Cellular responses to vasectomy

CYTOLOGY V199 - AP - 4993 / c6-295 / 04-03-00 10:08:13

Cellular Responses to Vasectomy Stuart W. McDonald Laboratory of Human Anatomy, University of Glasgow, Glasgow G12 8QQ, Scotland

A number of cell populations in the reproductive tract show a response to vasectomy. Some cell types show similar responses in man and all laboratory species, whereas others show marked species variations. This chapter describes these effects in a broadly chronological order and, in a general way, considers changes close to the site of vasectomy first and the longer term effects on the testis itself later. Following vasectomy, epididymal distension and sperm granuloma formation result from raised intraluminal pressure. The sperm granuloma is a dynamic structure and a site of much spermatozoal phagocytosis by its macrophage population. In many species, spermatozoa in the obstructed ducts are destroyed by intraluminal macrophages, and degradation products, rather than whole sperm, are absorbed by the epididymal epithelium. Humoral immunity against spermatozoal antigens following vasectomy is well established and there is evidence of modest T-lymphocyte activity. The role of lymphocytes in the reproductive tract epithelium and interstitium following vasectomy is poorly defined. In laboratory animals, there is evidence that pressure-mediated damage to the seminiferous epithelium can follow sperm granuloma formation and obstruction in the epididymal head. However, the contribution of lymphocytes and antisperm antibodies to testicular damage after vasectomy is far from clear. A number of studies have suggested that testicular changes may follow vasectomy in man but their validity and mechanism of occurrence require further study. KEY WORDS: Vasectomy, Testis, Epididymis, Ductus deferens, Sperm granuloma, Macrophages, Seminiferous epithelium, Epididymal epithelium, Orchitis, Antisperm antibodies, Immune complexes. 䊚 2000 Academic Press.

I. Introduction The effects of vasectomy are incompletely understood. For most men vasectomy gives few problems. Like all minor surgery, it carries the immediate International Review of Cytology, Vol. 199 0074-7696/00 $30.00

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Copyright 䉷 2000 by Academic Press All rights of reproduction in any form reserved.

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risks of bruising and infection. In the longer term, about 5% of patients experience pain or discomfort associated with distension and leakage of the epididymis and/or ductus (vas) deferens. For most patients, this settles with conservative management. A few men, however, suffer persistent and troublesome pain which may ultimately require further surgery, even excision of the epididymis and sometimes the testis as well (McDonald, 1996). Vasectomy gained widespread popularity as a method of contraception in the late 1960s and early 1970s. At that time there was little thought that it might be harmful to health. Over the past 30 years, there have been several scares about its safety, particularly in relation to cardiovascular disease, testicular cancer, and prostatic cancer (McDonald, 1997). The local effects of vasectomy on the reproductive tract in man are not known in detail. Because most men have few problems following the procedure, gross and microscopical examination are usually only carried out in individuals with marked symptoms and, almost certainly, distinct pathological changes. The degree of change in the reproductive tracts of the majority of vasectomized men who have no subsequent problems remains little documented. Knowledge of morphological and cellular changes following vasectomy has come largely from animal studies.

II. Structural Aspects A. The Effect of Interruption of the Ductus Deferens Vasectomy entails the removal of a small section of the ductus deferens and sealing the severed ends. It is soon followed by a rise in intraluminal pressure in the obstructed portion of the ductus deferens. This results from the continued production of spermatozoa and fluid by the testis and their transmission to the epididymis. The evidence for this comes from vasectomized hamsters in which Howards and Johnson (1979) directly measured the intraluminal pressure. They found marked elevation of the pressure in the obstructed ductus deferens and the adjacent tail of the epididymis. Intraluminal pressure in the head of the epididymis and in the seminiferous tubules, however, remained similar to that of control animals. The differential pressure along the epididymal duct may be related to peristaltic activity of the myoid cells surrounding the duct. It is likely that similar rises in intraluminal pressure occur in other species and that they are responsible for distension and leakage of the epididymis and ductus deferens. In all species, sperm leaking from the reproductive tract becomes localized in grossly visible inflammatory masses called sperm granulomas (Fig. 1).

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FIG. 1 Rat reproductive tract following vasectomy. The testis (T) appears healthy and the coils of the epididymal duct (E) contain sperm. The ligatures at the vasectomy site are shown at L. The empty ductus deferens and its accompanying blood vessels lie at D. A large granulomas (G) has formed in the tail of the epididymis. Two smaller granulomas (g1 & g2) have formed on the testicular side of the large granuloma. Reproduced from McGinn et al. (2000) by permission of the John Wiley & Sons, Inc.

Following vasectomy, the rabbit is particularly noted for showing gross distension of the excurrent ducts, especially of the cauda epididymidis and ductus deferens ( Jones, 1973). In the rabbit, leakage is generally a very late event (Alexander and Tung, 1977; Bedford, 1976). The rat, in contrast,

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displays little distension of its epididymal duct (Alexander, 1973) but invariably shows rupture and leakage of spermatozoa from the epididymis or obstructed ductus deferens within 3 weeks of the operation, provided the testes are healthy. The difference between rabbits and rats in their response to vasectomy seems to relate to the distensibility of the excurrent ducts. Any distension of the rat tract tends to occur in the cauda (Flickinger et al., 1990) and is reported prior to granuloma formation (Neaves, 1973; Feller et al., 1986). Mice seem to differ from rats in their response to vasectomy and little sperm granuloma formation is evident at 5 weeks after vasectomy (Croft and Bartke, 1976). A group of hamsters had granulomas by 5 months after vasectomy (Flickinger, 1981). Guinea pigs frequently do not show granulomas, even 3 years after vasectomy. Distension of the epididymal duct may occur in some guinea pigs (Figs. 2 and 3) but is minimal in others (Beaton et al., 1995). Humans show a mixed picture of rupture and distension. Lemack and Goldstein (1996) palpated granulomas at the vasectomy site on one or other side in 40% of patients. At vasectomy reversal, sperm granulomas were found at the vasectomy site in about 30–60% of human ductus deferens (Lee, 1986). Of Owen and Kapila’s vasovasostomy patients, 52% had granulomas, although most were small (Owen and Kapila, 1984). Shapiro and Silber (1979) mentioned that, in their large experience of vasectomy reversal, they always found some epididymal distension. Similarly, Errey and Edwards (1986) described the ductus deferens and epididymis to be typically grossly dilated at vasectomy reversal. Of the human epididymides studied by Pardanani et al. (1976), 68% appeared full, turgid, and distended. Information is scant on the incidence of epididymal granulomas in man but ultrasound studies suggest that they are probably common ( Jarvis and Dubbins, 1989; McMahon et al., 1992).

