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Sperm Granulomas: An Adverse Effect of Vasectomy
Vol. 110, October Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright © 1973 by The Williams & Wilkins Co.
SPERM GRANULOMAS: AN ADVERSE EFFECT OF VASECTOMY ARNOLD M. KWART AND DONALD S. COFFEY
From the James Buchanan Brady Urological Institute, The Johns Hopkins Hospital and the Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins School of Medicine, Baltimore, Maryland
Sperm granulomas have been previously described secondary to human sterilization by vasectomy and several large clinical studies report the frequency to be from 5 to 18 per cent. 1 -• However, the occurrence of these lesions has not deterred the popularity of this sterilization procedure nor do granulomas have a known deleterious effect on the patient. The present frequency of human vasectomy demands a clearer insight into the full etiology and implications of these lesions. This information is not readily available because of the generally poor followup data on vasectomized patients. Previous experimental animal studies on the formation of spermatic granulomas have been restricted to resection of the vas deferens without ligation. 4 • 5 In these animals the drainage of the open vas deferens produced a sperm granuloma similar to that observed in vasectomized animals•· 7 and patients. 3 This fact has led to the general concept that sperm granulomas occur in cases in which sperm leaks at operation or subsequent to the accumulation of intraluminal pressure against the suture, resulting in the severance of the vasal wall. We report herein the first long-term animal study of the incidence and mechanism of formation of sperm granulomas in rats following vas ligation. MATERIALS AND METHODS
We used Sprague-Dawley male rats, either prepubertal (40 days, 50 to 70 gm.) or adult (120 days, 250 to 300 gm.). Animals resided in individual Accepted for publication March 23, 1973. Supported by a grant from the Rockefeller Foundation, United States Public Health Service Training Grant AM-056-74-011 and the Louise and Gustavus Pfeiffer Foundation of New York, New York. 1 Glassy, F. J. and Mostofi, F. K.: Sperrnatic granulornas of the epididyrnis. Arner. J. Clin. Path., 26: 1303, 1956. 2 Banerji, T. P.: A study of male sterilization at. Karnpur. Report. on 202 cases of vasectomy. J. Indian Med. Ass., 36: 578, 1961. 3 Schmidt, S. S.: Technics and complications of elective vasectomy. Fertil. Steril., 17: 467, 1966. • Russell, M. and Friedman, N. B.: Studies in general biology of sperm: experimental production of sperrnatic granuloma. J. Urol., 65: 650, 1951. 'Mullaney, J.: Experimental production of sperrnatic granulorna in rats. Nature, 194: 487, 1962. • Poynter, H.: Testis hormone secretion in rat under conditions of vasectomy or isolation. Anat. Rec., 74: 355, 1939. 7 Smith, G.: The effects of ligation of the vasa efferentia and vasectomy on testicular function in the adult rat. J. Endocr., 23: 285, 1962. 416
cages. The animals were anesthetized with pentobarbital and a midline, lower abdominal incision was used to expose the genital tract. Resection of the vas deferens with proximal and distal ligation was performed with 7-zero silk in the prepubertal and 4-zero silk in the postpubertal animals. The testes occupied the scrotum in anatomic position. When indicated animals were mated by placing 2 female rats with each male rat. We determined direct sperm counts in testicular and epididymal tissue according to the method of Amann and Lambiase. 8 The rate of testicular 3 H-testosterone formation was determined by incubation of testicular minces with "H-cholesterol by a modification of the method of Hall and associates.• Thin layer chromatography on silica gel with a solvent system of 9: 1 chloroform to ether was used to isolate the labeled testosterone. The testosterone was acetylated by the procedure of Harper and associates and rechromatographed on alumina oxide gel using 7: 3 cyclohexane to ethyl ether as the solvent. 10 The tissue content of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) was determined by a modification of the technique of Hutchinson and associates 11 as reported by Coffey and associates. 12 Tissue proteins were obtained by the method of Folin-Ciocalteau on a 0.lM NaOH extraction at 70 degrees for 10 minutes. Neutral buffered formalin was used to fix the histologic sections except for the testicular tissue which was fixed in Bouin's solution for 8 hours before placement in 70 per cent ethyl alcohol. Hematoxylin and eosin were used to stain the sections. RESULTS
Experience in our laboratory with more than 100 vasectomized rats has indicated that sperm granu' Amann, R. P. and Lambiase, J. T., Jr.: The male rabbit. 3. Determination of daily sperm production by means of testicular homogenates. J. Anirn. Sci., 28: 369, 1969. • Hall, P. F., Irby, D. C. and De Krester, D. M.: Conversion of cholesterol to androgens by rat testis: comparison of interstitial cells and seminiferous tubules. Endocrinology, 84: 488, 1969. 10 Harper, M. E., Pierrepont, C. G., Famy, A. R. and Griffiths, K.: The metabolism of steroids in the canine prostate and testis. J. Endocr., 49: 213, 1971. 11 Hutchinson, W. C., Downie, E. D. and Murro, H. N.: Factors affecting the Scheider procedure for estimation of nucleic acids. Biochern. Biophys. Acta, 55: 561, 1962. 12 Coffey, D.S., Shirnazaki, J. and Williams-Ashman, H. G.: Polymerization of deoxyribonucleotides in relation to androgen-induced prostatic growth. Arch. Biochern., 124: 184, 1968.
