British Journal of Urology (1975), 47, 459-462 0
The Nitrofurans as Sperm Immobilising Agents: their Tissue Toxicity and their Clinical Application PETER s. ALBERT, DAVID T. MININBERG and JOSEPH E. DAVIS
Department of Urology, New York Medical College, New York City
Freund and Davis (1969) have shown that the disappearance rate of sperm following bilatera1 vasectomy and azoospermia can be predicted by an exponentially decreasing curve. Marshall and Lyon (1972) examined 200 cases of vasectomy, analysing every fourth ejaculate postoperatively. After 12 ejaculations only 65.5 % of the patients were sperm-free. After 24 ejaculations 2.5 % of the patients still had sperm in their seminal fluid. Although Freund and Davis noted a 35% decrease in the sperm count from the patient's previous ejaculate, Marshall and Lyon noted that more than 10 ejaculations were necessary to reach azoospermia in 35 % of their cases. They also found that younger and sexually active patients cleared their ductal reservoir of sperm with fewer postoperative ejaculations. Vas irrigations with water have been used by Craft and McQueen (1972) at the time of vasectomy to reduce their postoperative sperm counts. They were successful in decreasing the sperm counts from 34 % at 12 weeks postoperatively in the non-irrigated patients to 16% with positive counts in the irrigated group. Urquhart-Hay (1973) has successfully used euflavine solution as a vas irrigant to achieve immediate postoperative sterility in 81 patients. By irrigating the vas deferens with a sperm-immobilising solution, sterility would be attained in the immediate postoperative period. Prior and Ferguson (1950) demonstrated that certain nitrofuran compounds have the ability to depress spermatogenesis in rats. Nelson and Steinberger confirmed this in 1952 and found that nitrofurans halted spermatogenesis at the primary spermatocyte stage. Dokov and Timmermanns (1970) have shown that nitrofurans also cause metabolic alterations in the spermatogonium and interstitial tissue enzyme concentrations. Nelson and Bunge (1957) have reported that Furadantin given orally (in humans) in doses of 10 mg/kg/daily may produce temporary spermatogenic depression. Our work involved the investigation of the nitrofurans (more specifically, nitrofurantoin sodium and nitrofurazone) as sperm-immobilising agents, and evaluation of their tissue toxicity and their possible application in conjunction with vasectomy. Materials and Methods Part I Using calibrated droppers, varying concentrations (from 0.5 mg/ml to 40 mg/ml) of nitrofurantoin sodium and nitrofurazone were incubated with freshly ejaculated human spermatozoa at 37°C.Percentage motility of the solution was then assessed at 5-, lo-, 20- and 30-minute intervals and compared to control solutions comprised of spermatozoa and saline of matched concentrations assessed at the same intervals. A total of 24 ejaculates were analysed from different donor sources.
Part IZ Tissue toxicity studies were performed using 36 sexually mature Hartley strain male guinea-pigs. Under Nembutal anaesthesia intraperitoneally given on a weight basis, the guinea-pig's scrotal area on one side was shaved with animal shears and prepped with betadine solution. A 2-cm upper scrotal incision was made. The subcutaneous tissue was divided by blunt dissection and the testis in its coverings was then delivered into the operative field. The vas deferens was then isolated between two 3-0 Tewdek ligatures and an incision was made at a 45" angle to the vas 47/4-~*
459
460
BRITISH JOURNAL OF UROLOGY
with a fine iris scissors and the vas was cannulated with PE 50 Teflon catheter. The distal vas deferens was then flushed with a solution of nitrofurantoin sodium and/or nitrofurazone in a concentration 3 times greater than is necessary to obtain sperm immotility in v i m . The vas was then divided and the distal vas was anchored to the perivasal tissues for later identification. The incision was closed with a 2-0 chromic interrupted suture. At intervals of 1, 2, 4, 6, 8 and 12 weeks the guinea-pigs were sacrificed and their vasa were completely dissected. Specimens were sampled at different levels of the flushed distal vas segment. These were studied histologically and compared to the non-operated control side.
