patients: simplified new equipment and technique. J Urol 1990;143:305–7. 10. VerVoort SM. Infertility in spinal-cord injured male. Urology 1987;29: 157– 68. 11. Hirsch IH, Sedor J, Callahan HJ, Staas WE. Antisperm antibodies in seminal plasma of spinal cord-injured men. Urology 1992;39:243–7. 12. Lim TC, Mallidis C, Hill ST, Skinner DJ, Carter PD, Brown DJ, Baker HWG. A simple technique to prevent retrograde ejaculation during assisted ejaculation. Paraplegia 1994;32:142–9. 13. Linsenmeyer TA. Male infertility following spinal cord injury. J Am Paraplegia Soc 1991;14:116 –21. 14. De Lamirande E, Hassouna M, Leduc BE, Gagnon C, Iwasaki A. Increased reactive oxygen species formation in semen of patients with spinal cord injury. Fertil Steril 1995;53:637– 42. 15. Wilton LJ, Temple-Smith PD, Baker HWG, de Kretser DM. Human male infertility caused by degeneration and death of sperm in the epididymis. Fertil Steril 1988;49:1052– 8. 16. Brackett NL, Padron OF, Lynne CM. Semen quality of spinal cord injured men is better when obtained by vibratory stimulation versus electroejaculation. J Urol 1997;157:151–7. 17. Brackett NL, Bloch WE, Lynne CM. Predictors of necrospermia in men with spinal cord injury. J Urol 1998;159:844 –7. 18. Siosteen A, Frossman L, Steen Y, Sullivan L. Whickstrom I. Quality of semen after repeated ejaculation treatment in spinal cord injury men. Paraplegia 1990;28:96 –104. 19. Mallidis C, Lim TC, Hill ST, Skinner DJ, Brown DJ, Johnston WIH, et al. Collection of semen from men in the acute phase of spinal cord injury. Lancet 1994;343:1072–3. 20. World Health Organization. WHO manual for the examination of human semen and semen-cervical mucus interaction. Cambridge, UK: Cambridge University Press, 1992. 21. Liu DY, Baker HWG. Tests of sperm function in vitro. Fertil Steril 1992;58:465– 83. 22. Mallidis C, Baker HWG. Fine needle tissue aspiration biopsy of the testis. Fertil Steril 1994;61:367–75. 23. Brindley GS. Deep scrotal temperature and the effect on it of clothing, air temperature, activity, posture and paraplegia. Br J Urol 1982;54: 49 –55. 24. Bedford JM. Effects of elevated temperature on the epididymis and testis: experimental studies. In: Zorgniotti AW, ed. Temperature and environmental effects on the testis. New York: Plenum Press, 1991;19 –32. 25. Bedford JM. The status and the state of the human epididymis. Hum Reprod 1994;11:2187–99. 26. Brackett NL, Lynne CM, Weizman MS, Bloch WE, Padron OF. Scrotal and oral temperatures are not related to semen quality or serum gonadotropin levels in spinal cord-injured men. J Androl 1994;15:614 –19. 27. Hovatta O, von Smitten K. Sperm aspiration from vas deferens and in-vitro fertilization in cases of non-treatable anejaculation. Hum Reprod 1993;8:1689 –91. 28. Buch JP. Greatly improved sperm motility from vas deferens sperm retrieval: a case for accessory gland related subfertility in spinal cord injured men. Case report. Paraplegia 1994;32:501– 4. 29. Aitken RJ, Baker HWG. Seminal leucocytes: messengers, terrorists, or good Samaritans? Hum Reprod 1995;10:1736 –9. 30. Aird IA, Vince GS, Bates MD, Johnson PM, Lewis-Jones ID. Leukocytes in semen from men with spinal cord injuries. Fertil Steril 1999;72: 97–103.