B. The Sperm Granuloma 1. Sperm Granulomas In all species, when the epididymis or ductus deferens ruptures, extravasated spermatozoa form a cream colored nodule at the site of leakage. That the escaped spermatozoa remain localized is probably due to their being contained deep to the serosa covering the epididymis and ductus. The nodule forms a chronic inflammatory mass and is known as a sperm granuloma. A sperm granuloma commonly forms at the obstructed end of the ductus deferens on the testicular side of the vasectomy site. They also occur in the epididymis regardless of whether or not one is present at the vasectomy site.

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FIG. 2 Guinea pig left testis and epididymis 3 years after left unilateral vasectomy. The testis (T) appears healthy. The coils of duct in the epididymal tail (E) are distended. The head and body of the epididymis lie in the fat body (F). An arrow points to the vasectomy site. Reproduced from Aitken et al. (1999) by permission of the John Wiley & Sons, Inc.

Sperm granulomas are rounded or irregular in shape and range in diameter from about 1 mm to 1 cm or more, even in small species such as the rat. In the rat, single vasal granulomas generally increase in size for the first few months at least (Lopes and Hayashi, 1981). When first formed, they are soft but seem to become firmer with time because of fluid resorption (personal observation). Sperm granulomas consist of a central mass of degenerating spermatozoa, surrounded by a layer of epithelioid macrophages, surrounded in turn by a layer of loose vascular connective tissue rich in lymphocytes and plasma cells (Fig. 4). They closely resemble the granulomas of tuberculosis, except that their central content consists of

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FIG. 3 Guinea pig left testis 3 years after control sham operation. The testis (T) and epididymal tail (E) are healthy. One of the loose ligatures around the ductus deferens is visible (arrow). The head and body of the epididymis lie in the fat body (F). Reproduced from Aitken et al. (1999) by permission of the John Wiley & Sons, Inc.

spermatozoa rather than caseation (Mullaney, 1962). Berg (1954) noted that spermatozoa contain fatty acids similar to the mycolic acid released by tubercle bacilli, and this may explain the close resemblance. Sperm granulomas are important sites of spermatozoal destruction (Tait et al., 1999) and presentation of spermatozoal antigens to the immune system (Lewis and McDonald, 1992), However, in all species, serum antisperm antibodies are frequent and seem not to be dependent on the presence of

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FIG. 4 Typical light micrograph of the wall of a sperm granuloma 3 months after vasectomy in the rat, showing the central sperm mass (S), macrophage layer (M), and the connective tissue layer (C). The macrophages possess euchromatic nuclei with prominent nucleoli and contain dark vacuoles in their cytoplasm. Occasional giant cells are present. Lymphocytes and plasma cells infiltrate the connective tissue layer. Resin; Toluidine blue. ⫻ 250. Reproduced from Tait et al. (2000) by permission of the John Wiley & Sons, Inc.

sperm granulomas; individuals without granulomas can show antisperm antibodies (Alexander and Schmidt, 1977; Alexander and Tung, 1977). 2. Failure of Vasectomy In clinical practice, a range of techniques is used to ensure that, at vasectomy, the cut ends of the ductus deferens are effectively closed. Inadequate occlusion may result in spontaneous reanastomosis of the ductus and failure of vasectomy. There are, of course, several other reasons why vasectomy may appear to fail: sexual infidelity, failure to obstruct and divide the ductus deferens, and intercourse before spermatozoal clearance. True failure is rare, less than 0.1 per 100 women years (Vessey et al., 1982), although one study claimed to find spermatozoa in ejaculates of 9% of men with vasectomies of long standing (Lemack and Goldstein, 1996). Nevertheless, it is well documented that the reproductive tract can reestablish itself across the vasectomy site. The mechanism by which this occurs is not wholly

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understood but may be related to sperm granulomas bridging the gap between the cut ends of the ductus. Frequently, a mass of loose connective tissue with a content of multiple regenerating tubules is seen at the vasectomy site. This lesion is called vasitis nodosa and is regarded as an attempt at repair by the ductus deferens. The regenerating tubules are thought to establish connections with the lumens of both portions of the ductus and restore the passage of spermatozoa (Taxy et al., 1981). 3. Sperm Granulomas—An Advantage or Disadvantage? In the late 1970s, there was much controversy as to whether the formation of sperm granulomas was advantageous or disadvantageous to the patient. Schmidt (1979) recognized that sperm granulomas could be painful but it became clear that many patients had symptomless granulomas ( Jarvis and Dubbins, 1989). The painful ones may have formed adjacent to and irritated local nerves (Chen and Ball, 1991; Choa and Swami, 1992). It is also generally believed that sperm granulomas dissipate pressure within the lumen of the obstructed reproductive tract. Firm evidence for this was provided by Howard and Johnson’s (1979) studies on pressure within the excurrent ducts of vasectomized hamsters and guinea pigs. Recent studies by my own group on the rat (McGinn et al., 2000), however, suggested that the situation may be more complex because we found examples of epididymal ducts with areas of luminal distension and epithelial thinning but no communication with their associated granulomas. This suggested intermittent spermatozoal leakage interspersed with periods of raised intraluminal pressure (see later). The prognosis for fertility following vasectomy reversal is better in men with a vasal granuloma (Silber, 1978). Presumably its presence, for a time at least, protects the epididymis by reducing disruption of the epididymal architecture through distension and granuloma formation there. Occasionally workers have fallen into the trap of seeing degenerated testes and absent granulomas and falsely concluding that the testis had succumbed because intraluminal pressure was raised because of a failure of granuloma formation (Kuwahara and Frick, 1975; Sun et al., 1992). In these papers, it was much more probable that the granuloma had not formed because the testis had degenerated prior to a rise in intraluminal pressure. 4. Life History of the Sperm Granuloma In the rat, sperm granulomas start to form by 2 weeks after vasectomy. My group carried out a scanning and transmission electron microscopical study on sperm granulomas of Albino Swiss rats sacrificed 14 days after vasectomy (Caldwell et al., 1996). Our findings were consistent with the origin of the macrophages from monocytes. The young macrophages were most