417
SPERM GRANULOMAS: ADVERSE EFFECT OF VASECTOMY
loma 1s invariably present after vasectomy. To determine if the granulomas occurred by leakage of sperm at the time of the operation or before the healing of the ligated site, we designed an experiment using vasectomized prepubertal animals. Immature 40-day-old animals were autopsied and determined to be free of sperm by histological examination of epididymal and testicular tissue. In this prepubertal series, 24 animals were subjected to either sham, bilateral or unilateral vasectomy as indicated in table 1. This prepubertal vasectomy series was compared to 30 adult animals which were sham or bilaterally vasectomized. Both series were autopsied 6 months post-vasectomy. All animals in both series receiving vasectomy presented sperm granulomas on the ligated vas. Table 1 indicates the location of these granulomas and the percentage of animals with multiple granulomas in each vas. All postpubertal vasectomized animals had granulomas at the site of vasectomy. Although all prepubertal vasectomized animals had granulomas, only 46 to 50 per cent occurred at the site of ligation. This observation appears to eliminate leakage of sperm at the operation and suture as the predisposing factor in granuloma formation in the prepubertal vasectomized animals. In addition, another series of postpubertal vasectomized animals had their ·vas occluded by fulguration or ligation with either 4-zero silk, plain catgut or chromic sutures and this did not alter the 100 per cent occurrence of granulomas at the site of ligation. The incidence of granulomas proximal to the site of ligation was also higher m the prepubertal group. In many cases the granulomas occurred in the tail and body of the epididymis (table 1). The average size of all granulomas 6 months following vasectomy was 0.3 to 0.7 gm. A typical lesion is depicted in part A of the figure. In several cases these lesions were very large and approached testicular size (part B of figure). Histology of a typical lesion indicated that it contained massive accumulations of intact spermatozoa (part C of figure). This suggested that following vasectomy the sperm were not completely reabsorbed by the epididymis and were transported and accumulated in the vas deferens. Indeed, it was possible to remove the soft greenish content of the sperm granuloma and to perform a direct sperm count, which indicated that an average lesion at 6 months could contain from 0.8 to 1.8 billion sperm (table 2). Since a single ejaculate of the rat contains 10 to 100 million sperm 13 one of the typical granulomas (17.9 times 10 8) contained from 20 to 200 times this amount of sperm. Since this granuloma accumulated m 6 months and represents the output of only 1 testis, it suggests that the bulk of the rat sperm may not be fully reabsorbed by the epididymis following vasectomy. 13 Lawson, R. L., Krise, G. M. and Sorensen, A. M., Jr.: Electroejaculation and evaluation of semen from the albino rat. J. Appl. Physiol., 22: 174, 1967.
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KW ART AND COFFEY
SPERM GRANULOMAS: ADVERSE EFFECT OF VASECTOMY
419
A, typical location of granulomatous lesions at site of vasectomy. B, example of large granulomas at site of vasectomy and within tail of epididymis. C, early granulomatous inflammation surrounding central mass of sperm. Lesion at site of vasectomy. D 1 , pyogenic granuloma with polymorphonuclear neutrophils invading sperm mass. Increased granulomatous inflammation. H & E, reduced from x 64. D2, histiocytes engulfing spermatozoa during pyogenic phase. Reduced from x 160. E, pronounced granulomatous inflammation surrounding markedly diminished central sperm mass. Reduced from x 64.