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Necrospermia and chronic spinal cord injury Con Mallidis, Ph.D.,a,b,c Terence C. Lim, M.D.,d Steven T. Hill, M.D.,d Deborah J. Skinner, R.N.,d Douglas J. Brown, M.D.,d W. Ian H. Johnston, M.D.,b and H. W. Gordon Baker, M.D., Ph.D.a,b University of Melbourne and Reproductive Biology Unit, Royal Women’s Hospital and Melbourne IVF, Carlton, Victoria, Australia; University of Southern California School of Medicine, Los Angeles, California; and Victorian Spinal Cord Service, Austin and Repatriation Medical Centre, Melbourne, Victoria, Australia
Received November 1, 1999; accepted February 22, 2000. Reprint requests: H. W. Gordon Baker, M.D., Ph.D., Professorial Unit, Royal Women’s Hospital, Grattan Street, Carlton, Victoria 3053, Australia (FAX: 039347-1761; E-mail: g.baker @obgyn-rwh.unimelb.edu .au). a Department of Obstetrics and Gynecology, University of Melbourne. b Reproductive Biology Unit, Royal Women’s Hospital and Melbourne IVF. c University of Southern California School of Medicine. d Victorian Spinal Cord Service, Austin and Repatriation Medical Centre. 0015-0282/00/$20.00 PII S0015-0282(00)00650-6
Objective: To determine whether improvement in quality of semen over 4 consecutive days of electroejaculation in men with chronic spinal cord injury (SCI) was consistent with epididymal necrospermia. Design: Prospective study of a random sample of men with SCI. Setting: A southeastern Australian SCI management center in collaboration with the specialist andrology service of a university-based department of obstetrics and gynecology in a tertiary referral hospital. Patient(s): Nine men with chronic spinal cord injury. Intervention(s): Semen samples were obtained by using electroejaculation, and testicular biopsy samples were obtained by using fine-needle tissue aspiration. Main Outcome Measure(s): Semen analysis was performed according to World Health Organization criteria. Testicular biopsy and electron microscopy were done by using standard techniques. Result(s): During up to 4 days of consecutive-day electroejaculation, sperm motility and viability in semen obtained from men with chronic SCI increased by an average of 23% on days 2 and 3. The severity of the degenerative changes and the numbers of spermatozoa affected on day 1 became less marked by day 4. The changes were not present in late spermatids obtained from testicular biopsies. Conclusion(s): The asthenospermia of chronic SCI is similar to epididymal necrospermia and can be improved by consecutive-day electroejaculation. (Fertil Steril威 2000;74:221–7. ©2000 by American Society for Reproductive Medicine.) Key Words: Spinal cord injury, necrospermia, electroejaculation, epididymis, infertility
One of the major consequences of spinal cord injury (SCI) in male patients is infertility. Although reflex erections are common, ejaculation occurs rarely. It is estimated that less than 5% of men with chronic SCI can procreate without medical intervention (1). Studies in the past 40 years have shown that ejaculation can be stimulated by applying a vibrator to the ventral surface of the glans penis (vibration ejaculation) (2, 3) or an electrical current to the region of the prostate and seminal vesicles through a probe in the rectum (electroejaculation) (4, 5). These techniques have been widely used clinically; however, until the advent of intracytoplasmic sperm microinjection (ICSI), pregnancy rates were low despite improvements in equipment and the use of modern assisted reproduction techniques (6 –9).
The quality of semen obtained by assisted ejaculation from men with chronic SCI is almost always poor. It is characterized by small volumes, variable sperm concentrations, and, in particular, poor sperm motility (5, 6). This pattern of semen abnormality has been attributed to many factors, including stasis of accessory sex organ fluid, testicular hyperthermia, excessive reactive oxygen species generation, recurrent urinary tract infections, abnormal spermatogenesis, chronic medication, type of bladder management, changes in the hypothalamic-pituitary-testicular axis, sperm autoimmunity, general poor health, preexisting infertility, retrograde ejaculation, and urine contamination during assisted ejaculation (10 – 14). Although each of these factors could contribute to the overall deterioration in semen 221
quality, the mechanisms responsible for the semen defects in chronic SCI are poorly defined.
technique was explained to the patient and a consent form was completed.
The quality of semen produced by assisted ejaculation from men with chronic SCI is similar to that seen with epididymal necrospermia, a rare condition affecting fewer than 1 in 200 men seen for infertility without SCI (15). Characteristics of this condition are sperm motility ⬍20% and low sperm viability (assessed by dye exclusion) ⬍30%; however, these characteristics tend to improve with frequent ejaculation. The more commonly encountered forms of asthenospermia associated with defective spermatogenesis or specific cilial defects usually have sperm viability ⬎50%.