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commonly seen in the inner part of the macrophage layer. Transmission electron microscopy indicated that phagocytosis occurs when the young macrophages resemble monocytes (Fig. 5). The scanning electron micro-

FIG. 5 Transmission electron micrograph showing 3 monocyte-like cells in the wall of a newly forming sperm granuloma 14 days after vasectomy. Each has a lobulated nucleus and a folded surface. One is ingesting spermatozoal fragments (arrows) and is therefore classified operationally as a macrophage. Bar ⫽ 10 애m. Reproduced from Caldwell et al. (1996) by permission of the John Wiley & Sons, Inc.

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graphs demonstrated that such cells have a folded surface showing short ridges. These cells appeared to be wrapped around the spermatozoa, which are too large to be engulfed (Fig. 6). Macrophages closer to the connective tissue layer of the granuloma wall were larger and their contents indicated that considerable phagocytosis of spermatozoa was already established (Fig. 7). It is likely that the monocytes had entered the granuloma wall from the bloodstream via the numerous fine blood vessels seen grossly and histologically in the wall. Presumably they had passed between the more differentiated macrophages to reach a position adjacent to the degenerating spermatozoa. Many of the most peripherally placed macrophages of the granuloma wall had the appearances of epithelioid macrophages. By studying serial wax sections of epididymides several months after vasectomy in the rat, my group (McGinn et al., 2000) was able to comment on three further aspects of the life history of sperm granulomas: (i) the sites of tubular leakage and granuloma formation, (ii) the central sperm mass, and (iii) the macrophage layer. We were able to find sites of discharge of spermatozoa from the epididymal duct to the sperm granuloma in only two of our seven serially sectioned left epididymides collected 6–9 months after left unilateral vasectomy (Fig.

FIG. 6 Scanning electron micrograph of inner surface of a newly formed rat sperm granuloma wall following washing. Macrophages wrapped around spermatozoa. The sickle-shaped heads of rat spermatozoa are shown (arrows). Bar ⫽ 10 애m. Reproduced from Caldwell et al. (1996) by permission of the John Wiley & Sons, Inc.

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FIG. 7 Transmission electron micrograph of a typical macrophage (M) in the wall of a newly formed sperm granuloma in rat. Its nucleus is euchromatic, and the nucleolus is prominent. A fragment of a sperm head (H) and several profiles of sperm tails (arrows) are visible in the cytoplasm. A monocyte-like cell (m) lies nearby. Bar ⫽ 10 애m. Reproduced from Caldwell et al. (1996) by permission of the John Wiley & Sons, Inc.

8). The sections were 7 애m thick and every fifth section was mounted. We were, therefore, confident that no sites of entry of sperm into granulomas has been missed because of histological technique. At the two sites of continuity of the epididymal duct with the central sperm mass of the granuloma, the epididymal epithelium thinned. The epithelial cells became less

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FIG. 8 Profile of epididymal duct (E) of vasectomized rat leaking into a sperm granuloma (G). The granuloma has a homogeneous content of spermatozoa, although some leukocytes (L) lie near the site of leakage. The macrophage layer of the granuloma wall (M) bounds the sperm mass. Peripheral to the macrophages, the connective layer (C) is infiltrated by lymphocytes and plasma cells. H & E. ⫻ 80. Reproduced from McGinn et al. (2000) by permission of the John Wiley & Sons, Inc.

regularly shaped and gradually merged with the macrophage layer of the granuloma wall. It was particularly interesting that five of the specimens showed no continuity between the epididymal duct and the central sperm masses of the granulomas. Only one of these rats showed a vasal granuloma which may have been draining spermatozoa. The presence of multiple epididymal granulomas shows that there had been abundant spermatozoal leakage but

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that the breaches in the epididymal epithelium must have sealed off and healed. Lying close to granulomas, we sometimes observed profiles of the epididymal duct with thin epithelium but no actual breach. In three of the five specimens, however, we found localized distended regions of the epididymal duct with thinned and broken epithelium but no significant spermatozoal escape (Fig. 9). The sperm appeared to be retained by the connective tissue elements beyond the epithelium. Two of these three

FIG. 9 Part of a profile of a distended region of the epididymal duct in a vasectomized rat. The epithelium (E) is thinned and locally breached (arrows). At the breach, spermatozoa contact adjacent connective tissue (C). Neutrophils (N) are invading the luminal sperm. PAS & H. ⫻ 225. Reproduced from McGinn et al. (2000) by permission of the John Wiley & Sons, Inc.

