STERILITY IN MALE ANIMALS INDUCED BY INJECTION OF CHEMICAL AGENTS INTO THE VAS DEFERENS*
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1 FERTILITY AND STERILITY Copyright 1973 by The Williams & Wilkins Co. Vol. 24, No. 11, November 1973 Printed in U.S.A. STERILITY IN MALE ANIMALS INDUCED BY INJECTION OF CHEMICAL AGENTS INTO THE VAS DEFERENS* COY FREEMAN, M.D., AND DONALD S. COFFEY, PH.D. The James Buchanan Brady Urological Institute, The Johns Hopkins Hospital, and Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 2125 The side effects of vasectomy are still under investigation; nevertheless, voluntary surgical sterilization is one of the most popular and reliable methods of population control. It is estimated that 1 million American males will seek a vasectomy this year alone. 1 In addition, vast numbers of vasectomies are being done for population control in more heavily populated areas of the world. In areas where surgical personnel and minor surgical facilities are limited, mobile sterilization camps have been organized. 1 An acceptable method of male sterilization not requiring an operative procedure would be of immediate benefit in such population control programs. Certain risks, such as bleeding and infection, common to all surgical procedures, would be reduced or eliminated. In addition, some men who fear surgical operations on their genitalia, perhaps mistakenly associating the procedure with castration, might find a nonsurgical sterilization procedure more acceptable. We have endeavored to develop a simple, nonsurgical technic for achieving male sterility. This technic consists of inducing obstruction of the vas deferens by injecting sclerosing chemical agents through the skin of the scrotum directly into the vas. We have previously reported initial success Received June 6, * Supported by National Institutes of Health Training Grant AM and by Research Contract AID-CSD-368 from the University of Minnesota Program for Applied Research on Fertility Regulation. using 95% ethanol injections into rats. 2 This communication deals with a wider variety of chemical agents in rats, dogs, and human cadavers. METHODS Mature, male Sprague-Dawley rats (25-35 gm.) were supplied by Charles River Breeding Laboratories. Animals were housed in individual cages. Injections were made into the vas deferens of rats through a small, low midline abdominal incision, using pentobarbital anesthesia. A 25-gauge needle was inserted into the lumen of the vas in the retrograde direction at about 1.5 cm. "from its distal end. With a microsyringe, 25 or 5 ~l. of the agent to be tested were injected into each vas deferens. Ninety-five per cent ethanol, 1% silver nitrate, 36% acetic acid, 3.6% formaldehyde, 3% sodium tetradecyl sulfate, 5% sodium morrhuate, 5% potassium permanganate, 3.6% formaldehyde in 9% ethanol, and, for control,.9% sodium chloride have been tested in this manner (Table 2). Two weeks following treatment, the male rats were transferred to individual cages containing two mature female rats for exposure to continuous mating. Surgical exposure through the upper scrotum also was used in initial trials in dogs. Later, satisfactory injections were made into the vas deferens of some dogs, through the skin of the scrotum, without surgical exposure (Table 3). Agents injected have included 95% ethanol, 1% silver nitrate solution, and 3.6% formaldehyde in 9% ethanol. Injections of either 884..