420
KW ART AND COFFEY
It is apparent that this process could not continue indefinitely without the continued formation of massive lesions. It has already been noted by others that these granulomas in rats can decrease in size with time. 7 We confirmed this latter finding and observed a marked change in the histological pattern with more pronounced granulomatous inflammation of the lesion (parts C, D,, D2 and E of figure). These lesions reflect a spectrum of changes in the state of granulomatous inflammation that can be described initially as an accumulation of large masses of sperm which are bordered by a rim of epithelioid histiocytes, this rim in turn is encompassed by a perivascular accumulation of round cells. The lesion is limited by a fibrous capsule (part C of figure). The lesion then appears to progress to a pyogenic granuloma with a marked influx of polymorphonuclear neutrophils into the mass of sperm (part D 1 of figure). This is accompanied by a destruction of the central mass and a more pronounced histiocytic response with active phagocytosis of sperm by the histiocytes (part D 2 of figure). This process results in a smaller lesion (in some cases microscopic) which is a classical granuloma containing large numbers of epithelioid histiocytes with Langhans' giant cells (part E of figure). The sperm mass is considerably diminished at this point. This pathological process described for sperm granulomas in rats following vas ligation is similar to that observed by Glassy and Mostofi in humans with idiopathic spermatic granulomas of the epididymis. 1 This process is also suggestive of the lesions following human vasectomy mentioned by Schmidt." In our experiments it was possible to observe multiple granulomas in a rat vas at different stages of inflammation. This suggested that as some lesions became quiescent they were accompanied by other granulomas foci which became the reservoir of sperm accumulation. In some cases separate lesions appeared in the epididymal size (table 3). If granulomas were to continue to extend into the efferent ducts of the testes they could disrupt testicular function because others have shown that ligation of the vasa efferentia produces degeneration of the seminiferous tubules. 7 We did not observe this histologically and no alterations were seen in testicular function. It is important to note that following vasectomy TABLE
Lesion
1 2
2. Composition of 2 typical granulomas at 6 months post-vasectomy* Total Weight (mg.) 406 898
Weight Empty Capsule (mg.)
Weight Central Mass (mg.)
Total Sperm Count in Central Mass
102
304 729
8.4 X 10 8 17.9x10'
170
* Two typical size granulomas (similar to part C of figure) were selected for analysis.
some form of an equilibrium must occur ultimately between sperm production and sperm reabsorption. This is of particular significance since in our experiments testicular function and sperm formation were not altered 6 months following vasectomy. This was determined by measurements of sperm counts in the epididymis and testes, rate of testosterone synthesis, testicular content of DNA, RNA and protein (table 4) and comparative histology of the genital organs. Indeed, prepubertal animals were bred and the unilaterally vasectomized rats were fertile. This would be consistent with previous studies which show no significant changes in testicular function following vasectomy.•. 7 • 14 Lesions cultured yielded no infectious etiology. DISCUSSION
Early studies on the effects of vasectomy have focused primarily on testicular function as reflected in changes in the gross size of sexual accessory glands and in the level of spermatogenesis. 6 • 7 • 14 Although spermatoceles were reported in several of these studies, they received minimal attention. Previous studies designed to evaluate sperm granulomas have not used vasectomy but instead have used animal models in which the vas was severed, not ligated, and allowed to drain from the open end directly in the scrotal sac. 4 •, 5 No previous reports have described the frequency, distribution and etiology of sperm granulomas in a vasectomized animal which might be applicable to a similar situation occurring in humans following vasectomy. The frequency and location of sperm granulomas in vasectomized patients have not been carefully studied. Studies have been made of idiopathic sperm granulomas of the epididymis and 2 of 60 patients had undergone vasectomy.' Other investigators described cases of spermatic granuloma after vasectomy. 2 • 3 It is the consensus that these lesions are not rare. It is important that the lesions we describe in the rat are always present after vasectomy, have been observed without exception in more than 100 rats and that the pathology of the lesions is similar to that described by Mostofi for human idiopathic epididymal granuloma. 1 The appearance of granuloma after human vasectomy rarely causes acutely serious clinical problems. This is probably because the granulomatous lesions become limiting in size through a more organized granulomatous inflammation producing diminution in gross size of the lesion. However, the clinical importance of these lesions may be the implication that the reabsorption of sperm through this inflammatory process might trigger alterations in the immunological states of the animal. Indeed, sperm are known to be strong autoantigens and "Kar, A. B., Chandra, H. and Kamboj, V. P.: Long-term effect of vasectomy on the gonad-pituitary system of rats. Acta Biol. Med. German., 15: 381, 1965.
421
SPERNi GRANULOTvIAS: ADVERSE EFFECT OF VASECri'0I\1Y TABLE
3. Effect of prepubertal and postpubertal vasectomy on size of rat genital organs* Genital Organ Weight Single Epididymis Body Weight (gm.)
Ventral Prostate
Seminal Vesicle
Without Granuloma
Testist
With Granuloma
%of Epididymides
Weight
%of Epididymides
4 6 8 7
100 82 67 100
0 221 ± 7 230 ± 24 0
0 18 33 0
122 ± 3 116 ± 4
100 75
0 250
0 25
Weight (mg. per 100 gm. body weight) Prepubertal vasectomy: Control, sham Bilateral vasectomy Right unilateral vasectomy
468 ± 16 466 ± 10 446 ± 13
138 ± 9 144 ± 11 148 ± 28
86 ± 4 89 ± 4 92 ± 5
367 342 (R) 331 (L) 334
Postpubertal vasectomy: Control, sham Bilateral vasectomy
493 ± 11 523 ± 9
101 ± 15 90 ± 6
69 ± 2 61 ± 2
339 326
± 13 ± 10 ± 15 ± 21 ± ±
11 9
137 ± 121 ± 104 ± 127 ±
±
115
* All animals were sacrificed 6 months post-vasectomy; all values plus or minus standard error of mean. t Each testis and epididymis was individually weighed. Epididymides containing granulomas were treated separately.