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epididymides were associated with functional testes. That the testes of two of the rats were producing spermatozoa and fluid but that these were not entering a granuloma suggests that drainage into granulomas may be intermittent rather than continuous, at least in rats after long-term vasectomy and with multiple granulomas. The local distension and epithelial thinning probably result from elevated intraluminal pressure, the product of the continued production of spermatozoa and fluid by the testis and the activity of the myoid cells of the tract. A further unexpected observation was that the two examples of extravasation of spermatozoa were into the granulomas of the epididymal tail rather than those of its body which were present in both individuals. We had previously assumed that, when multiple granulomas were present, the one closest to the testis would be the most recent and the one into which the sperm would be draining. The results suggest that granulomas in the epididymal body receive sperm only intermittently and that the adjacent epididymal duct remains patent. This observation also explains why, in my experience, granulomas of the epididymal tail are often large, whereas those in the epididymal body are always small. My group has also started to provide information about age changes within individual granulomas (McGinn et al., 2000). Those granulomas receiving spermatozoa from the epididymal duct exhibited a fairly even distribution of sperm heads in the central mass. However, in one of these, the spermatozoa farther from the site of leakage were more densely packed, suggestive of spermatozoal concentration by resorption of fluid. Many of the other granulomas showed clumping of spermatozoa (Fig. 10) which I suggest indicates further fluid resorption and stagnation of flow. We wondered whether the clumping of spermatozoa in some granulomas could represent a fixation artefact, as the material in the study was fixed by immersion. To test this hypothesis, we perfused two additional rats with fixative and found that the clumping was just as marked, indicating that the appearance was not due simply to delay in fixation. In addition, the following features noted on the serial sections in the main study suggested that the clumping was genuine. (1) In an individual specimen, clumping was marked in some granulomas and was not present in others. (2) In granulomas showing clumping, the appearance was just as marked at the periphery of the granuloma, readily accessible to the fixative, as in its center. (3) Spermatozoa in the epididymal duct did not show clumping. (4) Those granulomas shown to be receiving spermatozoa from a ruptured epididymal duct, and where spermatozoa were presumably relatively fresh, did not show clumping. The macrophage layer of the granuloma wall often showed villus-like projections into the central sperm mass (Fig. 10). They did not occur in all granulomas but were seen both in those with a homogeneous content and

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FIG. 10 Section of the wall of a sperm granuloma in a vasectomized rat. The central sperm mass (S) shows distinct ovoid clumps of spermatozoa. The macrophage layer (M) is thrown into villus-like folds whose connective tissue cores (C) contain numerous lymphocytes. PAS & H. ⫻ 110. Reproduced from McGinn et al. (2000) by permission of the John Wiley & Sons, Inc.

in those with clumped sperm. The connective tissue core of the projection was rich in lymphocytes and plasma cells. One of the granulomas in continuity with the epididymal duct showed this feature suggesting that it probably does not represent granuloma involution. We have noted this feature previously in granulomas at 3 months after vasectomy and found the lymphocytes to be mostly helper T-cells (Caldwell et al., 1996). The lessons of our study in the rat (McGinn et al., 2000) may also apply to vasectomized patients and are ones that urologists should heed. The results make clear that, when multiple granulomas form after vasectomy, the dynamics of spermatozoal flow through the epididymal duct are more complex than generally realized. They also show that the sperm granuloma is a changing structure and that the one closest to the testis is not necessarily that which is draining spermatozoa and presumably enlarging.

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5. Lymphocytes of the Epididymis and Sperm Granuloma Ritchie et al. (1984) demonstrated many intraepithelial lymphocytes in the normal epididymis and found that, in humans, they are mostly suppressor T-cells. The observation was confirmed by El-Demiry et al. (1985) and a role in the prevention of an immune response was suggested. El-Demiry et al. (1985) also found that in the human epididymal interstitium helper T-cells were the more common phenotype. Nashan et al., 1990, working in mouse, found helper T-cells in the interstitium but not the epithelium of healthy epididymides. After vasectomy, the number of helper Tlymphocytes increased in both locations. The number of cytotoxic/suppressor T-cells, however, seemed similar between vasectomized and control mice. Hooper et al. (1995) found that in the head of the epididymis of Wistar rats, helper T-lymphocytes and suppressor/cytotoxic lymphocytes were both more frequent in the interstitium than in the epididymal epithelium. Cytotoxic lymphocytes were significantly more common in the epididymal tail. Following vasectomy, within the epididymal head cytotoxic T-cells increased in number in the epithelium and there was a decrease in helper T-cells and macrophages in the interstitium. Clearly, these finding do not match those of Nashan et al. (1990) in the mouse. Even though I have not looked in detail, my impression from the vasectomized Albino Swiss rats on which I work is that there is little inflammatory cell infiltration in the epididymis at sites remote from sperm granulomas. I was, therefore, surprized to read the accounts by Nashan et al. (1990) and Hooper at al. (1995) of significant leukocyte invasion unassociated with granulomas. I had noticed that Flickinger et al. (1990, 1993) had similarly described epididymal leukocyte invasion but no granulomas. Initially, I thought they had simply failed to notice or report the granulomas but review of these papers has made me reconsider. The Lewis rat seems to be generally more immunogenic after vasectomy than other strains and perhaps they, along with Wistars, show inflammatory responses absent in Albino Swiss rats. The lymphocytes of the sperm granuloma wall which accumulate peripheral to the macrophage layer were mostly helper T-cells, in the rat at least (Caldwell et al., 1996). This observation suggests a role for the granuloma in the genesis of immune responses to spermatozoa rather than in their suppression. The lymphocytes were present in appreciable numbers by 3 weeks after vasectomy. Counts of the lymphocytes in random frozen sections up to 3 months after vasectomy indicated that more than 20% of the cells in the regions of the connective tissue layer sampled were Tlymphocytes. At all intervals, T-lymphocytes were markedly more numerous than B-lymphocytes. Jessop and Ladds (1995) carried out a study similar to ours but on the sperm granulomas of vasectomized rams. They also found helper T-lymphocytes to be about twice as frequent as cytotoxic/suppressor cells, a ratio which concurs with our own findings in the rat. Plasma cells were also numerous.