Results Part Z Nitrofurantoin sodium was found to be sperm-immobilising in the minimum concentration of 20 mg/ml while nitrofurazone exhibited a similar effect at 1 mg/ml. The controls, however, revealed no loss of motility during the same time intervals examined (Tables I to IV). All results were found to be consistent in the 5 : 1 and 10 : I sperm dilutions. Table I Effect of Nitrofurantoin Sodium on Human Spermatozoa in vitro at 37°C. 5 : 1 Dilution ~~
~
5 rnin 5 mglcc 10mg/cc 20 mg/cc 40 mg/cc Control o/
-
/o -
10min
20 rnin
30min
50%
50%
40%
40%
40% 30%
20% 20% 0 0
0
0 0
>50%
50%
0
0 0 45%
45%
Percentage motility.
Table 11 Effect of Nitrofurantoin Sodium on Human Spermatozoa in vitro at 37°C. 10 : 1 Dilution 5 min 5 mg/cc 10mg/cc 20 mg/cc 40 mg/cc Control
45%
40% 0 0 >50%
10min
20min
30min
40% 30%
30% 30% 0
20% 25% 0
0 45%
0 45%
0 0
50%
% = Percentage motility.
Part ZZ Histological sections consistently revealed that nitrofurantoin sodium and nitrofurazone were non-toxic to the vas deferens when used in concentrations up to 3 times greater than necessary for sperm immobilisation. There was no evidence of any vasal or perivasal inflammation at the earlier intervals. There was also no evidence of any delayed inflammatory or granulomatous vas resection in the later specimen groups.
THE NITROFURANS AS SPERM IMMOBILISING AGENTS
461
Table III Effect of Nitrofurazone Solution on Human Spermatozoa in vitro at 37°C. 5 : 1 Dilution 5 min
0*5mg/ml 25% lmg/ml 0 0 2mg/ml Control 60%
10min
20min
30min
20%
15%
0 50%
0 50%
10% 0 0 50%
0
0
% = Percentage motility.
Table IV Effect of Nitrofurazone Solution on Human Spermatozoa in vitro at 37°C. 10 : 1 Dilution
5 mg/ml
1 mg/ml 2 mg/ml Control
5 min
10min
20min
30min
25% 0 0 160%
20% 0 0
15% 0 0 50%
15% 0
50%
0 50%
% = Percentage motility.
Conclusion Our experiments have shown that the nitrofurans have the capability of causing sperm immobilisation when utilised in certain critical concentrations. Both drugs were also known to be nontoxic to the vas deferentia in concentrations exceeding that necessary to attain their immobilising effect (Albert et al., unpublished findings). Clinical Application Clinically, our findings have broad potential applications in the area of male reproductive physiology. Since these two nitrofurans have been shown in v i m to be sperm immobilising and have not exhibited any deleterious toxic effects to the vas or perivasal tissues, it is a logical extension that these drugs could be used as an intraoperative flush as an aid in producing a more rapid disappearance rate of sperm post vasectomy. Procedure Vasectomy was performed as an out-patient procedure in 24 patients in the following manner: The vas was isolated from the other elements of the cord, held between the thumb and forefinger and 1% lignocaine was infiltrated over the vas and skin surface. An incision was made directly down the vas deferens which was then secured with an Allis clamp. After the adventitia had been dissected away, 3 to 4 cm of vas was then exposed. The testicular side was then occluded with two Weck clips, and a longitudinal vasotomy incision was then made in the upper vas deferens. The vas was catheterised with a PE 50 Teflon catheter, which was then advanced 2 cm attached to a 6 ml syringe filled with nitrofurantoin and/or nitrofurazone solution and each side flushed with 3 cc of the solution. The catheter was then removed, a l c m of vas excised, and the upper vas occluded with 2 Weck clips. The wound was closed in a single layer of 3-0 interrupted chromic. There were no untoward reactions to the vas irrigations; in particular, there was no evidence of any vasitis.
462
BRITISH JOURNAL OF UROLOGY
The initial 2 consecutive postoperative ejaculates were examined within the first week after vasectomy. Semen specimens were later examined at 6 months and 1 year after operation to ensure that spontaneous reunion had not occurred. All specimens were examined immediately upon their receipt and also after a 10-minute period of incubation. No live spermatozoa were found in any of the specimens.