Patients
With necrospermia, electron microscopic examination of ejaculated spermatozoa shows severe degenerative changes. Most flagella cross-sections lack clearly defined axonemal microtubules, having been replaced by granular material. The outer dense fibers are poorly defined. The mitochondria contain granular material, and the membranes are not clear. Most sperm have lost the plasma membrane and acrosome. However, sperm in the lumen of seminiferous tubules and corpus epididymis appear normal, with clear normal axonemes and acrosomes (15). In the first description of epididymal necrospermia, frequent ejaculation (two ejaculates per day for 4 or 5 days) was shown to improve sperm motility, viability and the appearance of the sperm on electron microscopy, in particular the proportion of tail cross-sections with distinguishable microtubules and heads with acrosomes. On the basis of these findings, it was suggested that the basic defect is in epididymal sperm storage (15). Subsequently, several patients with this condition have been able to produce pregnancies by practicing frequent ejaculation. More recently, it was shown that the method and the type of specimen collected were factors in predicting the degree of necrospermia in men with SCI (16, 17). Vibration ejaculation was found to result in lower percentages of immotile sperm that were dead compared with electroejaculation. In addition, the total percentage of dead sperm was lower with use of vibration ejaculation. Antegrade ejaculation specimens of semen showed a higher percentage of live immotile sperm and a lower total percentage of dead sperm compared with retrograde ejaculation samples. Some patients with chronic SCI who have undergone repeated vibration ejaculation or electroejaculation have shown improvement in semen quality, although rarely to the levels found in healthy men (18). Thus we aimed to determine whether men with chronic SCI had a condition similar to epididymal necrospermia.
To be included in the study, patients had to be 16 – 40 years of age, have SCI of at least 6 months’ duration, and have no known or suspected testicular dysfunction (e.g., past undescended testes or infertility). The aim was to perform electroejaculation on 4 consecutive days for semen analysis and cryopreservation. We also performed electron microscopy of the sperm obtained on day 1 and day 4 and of spermatids obtained from testicular biopsy samples when possible.
Electroejaculation The technique used for electroejaculation has been described in detail elsewhere (12). Briefly, the instrument used was the CGS Electrojector (Ratek Industries P/L, Melbourne, Victoria, Australia), a rechargeable, battery powered, electrical stimulator that provides a progressively increasing sine-wave current at 20 Hz to a maximum of 16 V and 500 mA. The stimulator is fitted with a blunt-end Delrin probe that houses three anteriorly placed stainless steel longitudinal bar electrodes. A thermocouple in the middle electrode monitors temperature at the electrode–mucosal interface. The temperature is read on an LED screen built into the stimulator casing. To prevent retrograde ejaculation, a nontoxic all-silicone Foley catheter was inserted into the bladder. Lubricating gels were avoided because they are known to be toxic to sperm. Once the catheter was placed, the balloon was inflated with 10 mL of normal saline. Urine was drained into a collection bag. An assistant applied constant, gentle traction to the catheter to tamponade the bladder neck (12). After proctoscopy to detect preexisting lesions, the rectal probe was inserted. The stimulator output was progressively increased toward the maximum output or until emission occurred. The temperature was kept below 40°C. Antegrade emission occurred through the urethra and around the catheter. The semen was collected in a warm, sterile, plastic jar. The proctoscope was reintroduced to check the condition of the rectal mucosa and the catheter was removed.
Semen Analysis
MATERIALS AND METHODS
On liquefaction, the ejaculate volume was measured in a graduated tube. The presence of gelatinous bodies and mucus streaks and the color and consistency of the sample were noted. Sperm concentration was measured in a hemocytometer chamber using an appropriate dilution. The percentage of dead sperm was determined by using the eosin Y wet method, which is based on the principle that dead sperm with damaged plasma membranes take up stain (20).
The research and ethics committees of the Austin & Repatriation Medical Centre and Royal Women’s Hospital approved the study. Before the procedure was begun, the
The percentage of motile sperm was determined by microscopic examination (⫻400) of 7 L of semen placed on a warm (37°C) glass slide and covered with a glass coverslip
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(22 mm ⫻ 22 mm). At least 200 sperm were counted and graded according to World Health Organization criteria. Nonmotile sperm were assigned a score of zero. Motile sperm were graded as 1 (no forward progression), 2 (forward progression), or 3 (rapid, linear forward progression). Computer-assisted semen analysis was not used because the low motility and dense background debris make the results unreliable. Sperm morphology was assessed from smears stained by using the Shorr procedure, and 200 sperm were graded according to the previously described criteria (21). The statistical significance of changes in semen analysis results was determined by using paired t-tests.