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C. Macrophages 1. Macrophages of the Granuloma Wall Several studies have provided clear evidence of phagocytosis of spermatozoa by macrophages of the wall of the sperm granuloma in a number of species (Flickinger, 1975; Bedford, 1976; Galle and Friend, 1977; Chapman and Heidger, 1979; Kennedy and Heidger, 1980; Lopes and Hayashi, 1981; Caldwell et al., 1996). However, only a few studies have given a detailed ultrastructural description of the macrophages: Galle and Friend (1977) in guinea pigs, Chapman and Heidger (1979) in monkeys and man, Kennedy and Heidger (1980) in Sprague-Dawley rats, Tait et al. (2000) in Albino Swiss rats. They all found that the macrophages form a distinct layer in the granuloma wall, external to the central mass of extravasated spermatozoa. The findings by my own group (Tait et al., 2000) at 3 months after vasectomy in the Albino Swiss rat seem typical. Transmission electron micrographs of the macrophages close to the spermatozoa showed much phagocytosis (Fig. 11). Many of the engulfed sperm tails were surrounded

FIG. 11 Transmission electron micrograph of several macrophages in the wall of a sperm granuloma 3 months after vasectomy in the rat. Many cross-sectional profiles of spermatozoal tails are seen in their cytoplasm. Microridges are a striking feature of much of the cell surfaces. They vary in length but are of uniform thickness and most interdigitate with those on adjacent cells. Bar ⫽ 5 애m. Reproduced from Tait et al. (2000) by permission of the John Wiley & Sons, Inc.

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by a membrane. In these, the internal features of the flagellum of the sperm tail often remained in good condition. Others, however, had lost their membranes and the components of the sperm tails were degenerating. In these young granulomas, monocytes were frequent (Fig. 12). Many of the macrophages had two, three, or more oval, euchromatic nuclei. Farther from the sperm mass, we found the largest macrophage forms, giant cells of the foreign body and Langhans types (Fig. 13). Interestingly, they retained evidence of phagocytic activity in the form of sperm fragments (Fig. 14). It is generally held that giant cells have little phagocytic activity. Sperm fragments were certainly relatively fewer than in the smaller macrophages. Perhaps their presence reflects resistance to breakdown, said to be caused by disulfide bonds in spermatozoal components (Bedford, 1976). In addition to these general and widely reported features, we noticed that macrophages, both mono- or multinucleate, frequently showed numerous surface microridges that interdigitated with those of neighboring cells (Fig.

FIG. 12 Transmission electron micrograph of a monocyte in the macrophage layer of a sperm granuloma 3 months after vasectomy in the rat. It has a horseshoe-shaped nucleus and finely granular cytoplasm containing occasional mitochondria, rough endoplasmic reticulum, and lysosomes. Bar ⫽ 1 애m. Reproduced from Tait et al. (2000) by permission of the John Wiley & Sons, Inc.

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FIG. 13 Transmission electron micrograph of a giant cell of the Langhans type in the periphery of the macrophage layer of a sperm granuloma 3 months after vasectomy in the rat. It has many irregularly shaped euchromatic nuclei (N). Longitudinal sections of sperm tails are present in the cytoplasm (arrows). The outer surface of the cell shows several layers of folds (arrow heads). Bar ⫽ 5 애m. Reproduced from Tait et al. (2000) by permission of the John Wiley & Sons, Inc.

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FIG. 14 High power electron micrograph of cytoplasm of the giant cell shown in Fig. 7. It contains mitochondria (m), lysosomes (1), rough endoplasmic reticulum (r) and sperm tails (arrows), which show various degrees of disintegration. Bar ⫽ 0.5 애m. Reproduced from Tait et al. (2000) by permission of the John Wiley & Sons, Inc.

15). The microridges varied in length, but were of uniform thickness and cytoplasmic content, and were rarely branched. They have been reported previously in the sperm granulomas of men and monkeys (Chapman and Heidger, 1979) and of Sprague-Dawley rats (Kennedy and Heidger, 1980), as well as at sites of macrophage infiltration of the epididymal interstitium (Flickinger et al., 1993). They have also been documented by scanning and transmission electron microscopy in other types of granuloma (Papadimitriou et al., 1973; Papadimitriou and Archer, 1974; Parakkal et al., 1974). Tait et al. (2000) also noted mononucleate cells in the connective tissue external to the macrophage layer. Their cytology suggested that they were macrophages but they showed much less phagocytic activity than those of the main macrophage layer and had distinctive aggregations of moderately electron-dense droplets. They probably corresponded to the ovoid cells rich in periodic acid-Schiff positive granules seen in our light microscopical material (McGinn et al., 1999). Probably they were macrophages which had become redundant because of their distance from the spermatozoal mass.

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FIG. 15 High power electron micrograph showing interdigitating microridges from two adjacent macrophages in a rat sperm granuloma 3 months after vasectomy. Sperm tails can be identified in the cytoplasm (arrows). Bar ⫽ 1 애m. Reproduced from Tait et al. (2000) by permission of the John Wiley & Sons, Inc.

2. Intraluminal Macrophages Even though sperm granulomas are clearly very important centers of spermatozoal phagocytosis following vasectomy, they are not the only sites of such disposal. In vasectomized men (Phadke, 1964; Ball and Mitchinson, 1984) and most species used in experimental work (monkeys: Alexander, 1972; rabbits: Bedford, 1976, Alexander and Tung, 1977; hamsters: Bedford, 1976, Flickinger, 1982), macrophages invade the epididymal lumen and phagocytose spermatozoa. It is generally presumed that the degradation products are absorbed by the lining epithelium. My own group has examined intraluminal macrophages in human and guinea pig material after vasectomy. The human epididymides came form patients with long-term pain after vasectomy, and we have only viewed them in wax sections (unpublished). Macrophages containing phagocytosed sperm heads were numerous and striking. The macrophages varied in size, the smaller ones being about 15–20 애m in diameter. It was often difficult to determine whether the intraluminal macrophages had more than one

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nucleus, but giant cells were rare. The nuclei were oval and euchromatic and the cytoplasm was foamy and granular. In the guinea pig (Aitken et al., 1999), we found macrophages in the testis and epididymis which were about 20–40 애m in diameter and had eccentric ovoid euchromatic nuclei (Figs. 16 and 17). Cellular profiles with two nuclei were common. In some cross-sectional profiles of seminiferous tubules, the macrophages had moderately basophilic cytoplasm and contained numerous phagocytosed sperm heads. In other profiles, the cytoplasm had a dense content of periodic

FIG. 16 Degenerated profile of a seminiferous tubule from the left testis of a guinea pig 3 years after left unilateral vasectomy. It contains spermatozoa, macrophages with intracytoplasmic sperm heads, and sloughed epithelial cells. Resin; Azur II. ⫻ 450. Reproduced from Aitken et al. (1999) by permission of the John Wiley & Sons, Inc.