TABLE
4. Six-month effects of prepubertal vasectomy on testicular function* Organ Sperm Count:j:
'H-Testosterone Production From 'H Cholesterol§
Testicular Content
Fertilityt Single Epididymis Testis
Without Granuloma
With Granuloma
(10' sperm per 100 mg. tissue) Control, sham (n - 9) Bilateral vasectomy (n - 6) Unilateral vasectomy (n - 9) Ligated (rt.) Intact (lt.)
8/9 0/6 9/9
DNA
RNA
Protein
(µg. per 100 mg. testis)
(10' cpm per testis per hour x) 7.4 ± 5 120 180 9.2 ± 3
19.2 ± 1.2 18.1 ± 0.7
50.1 ± 2.3 37.3 ± 1.4
None 92.2 ± 50
155± 4 149 ± 6
279 ± 913,080 270 ± 7 2,860
17.3 ± 0.9 17.9±1.2
41.0 ± 9.7 40.0 ± 4.9
80.7 ± 40 None
157 ± 6 164 ± 7
282 ± 9 3,060 ± 240 292 ± 7 3,130 ± 260
± ±
J
6.6 ± 4 5.2 ± 3
* Prepubertal animals from table 1; all values are plus or minus standard error of mean.
t Fertility was determined by production of offspring.
:j: Each testis and epididymis was assayed separately. Epididymides containing granulomas were treated separately.
§ Testicular tissue was minced and incubated with 'H-cholesterol for 1 hour and testosterone was isolated chromatographically.
humoral antibodies to sperm have been observed in rats, 15 monkeys 16 and man 17 after vasectomy. Long-term (up to 7 years) vasectomy in the rhesus monkey results in an autoimmune response to spermatozoa, which is measured by the presence of several types of sperm agglutinins in the serum and the formation of antigen-antibody reactions in the basal lamina of the epididymis as measured by direct fluorescent antibody techniques. 16 The clinical significance of this type of derangement in the immunological SY.Stem has not been resolved. For example the involvement of delayed hypersensitivity and other immunological parameters should be correlated with the development of these gr:mu1' Riimke, P. and Titus, M.: Spermagglutinin formation in male rats by subcutaneously injected syngeneic epididymal spermatozoa and by vasoligation or vasectomy. J. Reprod. Fertil., 21: 69, 1970. 16 Alexander, N. J.: Vasectomy: long-term effects in the rhesus monkey. J. Reprod. Fertil., 31: 399, 1972. 17 Ansbacher, R.: Sperm-agglutinating and spermimmobilizing antibodies in vasectomized men. Fertil. Steril., 22: 629, 1971.
lomas and whether this might alter deleteriously the immune state and competency of the animal. SUMMARY
Spermatic granulomas were always present 6 months after prepubertal or postpubertal vasectomy in the rat. Neither sperm leakage at the operation nor suture material is a necessary predisposing factor in granuloma formation. These granulomas contain massive accumulations of sperm (0.8 to 1.8 billion) which may account for a considerable portion of the testicular output of sperm during this 6-month period. This might suggest minimal reabsorption of sperm in the epididymis following vasectomy. Although granulomas were most frequently found at the site ofligation, individual granulomas were observed proximally in the vas deferens and epididymis. A spectrum of granulomatous inflammation was observed in these lesions, including the ultimate reabsorption of the sperm by histiocytic
422
KWART AND COFFEY
phagocytes. These lesions are discussed in relation to immunological considerations. ADDENDUM
Since this report was submitted for publication Sackler and associates have reported on the adverse effects of vasectomy on testicular function in the rat. 18 They reported a 30 per cent decrease in wet weight of the testes 28 weeks post-vasectomy and an 18 per cent decrease in urinary 17-ketosteroids at 15 weeks. In the pairs of testes of vasectomized animals, 56 per cent were small, less turgid or soft and were an abnormal purplish color. Sackler, A. M., Weltman, A. S., Pandhi, U. and Schwartz, R.: Gonadal effects of vasectomy and vasoliga·· tion. Science, 179: 293, 1973. 18
No histological data were presented and no mention was made of alterations in spermatogenesis or location of these testes at autopsy. Although they equate a lowered urinary 17-ketosteroid level with decreased testicular function, no mention was made of testosterone production or alteration in size of the sexual accessory tissue. Our study does not confirm the findings of diminished testicular function in either mature or immature vasectomized animals during a comparable time span. The discrepancy in these observations might be caused by errors in ligating the vascular supply to the testes or the possible abdominal location of the testes. Drs. Robert A. Squire and Joseph C. Eggleston were consultants for the histological study and Dr. Michael Bedford provided insight into the problem.