Fine-Needle Tissue Aspiration Biopsy Fine-needle tissue aspiration biopsy was used to obtain testicular tissue specimens from 5 patients. In 2 patients, they were obtained ⬎1 month before the daily electroejaculation studies; in 3 patients, they were obtained on the last day of the electroejaculation studies (22). The skin of the scrotum was prepared by applying an aqueous solution of 0.1% chlorhexidine. The positions of the epididymis and vas deferens were determined, and if the testis was retroverted, care was taken to avoid puncturing them. The testis was held in one hand, and a 20-gauge modified Turner needle (Portland Surgical Products, Portland, Victoria, Australia) was passed through the skin of the scrotum and into the substance of the testis to a depth of approximately 0.5 cm. The stylet was removed and a 20-mL plastic syringe with the plunger at the 5-mL mark was attached to the hub of the needle. The plunger was withdrawn to the 10-mL mark, and as pressure was maintained, the needle was advanced further into the testis. A quick thrusting movement was used to cut into the substance of the testis. The needle was pushed in three times in different directions to maximize the quantity of tissue recovered. The needle was then slowly removed from the testis and scrotal skin back pressure on the syringe was maintained. The skin was examined at the point of puncture because tubules sometimes came out of the needle and remained in the needle track. If possible, this tissue was recovered with forceps. The needle was detached, and its contents were expelled into a plastic Petri dish containing heparinized minimum essential medium. The syringe was also washed out with the medium to recover tissue aspirated into the barrel. Biopsy was then repeated on the other side. The tissue obtained was cut in half; one section was processed for histologic assessment and the other was prepared for electron microscopy. For standard histology, samples were embedded in wax and sections 3 m in thickness were cut and stained with a modified hematoxylin-eosin schedule (22).
Electron Microscopy Samples were processed by using the method described by Wilton et al. (15). To assess changes in sperm ultrastructure, 0.5-mL aliquots of the day 1 and day 3 or 4 specimens FERTILITY & STERILITY威
were processed for electron microscopy. A total of 15 semen samples (9 first and 6 final ejaculates) from nine patients were obtained. Each was centrifuged at 300 ⫻ g for 10 minutes and the seminal plasma was removed. The pellet was resuspended in warm normal saline and recentrifuged; the supernatant was then removed and the pellet was fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. Ejaculates obtained from four infertile patients were processed in tandem with ejaculates from patients with chronic SCI to act as controls for possible artifacts resulting from the processing procedure. Testicular tissue was obtained from 5 patients. Each sample was fixed by placing it into 5.5% glutaraldehyde in 0.1M sodium cacodylate buffer. Each specimen was postfixed in 2% osmium tetroxide, washed in sodium cacodylate buffer, and dehydrated through a graded series of alcohols before embedding in Spurr’s resin (62% nonenyl succinic anhydride, 23% vinylcyclohexene dioxide, 14% DER 736, and 1% dimethylaminoethanol). Embedding consisted of immersing the specimens into 1:1 resin:acetone, then 100% resin. The resin was polymerized at 70°C for 12 hours. Survey sections 1 m in thickness were cut by using glass knives on a Reichert Ultracut (Reichart Electrotechnik, Heimsheim, Germany) ultra-microtome. These sections were placed onto glass slides, stained with 1% toluidine blue, and examined by using a light microscope. Sections 60 nm in thickness were then cut using a diamond knife (Diatome U.S., Fort Washington, PA), placed onto copper grids, stained with uranyl acetate and lead citrate, and examined by using the Philips EM400 transmission electron microscope (Philips, Nijmegen, The Netherlands). Both semen and tissue samples were examined, and the numbers of head and tail cross-sections with acrosomal caps and a clearly distinguishable microtubule pattern were counted.