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FIG. 17 Transmission electron micrograph showing macrophage in the lumen of a seminiferous tubule of the left testis of a guinea pig 3 years after left-sided vasectomy. It has an eccentrically placed euchromatic nucleus, and the cytoplasm contains numerous profiles of phagocytosed sperm heads and tails. Bar ⫽ 10 애m.

acid-Schiff positive granules but no sperm heads. The peripheral nuclear chromatin in these latter cells was slightly more condensed than in those with recognizable sperm heads suggestive of reduced cellular activity. Transmission electron microscopy confirmed the presence of sperm heads and tails in the cytoplasm of the cells showing intracytoplasmic sperm heads on light microscopy. The PAS positive material in the other macrophage forms resolved as granules of heterogeneous debris, much of it in a whorled pattern. In these latter macrophages, occasional profiles of sperm tails were

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recognized from the pattern of flagellar microtubules and fibers. In addition, the ultrastructural study revealed occasional monocyte-like cells and young macrophages extending pseudopodia around sperm tails, evidence of continued macrophage recruitment. The timing of intraluminal macrophage invasion relative to that of distension and granuloma formation has not been established. Alexander and Tung (1977), in vasectomized rabbits, observed macrophages in the epididymis prior to damage of the testis and their appearance there. It is also unknown if macrophage activity explains why some men show less marked tubular distension and sperm granuloma formation than others. This certainly seemed to be the reason why most of our guinea pigs showed neither distension nor granuloma formation 3 years after vasectomy. On gross inspection, these showed little difference from sham-operated control material but on histology the testicular and epididymal tubules were rich in intraluminal macrophages (Aitken et al., 1999; Beaton et al., 1995). Bedford (1976) reported macrophages in the epididymal lumen of vasectomized rabbits, hamsters, and rats. In the rabbit, they seemed to invade the epididymal duct close to granulomas and were able to spread widely through the epididymis. In the hamster, they occurred mostly in the epididymal head. The rat seems to show intraluminal leukocytes only at or near sites of epithelial thinning and granuloma formation, an observation confirmed by Alexander (1973) and McGinn et al. (2000). Generalized intraluminal invasion of macrophages of the type reported in rabbit and guinea pig, as well as in man, does not seem to be a feature of the rat. Intraluminal macrophages are not always confined to the epididymal and vasal portions of the vasectomized reproductive tract. Alexander (1972) reported macrophages in the lumens of the efferent ductules in the longer term after vasectomy in rhesus monkeys. Intraluminal macrophages featured in the testis, rete testis, efferent ductules, in addition to the epididymis, in the longer term following vasectomy in the rabbits studied by Alexander and Tung (1977). We have found the same in vasectomized guinea pigs (Aitken et al., 1999; Beaton et al., 1995). Flickinger (1982) found macrophages in the lumen of the efferent ducts and epididymis of vasectomized hamsters. Some workers have reported macrophages in the epididymal interstitium, but their relationship to those in the lumen is uncertain. Alexander and Tung (1977), in vasectomized rabbits, observed macrophages in the connective tissue of the epididymal head consistent with traffic in/out of the tubules. In a later study, Tung and Alexander (1980) saw macrophages in the efferent ductules and epididymal head of vasectomized monkeys which seemed to be migrating through the tubular wall and some containing spermatozoa were seen in the interstitium around the duct profiles. Kumar et al. (1990) also reported occasional macrophages with sperm remnants in the monkey

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epididymal interstitium after vasectomy. Macrophages have been observed in the epididymal epithelium of healthy nonvasectomized rats (Wang and Holstein, 1983). Flickinger et al. (1990, 1993) reported macrophages and other leukocytes in the interstitium of the epididymis in vasectomized Lewis rats, but their ultrastructural study (1993) showed spermatozoal remnants to be extremely rare in these cells. They considered that the cellular infiltrate was a chronic inflammatory response to antigenic material escaping from the epididymal lumen in soluble form.

III. Effects of Vasectomy A. Changes following Vasectomy 1. The Epididymal Epithelium Only a few studies have examined the effect of vasectomy on the epididymal epithelium. Glover and Nicander (1971) noted that the rabbit epididymal epithelium thins after obstruction. Some have commented that localized thinning occurs at sites of luminal distension: McGinn et al. (2000) in rats and Marsh and Alexander (1982) in the ductuli efferentes of rhesus macaque monkeys. My own group found local breaches of the epithelium, not necessarily in continuity with sperm in the interior of sperm granulomas (McGinn et al., 2000). In rats, when the testis degenerates following vasectomy, there is often a reduction in luminal cross section and a consequent heightening of the epithelium. The main controversy concerning the epididymal epithelium is whether it can phagocytose spermatozoa, particularly in the presence of obstruction as in vasectomy. Glover and Nicander (1971) reported the ability of the epithelium of the efferent ductules and epididymal head in rabbits to take up particles from the lumen. Alexander (1972) observed loss of ciliated cells in the ductuli efferentes in rhesus macaques but found no recognizable sperm fragments in the lining epithelium, an observation confirmed in a later study (Marsh and Alexander, 1982). Others, examining the epithelial cells of the epididymis following vasectomy in a number of species, describe increases in the size and number vacuoles/lysosomes (rat: Alexander, 1973; Heidger and Sawatzke, 1977; Flickinger et al., 1990; hamster: Flickinger, 1982; rabbit: Lohiya et al., 1988; monkey: Kumar et al., 1990). The consensus is that spermatozoa either degenerate in the lumen (Alexander, 1972; Alexander, 1973; Galle and Friend, 1977; Lohiya et al., 1988) or are phagocytosed by intraluminal macrophages (Kumar et al., 1990) but, by either means, are degraded be-