THE JOURNAL OF UROLOGY
Copyright © 1973 by The Williams & Wilkins Co.
SPERM GRANULOMAS: AN ADVERSE EFFECT OF VASECTOMY ARNOLD M. KWART AND DONALD S. COFFEY
From the James Buchanan Brady Urological Institute, The Johns Hopkins Hospital and the Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins School of Medicine, Baltimore, Maryland
Sperm granulomas have been previously described secondary to human sterilization by vasectomy and several large clinical studies report the frequency to be from 5 to 18 per cent. 1 -• However, the occurrence of these lesions has not deterred the popularity of this sterilization procedure nor do granulomas have a known deleterious effect on the patient. The present frequency of human vasectomy demands a clearer insight into the full etiology and implications of these lesions. This information is not readily available because of the generally poor followup data on vasectomized patients. Previous experimental animal studies on the formation of spermatic granulomas have been restricted to resection of the vas deferens without ligation. 4 • 5 In these animals the drainage of the open vas deferens produced a sperm granuloma similar to that observed in vasectomized animals•· 7 and patients. 3 This fact has led to the general concept that sperm granulomas occur in cases in which sperm leaks at operation or subsequent to the accumulation of intraluminal pressure against the suture, resulting in the severance of the vasal wall. We report herein the first long-term animal study of the incidence and mechanism of formation of sperm granulomas in rats following vas ligation. MATERIALS AND METHODS
We used Sprague-Dawley male rats, either prepubertal (40 days, 50 to 70 gm.) or adult (120 days, 250 to 300 gm.). Animals resided in individual Accepted for publication March 23, 1973. Supported by a grant from the Rockefeller Foundation, United States Public Health Service Training Grant AM-056-74-011 and the Louise and Gustavus Pfeiffer Foundation of New York, New York. 1 Glassy, F. J. and Mostofi, F. K.: Sperrnatic granulornas of the epididyrnis. Arner. J. Clin. Path., 26: 1303, 1956. 2 Banerji, T. P.: A study of male sterilization at. Karnpur. Report. on 202 cases of vasectomy. J. Indian Med. Ass., 36: 578, 1961. 3 Schmidt, S. S.: Technics and complications of elective vasectomy. Fertil. Steril., 17: 467, 1966. • Russell, M. and Friedman, N. B.: Studies in general biology of sperm: experimental production of sperrnatic granuloma. J. Urol., 65: 650, 1951. 'Mullaney, J.: Experimental production of sperrnatic granulorna in rats. Nature, 194: 487, 1962. • Poynter, H.: Testis hormone secretion in rat under conditions of vasectomy or isolation. Anat. Rec., 74: 355, 1939. 7 Smith, G.: The effects of ligation of the vasa efferentia and vasectomy on testicular function in the adult rat. J. Endocr., 23: 285, 1962. 416
cages. The animals were anesthetized with pentobarbital and a midline, lower abdominal incision was used to expose the genital tract. Resection of the vas deferens with proximal and distal ligation was performed with 7-zero silk in the prepubertal and 4-zero silk in the postpubertal animals. The testes occupied the scrotum in anatomic position. When indicated animals were mated by placing 2 female rats with each male rat. We determined direct sperm counts in testicular and epididymal tissue according to the method of Amann and Lambiase. 8 The rate of testicular 3 H-testosterone formation was determined by incubation of testicular minces with "H-cholesterol by a modification of the method of Hall and associates.• Thin layer chromatography on silica gel with a solvent system of 9: 1 chloroform to ether was used to isolate the labeled testosterone. The testosterone was acetylated by the procedure of Harper and associates and rechromatographed on alumina oxide gel using 7: 3 cyclohexane to ethyl ether as the solvent. 10 The tissue content of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) was determined by a modification of the technique of Hutchinson and associates 11 as reported by Coffey and associates. 12 Tissue proteins were obtained by the method of Folin-Ciocalteau on a 0.lM NaOH extraction at 70 degrees for 10 minutes. Neutral buffered formalin was used to fix the histologic sections except for the testicular tissue which was fixed in Bouin's solution for 8 hours before placement in 70 per cent ethyl alcohol. Hematoxylin and eosin were used to stain the sections. RESULTS
Experience in our laboratory with more than 100 vasectomized rats has indicated that sperm granu' Amann, R. P. and Lambiase, J. T., Jr.: The male rabbit. 3. Determination of daily sperm production by means of testicular homogenates. J. Anirn. Sci., 28: 369, 1969. • Hall, P. F., Irby, D. C. and De Krester, D. M.: Conversion of cholesterol to androgens by rat testis: comparison of interstitial cells and seminiferous tubules. Endocrinology, 84: 488, 1969. 10 Harper, M. E., Pierrepont, C. G., Famy, A. R. and Griffiths, K.: The metabolism of steroids in the canine prostate and testis. J. Endocr., 49: 213, 1971. 11 Hutchinson, W. C., Downie, E. D. and Murro, H. N.: Factors affecting the Scheider procedure for estimation of nucleic acids. Biochern. Biophys. Acta, 55: 561, 1962. 12 Coffey, D.S., Shirnazaki, J. and Williams-Ashman, H. G.: Polymerization of deoxyribonucleotides in relation to androgen-induced prostatic growth. Arch. Biochern., 124: 184, 1968.