RESULTS Semen Analyses
Nine men 22–36 (mean [⫾SD], 28 ⫾ 4) years of age underwent electroejaculation beginning 8 to 103 (54 ⫾ 40) months after injury (Table 1). Six patients had consecutive daily electroejaculation for 4 days, two for 3 days, and one for 1 day. Patient 1 had no ejaculate on day 2 and a low volume of semen with no sperm present on day 3. Patient 4 had semen suitable for storage on day 1 and was excluded from the rest of the study because he did not have necrospermia. Patient 8 had no sperm on day 3. Results of semen analysis are shown in Figure 1. On average, with successive ejaculations, the semen volume and sperm concentration decreased but the initially low sperm motility increased significantly on days 2 and 3 by 23% (Figure 1). In three of the seven patients, sperm motility remained low (⬍10%). Viability also increased on day 2. Sperm morphology was less affected (data not shown). Pa223
TABLE 1 Clinical details of nine men with chronic spinal cord injury.
Patients
SCI level
Frankel gradea
No. of months since injury
1 2 3 4 5 6 7 8 9
C5 C6 T4 T6 T8 T8 T10 T11 L2
B A A A A A A A B
8 20 11 103 83 19 82 103 61
No. of consecutive days with electroejaculation
Testicular biopsy result
3 4 4 1 4 4 4 3 4
N ND N/MD ND N N ND N-SL N
Note: N ⫽ normal; ND ⫽ not done; N/MD ⫽ normal one side, moderate hypospermatogenesis on the other side; N-SL ⫽ normal to slight hypospermatogenesis; SCI ⫽ spinal cord injury. a The Frankel grade is an index of the severity of SCI ranging from A, complete motor and sensory, and B, complete motor, incomplete sensory, to E, minimal long tract signs. Mallidis. Necrospermia and chronic SCI. Fertil Steril 2000.
tient 7 had initial rates of motility (24%) and live sperm (32%) that were slightly higher than usually seen with necrospermia. In this patient, previous motilities of sperm obtained by electroejaculation varied from 4% to 40%. However, marked improvements in motility (51%–55%) and viability (58%– 60%) were seen with repeated ejaculation, which is characteristic of necrospermia. In contrast, patient 9 had an initial live sperm percentage of 49% that decreased with repeated daily ejaculation; this patient also had little improvement in motility (1% on day 1 to 8% on day 2). Viability in other samples from this man ranged from 8% to 36%. Overall, the major gains in sperm motility and viability were achieved by day 2, although in three patients, improvement in motility and viability continued into day 3 of the consecutive-day semen collection process.
Testicular Histology Tubule wall thickness, tubule diameter, epithelial lining, and cell complement and proliferation were normal in three patients. One patient had slight hypospermatogenesis. In patient 5, one testis had normal spermatogenesis and the other had moderately severe hypospermatogenesis with few spermatogenic cells remaining and thickened tubule walls.
Electron Microscopy All nine day 1 samples showed marked degeneration of spermatozoa (Fig. 2). Sperm heads had either completely or partially disrupted acrosomal caps (Fig. 2A) and the midpieces and tails had poorly defined indistinct or fuzzy membranes, mitochondria, outer dense fibers, and axonemes (Fig. 2A, insert). The latter were also partially obscured by dense material. Samples after repeated ejaculation showed im224
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proved ultrastructure with fewer degenerative features (Fig. 2B). Although most sperm still had partially disrupted or detached acrosomal caps, absent plasma membranes, and mitochondrial changes, the microtubules were clearly distinguishable in some midpieces and flagellae (Fig. 2B, insert). Regardless of the state of spermatogenesis, all five biopsy samples contained ultrastructurally normal late spermatids with intact acrosomes and midpieces (Fig. 2C).