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yond recognition before their residues are absorbed by the epididymal epithelium (Flickinger, 1982; Kumar et al., 1990). Flickinger (1972), in the rat, described increased size and number of vacuoles in the epididymal epithelial cells, especially in the light cells of the epididymal tail. Possibly both principal and light cell types are concerned with uptake of spermatozoal degradation products (Flickinger et al., 1990). Reports of the presence of recognizable sperm fragments in epididymal epithelial cells are rare (Flickinger, 1972). Flickinger (1972) and Lohiya et al. (1988) mention seeing sperm fragments in epididymal epithelial cells of rat and rabbit, respectively. Interestingly, a paper on the appearance of epididymal epithelial cells cultured with spermatozoa makes no mention of spermatozoal phagocytosis by the epithelial cells (Moore et al., 1992). 2. Immune Response to Spermatozoa Spermatozoa are autoantigenic because they first form at puberty, long after immunological tolerance to other body components has formed. A variety of surface and internal spermatozoal components can stimulate immune responses, as can chemical moieties secreted by the excurrent ducts which bind to the spermatozoal surface. Normally spermatozoal antigens are isolated from the immune system by epithelial barriers along the reproductive tract. The occluding junctions are best developed between the Sertoli cells of the testis. Similar but less extensive regions of membrane fusion also connect epithelial cells along the epididymis. They are relatively weak, however, in the rete testis and ductuli efferentes. The very high development of these intercellular junctions between Sertoli cells, the socalled blood testis barrier, may reflect the large amounts of programmed cell death and degradation of residual autoantigenic cytoplasm occurring in the seminiferous epithelium (Fawcett, 1979). Many of the questions considered by Fawcett (1979) in his review of the effect of vasectomy on the blood-reproductive tract barriers remain inadequately answered 20 years later. Neaves (1973) in Holtzman rats examined the junctional complexes between Sertoli cells after vasectomy and also after ligation of the efferent ductules. All the rats had healthy testes following vasectomy and there was no alteration in the morphology of the intercellular junctions nor of their permeability to lanthanum. In contrast, after efferent ductule ligation, the junctional complexes seemed morphologically intact but they became completely permeable to lanthanum. A further study on Lewis rats indicated the blood-testis barrier to be intact in apparently healthy tubules but to be permeable to lanthanum in degenerated tubules (Neaves, 1978). In contrast, Castro and Seiguer (1974) reported the barrier to be intact in degenerated testes following isoimmunization with testis homogenate and after vasectomy.

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Serum antisperm antibodies are well documented in vasectomized men (Samuel et al., 1975; Ansbacher et al., 1976). Consideration of the spermatozoal structural and binding antigens is a large topic beyond the scope of this chapter. The subject has been reviewed recently by Mazumdar and Levine (1998), Hjort (1998), and Naz and Menge (1994). Evidence from the rat suggests that the immune response following vasectomy is mediated via the regional lymphatics and lymph nodes rather than via the blood vessels and spleen (McDonald and Scothorne, 1986; McDonald and Halliday, 1992). In the rat at least, the first regional lymph node draining the testis, epididymis, and vasectomy site shows histological evidence of a humoral immune response, although it varies between individuals, even from the same inbred strain. The morphological criteria used were an increase in weight or in volume of the node, in the number and size of germinal centers, and in the width and cellularity of the medullary cords. The response developed slowly. It was not detectable 1 week after operation, and it was not until the ninth week that all three signs were present, and then only in some animals. The lymph node changes were maximal at 3 months after vasectomy. By 6 and 9 months after vasectomy, the response had waned despite the continued presence of sperm granulomas (McDonald and Scothorne, 1989). Although some nodes remained enlarged at both 6 and 9 months, the germinal center activity fell between these two intervals, while the cellularity of the medullary cords was similar at 6 and 9 months but less than that at 3 months. The regressive changes need not necessarily imply a fall in antibody production but they did, in a general way, correspond to the gradual reduction of circulating antisperm antibodies noted, in some other strains of rats, by Kosuda and Bigazzi (1979) after 3 months. In a subsequent paper (McDonald and Halliday, 1992), we showed slight enlargement of the deep cortex, the thymus-dependent region of the node, at 3 months after vasectomy indicative of a cell-mediated response. In the same animals no morphological changes were detected in the white pulp of the spleen. Kosuda and Bigazzi (1979) reported circulating antisperm antibodies in five out of ten inbred strains of rats. In each of the reacting strains, they found that the response developed slowly, and that the incidence of positive individuals did not reach a maximum until 3 months or even longer after vasectomy. This wide variation in serological response of different strains showed the importance of genetic factors, but the absence of an antibody response after vasectomy in 20% of rats of even the most reactive strain (Lewis) indicated that there were other variables, perhaps differences between individuals in the rate of spermatozoal breakdown and therefore of antigenic stimulation.