Comment The irrigation procedure adds little time to the standard vasectomy and can easily be performed as an out-patient procedure. It minimises time-consuming postoperative sperm analyses. In underdeveloped countries where postoperative follow-up examinations are more difficult to obtain, this procedure would provide an additional positive factor in an attempt to ensure sterility.
Summary Nitrofurantoin sodium and nitrofurazone in certain critical concentrations have the ability to cause sperm immobilisation. Intraoperative vas irrigation with these solutions in 24 patients have produced sterility in the immediate postoperative period.
References ALBERT, P. et al. Awaiting publication, Journal of Urology. CRAFT,I. and MCQUEEN, J. (1972). Effect of irrigation of the vas on postvasectomy semen counts. Lancer. 1, 515516. DOKOV. V. K. and TIMMERMANNS, L. (1970). Arret de la spermatogknese par certains antibiotiques: recherches expirimentales prkliminaires. Actu Urologicu Belgicu, 38, 277-287. M. and DAVIS, J. E. (1969). Disappearance rate of spermatozoa from the ejaculate following vasectomy. FREUND, Fertility and Sterility, 20, 163-170. MARSHALL, S. and LYON,R. (1972). disappearance from the ejaculate after vasectomy. Journa . . Variability . of sperm . .. of Urology, 107, 815-817. W. 0. and STEINBERGER, E. (1952). The effect of furadroxyl upon the testis of the rat. Anatomical Record, NELSON, 112. 367-368. NELSON,'^. 0.and BUNGE,R. G. (1957). Effect of therapeutic dosages of nitrofurantoin (Furadantin) upon spermatogenesis in man. Journal of Urology, 77, 275-281. PRIOR,J. T. and FERGUSON, J. H. (1950). Cytotoxic effects of nitrofuran on the rat testis. Cancer, 3, 1062-1072. D. (1973). Immediate sterility after vasectomy. British Medical Journal, 3, 378-379. URQUHART-HAY,
The Authors Peter S. Albert, MD, Clinical Instructor. David T. Mininberg, MD, FACS, Associate Professor of Urology. Joseph E. Davis, MD, FACS, Professor and Chairman of Urology.
The Nitrofurans as Sperm Immobilising Agents: their Tissue Toxicity and their Clinical Application PETER s. ALBERT, DAVID T. MININBERG and JOSEPH E. DAVIS
Department of Urology, New York Medical College, New York City
Freund and Davis (1969) have shown that the disappearance rate of sperm following bilatera1 vasectomy and azoospermia can be predicted by an exponentially decreasing curve. Marshall and Lyon (1972) examined 200 cases of vasectomy, analysing every fourth ejaculate postoperatively. After 12 ejaculations only 65.5 % of the patients were sperm-free. After 24 ejaculations 2.5 % of the patients still had sperm in their seminal fluid. Although Freund and Davis noted a 35% decrease in the sperm count from the patient's previous ejaculate, Marshall and Lyon noted that more than 10 ejaculations were necessary to reach azoospermia in 35 % of their cases. They also found that younger and sexually active patients cleared their ductal reservoir of sperm with fewer postoperative ejaculations. Vas irrigations with water have been used by Craft and McQueen (1972) at the time of vasectomy to reduce their postoperative sperm counts. They were successful in decreasing the sperm counts from 34 % at 12 weeks postoperatively in the non-irrigated patients to 16% with positive counts in the irrigated group. Urquhart-Hay (1973) has successfully used euflavine solution as a vas irrigant to achieve immediate postoperative sterility in 81 patients. By irrigating the vas deferens with a sperm-immobilising solution, sterility would be attained in the immediate postoperative period. Prior and Ferguson (1950) demonstrated that certain nitrofuran compounds have the ability to depress spermatogenesis in rats. Nelson and Steinberger confirmed this in 1952 and found that nitrofurans halted spermatogenesis at the primary spermatocyte stage. Dokov and Timmermanns (1970) have shown that nitrofurans also cause metabolic alterations in the spermatogonium and interstitial tissue enzyme concentrations. Nelson and Bunge (1957) have reported that Furadantin given orally (in humans) in doses of 10 mg/kg/daily may produce temporary spermatogenic depression. Our work involved the investigation of the nitrofurans (more specifically, nitrofurantoin sodium and nitrofurazone) as sperm-immobilising agents, and evaluation of their tissue toxicity and their possible application in conjunction with vasectomy. Materials and Methods Part I Using calibrated droppers, varying concentrations (from 0.5 mg/ml to 40 mg/ml) of nitrofurantoin sodium and nitrofurazone were incubated with freshly ejaculated human spermatozoa at 37°C.Percentage motility of the solution was then assessed at 5-, lo-, 20- and 30-minute intervals and compared to control solutions comprised of spermatozoa and saline of matched concentrations assessed at the same intervals. A total of 24 ejaculates were analysed from different donor sources.