DISCUSSION Our findings show that a condition consistent with epididymal necrospermia occurs in men with chronic SCI. Sperm motility and viability are low but improve with frequent ejaculation. The ultrastructural characteristics of degenerate spermatozoa in the ejaculate without such characteristics in the late spermatids in the testis are similar to those described in epididymal necrospermia (15). With daily ejaculation, motility and morphology of sperm from men with chronic SCI improved but did not return to normal. This observation is in keeping with the findings in able-bodied men with necrospermia (15). The rapid decrease in sperm output with daily ejaculation also suggests that epididymal storage is impaired in men with chronic SCI. We postulate that necrospermia occurs in SCI because of defective temperature regulation and reproductive tract stasis. Brindley (23) showed that intrascrotal temperatures are elevated in men with chronic SCI. Bedford (24) claimed that elevated scrotal temperature has the greatest effect on cauda epididymal function. It has long been known that the scrotal contents are maintained below core body temperature and that increasing the temperature to or slightly above body temperature produces semen abnormalities. Placing the testes or epididymides in the abdomen of rats, rabbits, and hamsters produces major disturbances in epididymal function, and Bedford (25) noted parallel effects in humans. In animals exposed to scrotal heating, the size of the tail of the epididymis is decreased and the rate of transit of sperm through the epididymis is increased (25). In the rat, sperm in the tail of the epididymis are reduced in number, motility, and viability; frequent ejaculation improves these abnormalities. Of note, although the overall fertility of the cauda cryptepididymal sperm is reduced, there is a decrease in capacitation time, with earlier hyperactivation, acrosome reaction and fertilization in vitro. The epididymal changes are reversible if the epididymis is returned to the scrotum within a few weeks. These results have led to speculation that the generally poor semen quality, rapid epididymal transit, small sperm reserves, and short capacitation times of humans compared with those of domestic and laboratory animals may result from interference with testicular cooling caused by clothing and the common habit of sitting with their legs crossed (24, 25). Vol. 74, No. 2, August 2000
FIGURE 1 Changes in semen with daily electroejaculation in nine men with chronic spinal cord injury. Sperm motility increased significantly on days 2 and 3 (mean [⫾SE] difference from day 1 to day 2: 23% ⫾ 6.9%, paired t ⫽ 3.32, P⫽.016; mean difference from day 1 to day 3: 23.3% ⫾ 6.7%, paired t ⫽ 3.50, P⫽.017). Sperm viability also increased on day 2 (24.9% ⫾ 6.8%, paired t ⫽ 3.63, P⫽.011).
Mallidis. Necrospermia and chronic SCI. Fertil Steril 2000.
Men with chronic SCI sit in wheelchairs and have scrotal temperatures about 1°C higher than those of clothed, sitting, able-bodied men (23). Men with SCI who have higher scrotal temperatures are less likely to have motile sperm in electroejaculation specimens (23). This finding was not confirmed by Brackett et al. (26); however, these investigators measured scrotal temperatures in men with SCI who were seated on an examination table with their trousers removed and after a 15-minute adaptation period, not while the men were sitting in their wheelchairs. Instead of or in addition to defective temperature regulation with chronic SCI, the contents of the vas may fail to clear completely because of neurologic dysfunction. Hence, the vas FERTILITY & STERILITY威
and perhaps the seminal vesicles may become packed with sperm, secretions, and cells involved in removal of the aging sperm. This effect would be somewhat akin to the finding of degenerating sperm in the epididymal tubules most distal from the testis in congenital agenesis of the vas. The semen containing the degenerating sperm and its products would be collected with the first assisted ejaculation procedure. Subsequent frequent ejaculation would clear the vasa, caudae epididymides, and seminal vesicles of accumulated degenerating sperm, allowing younger sperm to later appear in the ejaculate. This possibility is supported by reports that highly motile sperm can be obtained by aspirating or flushing the proximal end of the vas in men with chronic SCI (27, 28). 225
FIGURE 2 Transmission electron micrographs of spermatozoa from a man with chronic spinal cord injury who underwent daily electroejaculation for 4 days (overall magnification, ⫻20,000; magnification of inserts, ⫻40,000). (A), On day 1, the acrosome is missing and the midpiece structures (inset) are indistinct. (B), On day 4, the acrosome is better preserved and the midpiece (inset) has distinct microtubules and outer dense fibers, but the mitochondria and plasma membrane are still abnormal. (C), In the testis, the acrosomes and midpiece (inset) of late spermatids are normal.
Mallidis. Necrospermia and chronic SCI. Fertil Steril 2000.