2 November 1973 NONSURGICALLY INDUCED STERILITY IN MALES or 5 Ill. were made directly into the vas deferens. RESULTS AND DISCUSSION Ethanol was chosen first for intense investigation because of its known low toxicity once it has been diluted in body water. Thirty-two rats received injections of 95% ethanol into both vasa deferentia, or into one vas deferens with the other being ligated (Table 1). All of these rats were made sterile and have remained so for more than 8 months of continuous mating exposure with two mature females. In contrast, 12 of 16 rats that received injections of 5 Ill. of.9% sodium chloride solution remained fertile. In addition, five of six rats that received injections of 95% ethanol into one vas deferens, while the other was undisturbed, have remained fertile (Table 1). Several other agents have been tested for their ability to produce vas deferens obstruction and sterility in rats. Twenty rats that received 5-Ill. injections of 1% silver nitrate, 36% acetic acid, 3.6% formaldehyde, or 3% sodium tetradecyl sulfate were also made sterile and have remained so for 8 months. Of a small series of rats that received injections of either 5% sodium morrhuate or 5% potassium permanganate, some from each group remained fertile (Table 2). A later group of 11 rats received injections of only 25 Ill. of 3.6% formaldehyde in 9% ethanol, and have thus far remained sterile for 2 months of continuous mating exposure. Animals from each group were killed for comparative histologic studies. Figure 1 shows a cross-section of a rat vas deferens which had been injected with 5 Ill. of 95% ethanol 6 weeks prior to the animal's being killed. The region of the lumen has been completely replaced by scar tissue and sperm are completely absent. Similar obstruction with scar tissue was seen when silver nitrate, formaldehyde, acetic acid, or sodium tetradecyl sulfate was used. A normal rat vas deferens is shown for comparison (Fig. 2). Kwart and Coffey 3 have shown that sperm granulomas invariably form when the rat vas deferens is obstructed. Our work confirms this: all rats with vas obstruction, whether produced by chemically TABLE 1. Fertility of Rats 8 Months following a Single Injection of Ethanol or Saline into the Vas Deferens None None Ligated Ligated 15 None Ligated % NaCI.9% NaCI % ethanol 95% ethanol 22 95% ethanol None % ethanol Ligated 1 Single injections were made into each of the exposed vas deferens. Ligations were performed with 4- silk followed by surgical division. t Two weeks post-treatment, each animal was placed with two females for breeding for a period of 8 months. TABLE 2. Fertility of Rats 8 Months following a Single Injection of Chemical Sclerosing Agents into Each Vas Deferens Treatment* No treatment.9% sodium chloride 95% ethanol 3.6% formaldehyde in 9% ethanolt 1% silver nitrate 36% acetic acid 3.6% formaldehyde 3% sodium tetradecyl sulfate 5% potassium permanganate 5% sodium morrhuate Amount injected 1' No. of rats * See footnotes to Table l. t These eleven rats were exposed to mating for only 2 months No. fertile* Per- Treatment (5 1'1.)* No. No. centof fer- age Left vas Right vas rats tilet fertile Percentage fertile
3 886 FREEMAN AND COFFEY Vol. 24 induced scarring or by surgical ligation, were found to have sperm granulomas, often in the tail of the epididymis. It is the scarring obstruction in the vas deferens rather than the epididymal granuloma which is responsible for the sterility produced, however. Evidence for this is the fact that those animals which had received injections of sodium morrhuate or potassium permanganate were found to have epididymal granulomas, even though they remained fertile and were found to have sperm in their ejaculates obtained by electroejaculation immediately prior to killing. Because we were satisfied that certain chemical sclerosing agents were as effective as vasectomy in achieving sterility in rats, we turned our attention to dogs, which have a vas deferens anatomically similar to that of man. Initially, surgical exposure of the vas deferens was used for injection. Later, the same result was obtained in some dogs when injections were made directly through the skin of the scrotum, without surgical exposure (Table 3). Figures 3, 4, and 5, respectively, show the vas deferens of a normal dog, a dog vas deferens injected with 1% silver nitrate 2 months previously, and a dog vas deferens injected with 3.6% formaldehyde 2 months previously; all figures are of the same magnification. Each of the latter two dogs received 5-}L1. injections. In Fig. 4, the lumen has been replaced by scar tissue and the diameter of the vas is reduced after silver nitrate injection. In Fig. 5, the reduction in diameter of the vas is even more striking after 3.6% formaldehyde injection. FIG. 1. Rat vas deferens 6 weeks after injection of 95% ethanol. x