Part IZ Tissue toxicity studies were performed using 36 sexually mature Hartley strain male guinea-pigs. Under Nembutal anaesthesia intraperitoneally given on a weight basis, the guinea-pig's scrotal area on one side was shaved with animal shears and prepped with betadine solution. A 2-cm upper scrotal incision was made. The subcutaneous tissue was divided by blunt dissection and the testis in its coverings was then delivered into the operative field. The vas deferens was then isolated between two 3-0 Tewdek ligatures and an incision was made at a 45" angle to the vas 47/4-~*
459
460
BRITISH JOURNAL OF UROLOGY
with a fine iris scissors and the vas was cannulated with PE 50 Teflon catheter. The distal vas deferens was then flushed with a solution of nitrofurantoin sodium and/or nitrofurazone in a concentration 3 times greater than is necessary to obtain sperm immotility in v i m . The vas was then divided and the distal vas was anchored to the perivasal tissues for later identification. The incision was closed with a 2-0 chromic interrupted suture. At intervals of 1, 2, 4, 6, 8 and 12 weeks the guinea-pigs were sacrificed and their vasa were completely dissected. Specimens were sampled at different levels of the flushed distal vas segment. These were studied histologically and compared to the non-operated control side.
Results Part Z Nitrofurantoin sodium was found to be sperm-immobilising in the minimum concentration of 20 mg/ml while nitrofurazone exhibited a similar effect at 1 mg/ml. The controls, however, revealed no loss of motility during the same time intervals examined (Tables I to IV). All results were found to be consistent in the 5 : 1 and 10 : I sperm dilutions. Table I Effect of Nitrofurantoin Sodium on Human Spermatozoa in vitro at 37°C. 5 : 1 Dilution ~~
~
5 rnin 5 mglcc 10mg/cc 20 mg/cc 40 mg/cc Control o/
-
/o -
10min
20 rnin
30min
50%
50%
40%
40%
40% 30%
20% 20% 0 0
0
0 0
>50%
50%
0
0 0 45%
45%
Percentage motility.
Table 11 Effect of Nitrofurantoin Sodium on Human Spermatozoa in vitro at 37°C. 10 : 1 Dilution 5 min 5 mg/cc 10mg/cc 20 mg/cc 40 mg/cc Control
45%
40% 0 0 >50%
10min
20min
30min
40% 30%
30% 30% 0
20% 25% 0
0 45%
0 45%
0 0
50%
% = Percentage motility.
Part ZZ Histological sections consistently revealed that nitrofurantoin sodium and nitrofurazone were non-toxic to the vas deferens when used in concentrations up to 3 times greater than necessary for sperm immobilisation. There was no evidence of any vasal or perivasal inflammation at the earlier intervals. There was also no evidence of any delayed inflammatory or granulomatous vas resection in the later specimen groups.
THE NITROFURANS AS SPERM IMMOBILISING AGENTS
461
Table III Effect of Nitrofurazone Solution on Human Spermatozoa in vitro at 37°C. 5 : 1 Dilution 5 min
0*5mg/ml 25% lmg/ml 0 0 2mg/ml Control 60%
10min
20min
30min
20%
15%
0 50%
0 50%
10% 0 0 50%
0
0
% = Percentage motility.
Table IV Effect of Nitrofurazone Solution on Human Spermatozoa in vitro at 37°C. 10 : 1 Dilution
5 mg/ml
1 mg/ml 2 mg/ml Control
5 min
10min
20min
30min
25% 0 0 160%
20% 0 0
15% 0 0 50%
15% 0
50%
0 50%
% = Percentage motility.