Although our findings indicate that necrospermia is common in men with chronic SCI, the problem is clearly more complex, with many other mechanisms also operating to impair sperm function. Large numbers of polymorphonuclear leukocytes are almost always present in the ejaculate of these men. The leukocytes may be present because of infections in the accessory sex glands associated with catheterization or urinary infections. Leukocytes may further compromise sperm function by various mechanisms (29). High reactive oxygen species generation by semen and progressive gradient centrifugation from men with chronic SCI have been reported (14). However, this cannot be the only mechanism of sperm damage, as it has been shown that leukocyte counts in semen from men with SCI did not correlate with reduced sperm motility (30). Patient 7 had variable sperm motility in samples obtained by electroejaculation. Although the initial motility and viability of sperm were higher than usual in this patient, repeated ejaculation produced marked improvement in semen quality. Able-bodied men with epididymal necrospermia may also have variable semen analysis results. Sperm motility may be reduced for other reasons. For example, patient 9 had low motility but near-normal viability that decreased with repeated ejaculation, suggesting that he did not have necrospermia, although electron microscopy of the initial 226
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semen sample contained degenerate sperm suggestive of the condition. Of note, this man had low (below L2) and incomplete paraplegia. In addition, spermatogenesis can be reduced in some men with chronic SCI. Although none of the patients in our study had severe hypospermatogenesis in both testes, we have tried the repeated ejaculation procedure in other men with both SCI and severe spermatogenic disorders without success. There are also patients in whom it is difficult to obtain semen by using assisted ejaculation despite normal results of testicular biopsy. Finally, some men with SCI, such as patient 4 in our study, have reasonably good semen quality. It is possible that this particular patient had spontaneous ejaculation or emission unbeknownst to him; he was thus excluded from the study. The deterioration in genital tract function leading to reduced semen quality appears rapidly after SCI. We have been able to collect normal semen for cryopreservation in the acute phase of SCI once patients have recovered from spinal shock. However, the quality of the semen deteriorated after about 10 days, despite application of electroejaculation three times per week (19). This knowledge can be used to optimize the clinical management of fertility in men with SCI. Where possible, Vol. 74, No. 2, August 2000
normal semen should be collected and cryopreserved in the first 2 weeks after the injury. This should enable pregnancies to be produced by artificial insemination and avoid the need for more expensive interventions, such as the use of assisted reproductive technology. For men with SCI who require assisted ejaculation, part of the fertility work-up could include an approach similar to that described in our paper— electroejaculation or vibration ejaculation performed on 3 to 4 consecutive days. We already know that semen quality can be improved with repeated assisted ejaculation (7, 18). Our findings indicate that the quality of semen, in particular sperm motility, is improved on the second and third days of repeated assisted ejaculation, which allows the possibility of semen cryopreservation. Once this pattern of improvement in semen quality is known, these findings can be reproduced at a later date and be timed for the provision of fresh samples for artificial insemination or IVF. The previously cryopreserved semen can be considered as back-up in case of failure to obtain the expected semen sample. Men with chronic SCI who can produce semen by safe vibration ejaculation might be advised to perform regular ejaculation 2 to 3 times per week, with the aim of maintaining semen quality so that home insemination can be performed. Evidence indicates that semen quality is better when vibration ejaculation rather than electroejaculation is used (16). In summary, we report a practical approach to improving the quality of semen in men with chronic SCI. Use of electroejaculation on consecutive days can improve the quality of semen. However, the improvement in quality was not always good enough for artificial insemination. Thus, ICSI with sperm aspirated from the genital tract is a major advance in the treatment of infertility in men with chronic SCI, in which other methods of obtaining sperm have failed. The results raise further questions about the causes of poor quality of semen after SCI. Semen quality in men with SCI is similar to that seen in able-bodied men with epididymal necrospermia. Perhaps SCI can be viewed as an experiment in nature that allows clinical research to better elucidate the mechanisms of necrospermia in able-bodied persons. References 1. Talbot HS. The sexual function in paraplegia. J Urol 1955;77:91–100. 2. Bors E, Comarr AE. Neurological disturbances of sexual function with special reference to 529 patients with spinal cord injury. Urol Surv 1960;10:191–222. 3. Brindley GS. Reflex ejaculation under vibratory stimulation in paraplegic men. Paraplegia 1981;19:299 –302. 4. Brindley GS. Electro-ejaculation: its technique, neurological implication and uses. J Neurol Neurosurg Psychiatry. 1981;44:9 –18.