417
SPERM GRANULOMAS: ADVERSE EFFECT OF VASECTOMY
loma 1s invariably present after vasectomy. To determine if the granulomas occurred by leakage of sperm at the time of the operation or before the healing of the ligated site, we designed an experiment using vasectomized prepubertal animals. Immature 40-day-old animals were autopsied and determined to be free of sperm by histological examination of epididymal and testicular tissue. In this prepubertal series, 24 animals were subjected to either sham, bilateral or unilateral vasectomy as indicated in table 1. This prepubertal vasectomy series was compared to 30 adult animals which were sham or bilaterally vasectomized. Both series were autopsied 6 months post-vasectomy. All animals in both series receiving vasectomy presented sperm granulomas on the ligated vas. Table 1 indicates the location of these granulomas and the percentage of animals with multiple granulomas in each vas. All postpubertal vasectomized animals had granulomas at the site of vasectomy. Although all prepubertal vasectomized animals had granulomas, only 46 to 50 per cent occurred at the site of ligation. This observation appears to eliminate leakage of sperm at the operation and suture as the predisposing factor in granuloma formation in the prepubertal vasectomized animals. In addition, another series of postpubertal vasectomized animals had their ·vas occluded by fulguration or ligation with either 4-zero silk, plain catgut or chromic sutures and this did not alter the 100 per cent occurrence of granulomas at the site of ligation. The incidence of granulomas proximal to the site of ligation was also higher m the prepubertal group. In many cases the granulomas occurred in the tail and body of the epididymis (table 1). The average size of all granulomas 6 months following vasectomy was 0.3 to 0.7 gm. A typical lesion is depicted in part A of the figure. In several cases these lesions were very large and approached testicular size (part B of figure). Histology of a typical lesion indicated that it contained massive accumulations of intact spermatozoa (part C of figure). This suggested that following vasectomy the sperm were not completely reabsorbed by the epididymis and were transported and accumulated in the vas deferens. Indeed, it was possible to remove the soft greenish content of the sperm granuloma and to perform a direct sperm count, which indicated that an average lesion at 6 months could contain from 0.8 to 1.8 billion sperm (table 2). Since a single ejaculate of the rat contains 10 to 100 million sperm 13 one of the typical granulomas (17.9 times 10 8) contained from 20 to 200 times this amount of sperm. Since this granuloma accumulated m 6 months and represents the output of only 1 testis, it suggests that the bulk of the rat sperm may not be fully reabsorbed by the epididymis following vasectomy. 13 Lawson, R. L., Krise, G. M. and Sorensen, A. M., Jr.: Electroejaculation and evaluation of semen from the albino rat. J. Appl. Physiol., 22: 174, 1967.
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KW ART AND COFFEY
SPERM GRANULOMAS: ADVERSE EFFECT OF VASECTOMY
419
A, typical location of granulomatous lesions at site of vasectomy. B, example of large granulomas at site of vasectomy and within tail of epididymis. C, early granulomatous inflammation surrounding central mass of sperm. Lesion at site of vasectomy. D 1 , pyogenic granuloma with polymorphonuclear neutrophils invading sperm mass. Increased granulomatous inflammation. H & E, reduced from x 64. D2, histiocytes engulfing spermatozoa during pyogenic phase. Reduced from x 160. E, pronounced granulomatous inflammation surrounding markedly diminished central sperm mass. Reduced from x 64.