Conclusion Our experiments have shown that the nitrofurans have the capability of causing sperm immobilisation when utilised in certain critical concentrations. Both drugs were also known to be nontoxic to the vas deferentia in concentrations exceeding that necessary to attain their immobilising effect (Albert et al., unpublished findings). Clinical Application Clinically, our findings have broad potential applications in the area of male reproductive physiology. Since these two nitrofurans have been shown in v i m to be sperm immobilising and have not exhibited any deleterious toxic effects to the vas or perivasal tissues, it is a logical extension that these drugs could be used as an intraoperative flush as an aid in producing a more rapid disappearance rate of sperm post vasectomy. Procedure Vasectomy was performed as an out-patient procedure in 24 patients in the following manner: The vas was isolated from the other elements of the cord, held between the thumb and forefinger and 1% lignocaine was infiltrated over the vas and skin surface. An incision was made directly down the vas deferens which was then secured with an Allis clamp. After the adventitia had been dissected away, 3 to 4 cm of vas was then exposed. The testicular side was then occluded with two Weck clips, and a longitudinal vasotomy incision was then made in the upper vas deferens. The vas was catheterised with a PE 50 Teflon catheter, which was then advanced 2 cm attached to a 6 ml syringe filled with nitrofurantoin and/or nitrofurazone solution and each side flushed with 3 cc of the solution. The catheter was then removed, a l c m of vas excised, and the upper vas occluded with 2 Weck clips. The wound was closed in a single layer of 3-0 interrupted chromic. There were no untoward reactions to the vas irrigations; in particular, there was no evidence of any vasitis.
462
BRITISH JOURNAL OF UROLOGY
The initial 2 consecutive postoperative ejaculates were examined within the first week after vasectomy. Semen specimens were later examined at 6 months and 1 year after operation to ensure that spontaneous reunion had not occurred. All specimens were examined immediately upon their receipt and also after a 10-minute period of incubation. No live spermatozoa were found in any of the specimens.
Comment The irrigation procedure adds little time to the standard vasectomy and can easily be performed as an out-patient procedure. It minimises time-consuming postoperative sperm analyses. In underdeveloped countries where postoperative follow-up examinations are more difficult to obtain, this procedure would provide an additional positive factor in an attempt to ensure sterility.
Summary Nitrofurantoin sodium and nitrofurazone in certain critical concentrations have the ability to cause sperm immobilisation. Intraoperative vas irrigation with these solutions in 24 patients have produced sterility in the immediate postoperative period.
References ALBERT, P. et al. Awaiting publication, Journal of Urology. CRAFT,I. and MCQUEEN, J. (1972). Effect of irrigation of the vas on postvasectomy semen counts. Lancer. 1, 515516. DOKOV. V. K. and TIMMERMANNS, L. (1970). Arret de la spermatogknese par certains antibiotiques: recherches expirimentales prkliminaires. Actu Urologicu Belgicu, 38, 277-287. M. and DAVIS, J. E. (1969). Disappearance rate of spermatozoa from the ejaculate following vasectomy. FREUND, Fertility and Sterility, 20, 163-170. MARSHALL, S. and LYON,R. (1972). disappearance from the ejaculate after vasectomy. Journa . . Variability . of sperm . .. of Urology, 107, 815-817. W. 0. and STEINBERGER, E. (1952). The effect of furadroxyl upon the testis of the rat. Anatomical Record, NELSON, 112. 367-368. NELSON,'^. 0.and BUNGE,R. G. (1957). Effect of therapeutic dosages of nitrofurantoin (Furadantin) upon spermatogenesis in man. Journal of Urology, 77, 275-281. PRIOR,J. T. and FERGUSON, J. H. (1950). Cytotoxic effects of nitrofuran on the rat testis. Cancer, 3, 1062-1072. D. (1973). Immediate sterility after vasectomy. British Medical Journal, 3, 378-379. URQUHART-HAY,
The Authors Peter S. Albert, MD, Clinical Instructor. David T. Mininberg, MD, FACS, Associate Professor of Urology. Joseph E. Davis, MD, FACS, Professor and Chairman of Urology.