4 November 1973 NONSURGICALLY INDUCED STERILITY IN MALES 887 FIG. 2. Normal rat vas deferens. x 14. TABLE 3. Patency of Dog Vas Deferens 4-14 Weeks following Injection of Chemical Sclerosing Agents Agent injected No treatment Ligation Amount injected /ll. 95% ethanol 5 95% ethanol 5 3.6% formaldehyde 5 1% silver nitrate 5 1% silver nitrate 5 1% silver nitrate 5 (injected percutaneously) 3.6% formaldehyde in 1 9% ethanol Histologic findings Normal Obstructed; distension of epididymis Obstructed; intraluminal granuloma Obstructed; intraluminal granuloma Obstructed; <.5 cm. of vas scarred Grossly, this reduction in size was visible over a distance of approximately 2 cm. No sperm granulomas were found, either grossly or microscopically, in the vasa deferentia or epididymides of these dogs. The testes were normal in size and in microscopic appearance. In the human male, the vas deferens may be brought up easily to the skin of the scrotum, without any surgery, and held between the thumb and forefinger like a small suitcase handle. Figure 6 shows a 25-gauge needle inserted easily into the vas of a living human volunteer. Although no attempt has yet been made to produce human sterility by this method, injections of methylene blue dye in alcohol have been made through the skin of the scrotum into the vas deferens of several intact human
5 888 FREEMAN AND COFFEY Vol. 24 autopsy specimens. When the vas was removed after the injection, the dye was found to be confined within the sheath of the vas deferens, and the full thickness of the wall of the vas was stained. Thus it seems that male sterility may be achieved via direct injection into the vas deferens, achieving a result similar to that achieved by surgical division and ligation of the vas deferens. We believe that the potential advantages of vasoinjection, as opposed to vasectomy, are as follows: 1. The risk of postsurgical hemorrhage essentially will be eliminated. This is the most common immediate postoperative complication and is reported to occur in frequencies of.3% ("extensive hematoma") 4-5% The risk of postoperative infection will be diminished greatly. The consequences of infection may be particularly severe if postoperative bleeding has taken place. 3. This procedure should be more acceptable to the candidate for sterilization who has a fear of genital operative procedures. 4. No surgery or surgical equipment is required. 5. Once this procedure has been learned, it can be done quite rapidly at very low cost. FIG. 3. Normal dog vas deferens. x 45.
6 November 1973 NONSURGICALLY INDUCED STERILITY IN MALES 889 FIG. 4. Dog vas deferens 8 weeks after injection of 1% silver nitrate. x 45. FIG. 5. Dog vas deferens 8 weeks after injection of 3.6% formaldehyde. x Adequately trained paramedical personnel could be taught to do this procedure. 7. This technic seems particularly applicable to mass voluntary sterilization programs in less developed countries. Possible limitations to this procedure are the following: 1. At present this procedure appears to be permanent. We have seen no evidence of recanalization of the vas or return of fertility in animals which were successfully made sterile. However, these observations have taken place over a relatively short period of time when compared with human
7 89 FREEMAN AND COFFEY Vol. 24 methods of vas occlusion need carefully controlled studies. FIG. 6--:- Identification and isolation of human vas deferens; note ease of needle placement. reproductive life. This permanence must be confirmed in human volunteers. 2. The possibility of surgical reversibility is not known. It is anticipated that the amount of vas affected would depend upon the volume and nature of the material injected. 3. The failure rate of surgical vasectomy varies with the operator and the technic. Similar limitations undoubtedly would apply to this method. 4. Long-term adverse effects of all SUMMARY A new approach to rapid male sterilization has been developed in rats, dogs, and human cadavers. This procedure involves injection of chemical agents directly into the vas deferens, resulting in an obstructing scar within the lumen of the vas deferens. A wide variety of chemical agents has been tested in a large series of animals and several of these agents appear to produce complete blockage of the vas, as determined by mating studies, microscopic examination of ejaculates, and histologic findings in the vas deferens. It is believed that this technic can be developed into a rapid, nonsurgical method of producing human male sterility, with particular application to large scale voluntary sterilization programs in less developed countries. However, further testing will be required to demonstrate the permanency of this procedure. REFERENCES 1. SPEIDEL, J. J. Role of the urologist in population stabilization. Urology 1:277, FREEMAN, C., AND COFFEY, D. S. Male sterility induced by ethanol injection into the vas deferens. Int J Fertil. In press. 3. KWART, A., AND COFFEY, D. S. Adverse effects of vasectomy: Sperm granuloma. J Urol. In press. 4. KLAPPROTH, J. J., AND YOUNG, I. S. Vasectomy, vas ligation and vas occlusion. Urology 1:292, JOHNSON, D. S. Reversible male sterilization: Current status and future directions. Contraception 5:327, 1972.
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