420
KW ART AND COFFEY
It is apparent that this process could not continue indefinitely without the continued formation of massive lesions. It has already been noted by others that these granulomas in rats can decrease in size with time. 7 We confirmed this latter finding and observed a marked change in the histological pattern with more pronounced granulomatous inflammation of the lesion (parts C, D,, D2 and E of figure). These lesions reflect a spectrum of changes in the state of granulomatous inflammation that can be described initially as an accumulation of large masses of sperm which are bordered by a rim of epithelioid histiocytes, this rim in turn is encompassed by a perivascular accumulation of round cells. The lesion is limited by a fibrous capsule (part C of figure). The lesion then appears to progress to a pyogenic granuloma with a marked influx of polymorphonuclear neutrophils into the mass of sperm (part D 1 of figure). This is accompanied by a destruction of the central mass and a more pronounced histiocytic response with active phagocytosis of sperm by the histiocytes (part D 2 of figure). This process results in a smaller lesion (in some cases microscopic) which is a classical granuloma containing large numbers of epithelioid histiocytes with Langhans' giant cells (part E of figure). The sperm mass is considerably diminished at this point. This pathological process described for sperm granulomas in rats following vas ligation is similar to that observed by Glassy and Mostofi in humans with idiopathic spermatic granulomas of the epididymis. 1 This process is also suggestive of the lesions following human vasectomy mentioned by Schmidt." In our experiments it was possible to observe multiple granulomas in a rat vas at different stages of inflammation. This suggested that as some lesions became quiescent they were accompanied by other granulomas foci which became the reservoir of sperm accumulation. In some cases separate lesions appeared in the epididymal size (table 3). If granulomas were to continue to extend into the efferent ducts of the testes they could disrupt testicular function because others have shown that ligation of the vasa efferentia produces degeneration of the seminiferous tubules. 7 We did not observe this histologically and no alterations were seen in testicular function. It is important to note that following vasectomy TABLE
Lesion
1 2
2. Composition of 2 typical granulomas at 6 months post-vasectomy* Total Weight (mg.) 406 898
Weight Empty Capsule (mg.)
Weight Central Mass (mg.)
Total Sperm Count in Central Mass
102
304 729
8.4 X 10 8 17.9x10'
170
* Two typical size granulomas (similar to part C of figure) were selected for analysis.
some form of an equilibrium must occur ultimately between sperm production and sperm reabsorption. This is of particular significance since in our experiments testicular function and sperm formation were not altered 6 months following vasectomy. This was determined by measurements of sperm counts in the epididymis and testes, rate of testosterone synthesis, testicular content of DNA, RNA and protein (table 4) and comparative histology of the genital organs. Indeed, prepubertal animals were bred and the unilaterally vasectomized rats were fertile. This would be consistent with previous studies which show no significant changes in testicular function following vasectomy.•. 7 • 14 Lesions cultured yielded no infectious etiology. DISCUSSION
Early studies on the effects of vasectomy have focused primarily on testicular function as reflected in changes in the gross size of sexual accessory glands and in the level of spermatogenesis. 6 • 7 • 14 Although spermatoceles were reported in several of these studies, they received minimal attention. Previous studies designed to evaluate sperm granulomas have not used vasectomy but instead have used animal models in which the vas was severed, not ligated, and allowed to drain from the open end directly in the scrotal sac. 4 •, 5 No previous reports have described the frequency, distribution and etiology of sperm granulomas in a vasectomized animal which might be applicable to a similar situation occurring in humans following vasectomy. The frequency and location of sperm granulomas in vasectomized patients have not been carefully studied. Studies have been made of idiopathic sperm granulomas of the epididymis and 2 of 60 patients had undergone vasectomy.' Other investigators described cases of spermatic granuloma after vasectomy. 2 • 3 It is the consensus that these lesions are not rare. It is important that the lesions we describe in the rat are always present after vasectomy, have been observed without exception in more than 100 rats and that the pathology of the lesions is similar to that described by Mostofi for human idiopathic epididymal granuloma. 1 The appearance of granuloma after human vasectomy rarely causes acutely serious clinical problems. This is probably because the granulomatous lesions become limiting in size through a more organized granulomatous inflammation producing diminution in gross size of the lesion. However, the clinical importance of these lesions may be the implication that the reabsorption of sperm through this inflammatory process might trigger alterations in the immunological states of the animal. Indeed, sperm are known to be strong autoantigens and "Kar, A. B., Chandra, H. and Kamboj, V. P.: Long-term effect of vasectomy on the gonad-pituitary system of rats. Acta Biol. Med. German., 15: 381, 1965.
421
SPERNi GRANULOTvIAS: ADVERSE EFFECT OF VASECri'0I\1Y TABLE
3. Effect of prepubertal and postpubertal vasectomy on size of rat genital organs* Genital Organ Weight Single Epididymis Body Weight (gm.)
Ventral Prostate
Seminal Vesicle
Without Granuloma
Testist
With Granuloma
%of Epididymides
Weight
%of Epididymides
4 6 8 7
100 82 67 100
0 221 ± 7 230 ± 24 0
0 18 33 0
122 ± 3 116 ± 4
100 75
0 250
0 25
Weight (mg. per 100 gm. body weight) Prepubertal vasectomy: Control, sham Bilateral vasectomy Right unilateral vasectomy
468 ± 16 466 ± 10 446 ± 13
138 ± 9 144 ± 11 148 ± 28
86 ± 4 89 ± 4 92 ± 5
367 342 (R) 331 (L) 334
Postpubertal vasectomy: Control, sham Bilateral vasectomy
493 ± 11 523 ± 9
101 ± 15 90 ± 6
69 ± 2 61 ± 2
339 326
± 13 ± 10 ± 15 ± 21 ± ±
11 9
137 ± 121 ± 104 ± 127 ±
±
115
* All animals were sacrificed 6 months post-vasectomy; all values plus or minus standard error of mean. t Each testis and epididymis was individually weighed. Epididymides containing granulomas were treated separately.