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5. Brindley GS. The fertility of men with spinal injuries. Paraplegia 1984;22:337– 48. 6. Halstead LS, VerVoort SM, Seager SW. Rectal probe electrostimulation in the treatment of anejaculatory spinal cord injured men. Paraplegia 1987;25:120 –9. 7. Ohl DA, Bennett CJ, McCabe M, Menge AC, McGuire EJ. Predictors of success in electroejaculation of spinal cord injured men. J Urol 1989;142:1483– 6. 8. Bennett CJ, Seager SW, Vasher EA, McGuire EJ. Sexual dysfunction and electroejaculation in men with spinal cord injury: review. J Urol 1988;139:453–547. 9. Perkash I, Martin DE, Warner H Speck V. Electroejaculation in spinal cord injured patients: simplified new equipment and technique. J Urol 1990;143:305–7. 10. VerVoort SM. Infertility in spinal-cord injured male. Urology 1987;29: 157– 68. 11. Hirsch IH, Sedor J, Callahan HJ, Staas WE. Antisperm antibodies in seminal plasma of spinal cord-injured men. Urology 1992;39:243–7. 12. Lim TC, Mallidis C, Hill ST, Skinner DJ, Carter PD, Brown DJ, Baker HWG. A simple technique to prevent retrograde ejaculation during assisted ejaculation. Paraplegia 1994;32:142–9. 13. Linsenmeyer TA. Male infertility following spinal cord injury. J Am Paraplegia Soc 1991;14:116 –21. 14. De Lamirande E, Hassouna M, Leduc BE, Gagnon C, Iwasaki A. Increased reactive oxygen species formation in semen of patients with spinal cord injury. Fertil Steril 1995;53:637– 42. 15. Wilton LJ, Temple-Smith PD, Baker HWG, de Kretser DM. Human male infertility caused by degeneration and death of sperm in the epididymis. Fertil Steril 1988;49:1052– 8. 16. Brackett NL, Padron OF, Lynne CM. Semen quality of spinal cord injured men is better when obtained by vibratory stimulation versus electroejaculation. J Urol 1997;157:151–7. 17. Brackett NL, Bloch WE, Lynne CM. Predictors of necrospermia in men with spinal cord injury. J Urol 1998;159:844 –7. 18. Siosteen A, Frossman L, Steen Y, Sullivan L. Whickstrom I. Quality of semen after repeated ejaculation treatment in spinal cord injury men. Paraplegia 1990;28:96 –104. 19. Mallidis C, Lim TC, Hill ST, Skinner DJ, Brown DJ, Johnston WIH, et al. Collection of semen from men in the acute phase of spinal cord injury. Lancet 1994;343:1072–3. 20. World Health Organization. WHO manual for the examination of human semen and semen-cervical mucus interaction. Cambridge, UK: Cambridge University Press, 1992. 21. Liu DY, Baker HWG. Tests of sperm function in vitro. Fertil Steril 1992;58:465– 83. 22. Mallidis C, Baker HWG. Fine needle tissue aspiration biopsy of the testis. Fertil Steril 1994;61:367–75. 23. Brindley GS. Deep scrotal temperature and the effect on it of clothing, air temperature, activity, posture and paraplegia. Br J Urol 1982;54: 49 –55. 24. Bedford JM. Effects of elevated temperature on the epididymis and testis: experimental studies. In: Zorgniotti AW, ed. Temperature and environmental effects on the testis. New York: Plenum Press, 1991;19 –32. 25. Bedford JM. The status and the state of the human epididymis. Hum Reprod 1994;11:2187–99. 26. Brackett NL, Lynne CM, Weizman MS, Bloch WE, Padron OF. Scrotal and oral temperatures are not related to semen quality or serum gonadotropin levels in spinal cord-injured men. J Androl 1994;15:614 –19. 27. Hovatta O, von Smitten K. Sperm aspiration from vas deferens and in-vitro fertilization in cases of non-treatable anejaculation. Hum Reprod 1993;8:1689 –91. 28. Buch JP. Greatly improved sperm motility from vas deferens sperm retrieval: a case for accessory gland related subfertility in spinal cord injured men. Case report. Paraplegia 1994;32:501– 4. 29. Aitken RJ, Baker HWG. Seminal leucocytes: messengers, terrorists, or good Samaritans? Hum Reprod 1995;10:1736 –9. 30. Aird IA, Vince GS, Bates MD, Johnson PM, Lewis-Jones ID. Leukocytes in semen from men with spinal cord injuries. Fertil Steril 1999;72: 97–103.
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