TABLE
4. Six-month effects of prepubertal vasectomy on testicular function* Organ Sperm Count:j:
'H-Testosterone Production From 'H Cholesterol§
Testicular Content
Fertilityt Single Epididymis Testis
Without Granuloma
With Granuloma
(10' sperm per 100 mg. tissue) Control, sham (n - 9) Bilateral vasectomy (n - 6) Unilateral vasectomy (n - 9) Ligated (rt.) Intact (lt.)
8/9 0/6 9/9
DNA
RNA
Protein
(µg. per 100 mg. testis)
(10' cpm per testis per hour x) 7.4 ± 5 120 180 9.2 ± 3
19.2 ± 1.2 18.1 ± 0.7
50.1 ± 2.3 37.3 ± 1.4
None 92.2 ± 50
155± 4 149 ± 6
279 ± 913,080 270 ± 7 2,860
17.3 ± 0.9 17.9±1.2
41.0 ± 9.7 40.0 ± 4.9
80.7 ± 40 None
157 ± 6 164 ± 7
282 ± 9 3,060 ± 240 292 ± 7 3,130 ± 260
± ±
J
6.6 ± 4 5.2 ± 3
* Prepubertal animals from table 1; all values are plus or minus standard error of mean.
t Fertility was determined by production of offspring.
:j: Each testis and epididymis was assayed separately. Epididymides containing granulomas were treated separately.
§ Testicular tissue was minced and incubated with 'H-cholesterol for 1 hour and testosterone was isolated chromatographically.
humoral antibodies to sperm have been observed in rats, 15 monkeys 16 and man 17 after vasectomy. Long-term (up to 7 years) vasectomy in the rhesus monkey results in an autoimmune response to spermatozoa, which is measured by the presence of several types of sperm agglutinins in the serum and the formation of antigen-antibody reactions in the basal lamina of the epididymis as measured by direct fluorescent antibody techniques. 16 The clinical significance of this type of derangement in the immunological SY.Stem has not been resolved. For example the involvement of delayed hypersensitivity and other immunological parameters should be correlated with the development of these gr:mu1' Riimke, P. and Titus, M.: Spermagglutinin formation in male rats by subcutaneously injected syngeneic epididymal spermatozoa and by vasoligation or vasectomy. J. Reprod. Fertil., 21: 69, 1970. 16 Alexander, N. J.: Vasectomy: long-term effects in the rhesus monkey. J. Reprod. Fertil., 31: 399, 1972. 17 Ansbacher, R.: Sperm-agglutinating and spermimmobilizing antibodies in vasectomized men. Fertil. Steril., 22: 629, 1971.
lomas and whether this might alter deleteriously the immune state and competency of the animal. SUMMARY
Spermatic granulomas were always present 6 months after prepubertal or postpubertal vasectomy in the rat. Neither sperm leakage at the operation nor suture material is a necessary predisposing factor in granuloma formation. These granulomas contain massive accumulations of sperm (0.8 to 1.8 billion) which may account for a considerable portion of the testicular output of sperm during this 6-month period. This might suggest minimal reabsorption of sperm in the epididymis following vasectomy. Although granulomas were most frequently found at the site ofligation, individual granulomas were observed proximally in the vas deferens and epididymis. A spectrum of granulomatous inflammation was observed in these lesions, including the ultimate reabsorption of the sperm by histiocytic
422
KWART AND COFFEY
phagocytes. These lesions are discussed in relation to immunological considerations. ADDENDUM
Since this report was submitted for publication Sackler and associates have reported on the adverse effects of vasectomy on testicular function in the rat. 18 They reported a 30 per cent decrease in wet weight of the testes 28 weeks post-vasectomy and an 18 per cent decrease in urinary 17-ketosteroids at 15 weeks. In the pairs of testes of vasectomized animals, 56 per cent were small, less turgid or soft and were an abnormal purplish color. Sackler, A. M., Weltman, A. S., Pandhi, U. and Schwartz, R.: Gonadal effects of vasectomy and vasoliga·· tion. Science, 179: 293, 1973. 18
No histological data were presented and no mention was made of alterations in spermatogenesis or location of these testes at autopsy. Although they equate a lowered urinary 17-ketosteroid level with decreased testicular function, no mention was made of testosterone production or alteration in size of the sexual accessory tissue. Our study does not confirm the findings of diminished testicular function in either mature or immature vasectomized animals during a comparable time span. The discrepancy in these observations might be caused by errors in ligating the vascular supply to the testes or the possible abdominal location of the testes. Drs. Robert A. Squire and Joseph C. Eggleston were consultants for the histological study and Dr. Michael Bedford provided insight into the problem.