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Handley et al. (1990) found the specificity of the antisperm antibodies in the serum of their Lewis rats varied over a 10-month period after vasectomy. They also detected antibodies against cytoplasmic components of spermatids and an antigen which seemed to develop on the spermatozoal tail in transit through the epididymis (Handley et al., 1991). The antibodies were predominantly IgM in the first month and IgG in the second and third months after vasectomy (Flickinger et al., 1994). In a further study, Flickinger et al. (1995) found that Lewis rats showed a more intense antisperm antibody response than the Fischer strain despite similar spermatozoal antigens in both groups. Although the incidence and distribution of sperm granulomas were similar, the authors wondered if there might be differences in the method of antigen presentation. Information on cell-mediated immunity to spermatozoal autoantigens is scant but, in the vasectomized rat and sheep, helper T-lymphocytes are found in the granuloma wall (Caldwell et al., 1996; Jessop and Ladds, 1995). In vasectomized rats, the regional testicular lymph nodes show a little enlargement of the thymus-dependent cortex (McDonald and Halliday, 1992) and there are changes in the distributions of helper and cytotoxic T-lymphocytes in the epididymal epithelium and interstitium (Hooper et al., 1995). 3. Spermatozoal Antigen Presentation The manner in which antigen is presented to the regional lymph node(s) of the reproductive tract probably varies with the species. Ball et al. (1982) discovered spermatozoa in the para-aortic lymph nodes of a man having a staging laparotomy. The same workers (Ball and Setchell, 1983) subsequently studied vasectomized rams and boars and found large numbers of free spermatozoa in the testicular lymphatics intermittently between 1 week and 2–3 months after operation. In the regional lymph nodes, they found phagocytosis of spermatozoa by sinus macrophages and, in some cases, the development of a granulomatous reaction. In contrast, my study of rat material (McDonald and Scothorne, 1987) suggested that the regional lymph nodes of the rat reproductive tract usually had absent or few sperm heads. We did, however, find that in vasectomized rats with an epididymal granuloma, lymphatics in the epididymis adjacent to the granuloma contained numerous macrophages and lymphocytes, whereas these cells were absent from epididymal lymphatics of sham-operated controls and of vasectomized animals in which an epididymal granuloma had not developed (McDonald et al., 1991). The study did not allow estimation of the numbers of leukocytes transported from the granuloma to the regional testicular node, but the almost invariable presence of macrophages and lymphocytes in random profiles of lymphatics adjacent to a granuloma indicated that

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the cellular traffic was significant. Their abundance in lymphatic profiles adjacent to a granuloma, and their paucity in lymphatics remote from a granuloma, suggested very strongly that the cells came from the granuloma. 4. Testicular Degeneration The effect of vasectomy on the testis in man and animals is controversial. Many researchers and clinicians have believed that vasectomy has no effect on the testis. This is not so. Several groups have reported histological changes in testicular biopsies from vasectomized men (Derrick et al., 1974; Gupta et al., 1975; Fallon et al., 1978; Jenkins et al., 1979; Bigazzi et al., 1979; Choi and Reiner, 1983; Jarow et al., 1985; Mehrotra et al., 1985; Jarow et al., 1994). Despite the abnormalities, some of these men have been fertile following vasectomy reversal ( Jenkins et al., 1979; Jarow et al., 1985; Mehrotra et al., 1985). The presence of interstitial fibrosis in biopsy specimens collected at vasovasostomy may indicate a poor prognosis for subsequent fertility ( Jarow et al., 1985). The mechanism by which testicular changes occur following human vasectomy is unknown. Animal models have been particularly useful in illustrating possible ways in which the testis might be affected. It must be noted that testicular degeneration following vasectomy in laboratory animals can arise for nonspecific reasons, which may be related to surgical technique, such as cryptorchidism in animals with communication between the peritoneal cavity and processus vaginalis, infection, and damage to the testicular blood supply (Heller and Rothchild, 1974; McGlynn and Erpino, 1974; Neaves, 1974; Lekili et al., 1998). a. Temporary Depression of Spermatogenesis Temporary depression of spermatogenesis following vasectomy is reported in the dog and is perhaps the result of raised intraluminal pressure (Grewal and Sachan, 1968; Kothari et al., 1973; Vare and Bansal, 1973; Derrick et al., 1974; MacDougall et al., 1975). Normal function seems to return after a few weeks (Grewal and Sachan, 1968; Vare and Bansal, 1973; MacDougall et al., 1975). Most of the work on dogs is more than 25 years old. Sarrat et al. (1996) revisited this topic and, in contrast to previous work, found progressive degenerative changes in beagle testis in the first few months after vasectomy. The earliest morphological lesion seemed to be arrest of spermatid development, also reported by Derrick et al. (1974) as a temporary phenomenon. There is no evidence for transient depression of spermatogenesis in the rat or in other species with the possible exception of the hamster. Lue et al. (1997) found evidence of increased germ-cell apoptosis, especially of spermatocytes, 3 weeks after vasectomy. Hamasaki et al. (1991) found reduced spermatogenesis in some of their vasectomized hamsters at 2 weeks after vasectomy, and those at 4 and 8 weeks after operation showed testes

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similar to controls. They also noted abnormal spermatozoal forms by 2 weeks after vasectomy which seemed to increase in frequency thereafter. b. Obstruction of Head of Epididymis Following vasectomy, an association between degeneration of seminiferous tubules and sperm granuloma formation in the epididymal head has been reported in the vasectomized rabbit, hamster (Bedford, 1976), and rat (McDonald et al., 1996). It seems that granulomas in the epididymal head are unable to accommodate the spermatozoa and fluid produced by the testis. This may be because enlarging granulomas compress surrounding loops of the epididymal duct which is narrow in this region. The consequent rise in intraluminal pressure damages the seminferous epithelium and leads to tubular degeneration and collapse. In the rat (McDonald et al., 1996), there was good evidence that the atrophy was the result of elevated pressure because some testes were tense, blanched, and swollen to about twice their normal size. Their seminiferous tubules were distended. Clearly human testes, having a much tougher tunica albuginea, would not show such marked swelling after vasectomy, but the possibility that they show pressure-mediated damage has not been excluded. c. Autoimmune Orchitis For over 25 years, there has been general acceptance that, in certain species, the testis suffers autoimmune orchitis following vasectomy. The initial suggestion of an immunological mechanism for testicular degeneration came from the observation of bilateral testicular atrophy following unilateral vasectomy (Alexander, 1973), a phenomenon I and others have observed in rats (Chehval et al., 1995; McDonald et al., 1996), and the realization that, under certain experimental conditions, exposure of the immune system to spermatozoal autoantigens can induce allergic orchitis (Mancini, 1976). Two general mechanisms are proposed: (i) an assault on the seminiferous epithelium by sensitized lymphocytes and (ii) impairment of spermatogenic function by antisperm antibody deposition. Even though these mechanisms have entered the vasectomy literature, I have serious doubts about their validity. d. Lymphocytic Attack It is generally held that the guinea pig suffers a cellular autoimmune orchitis after vasectomy. Alexander (1973) was the first to speak of autoimmune hypospermatogenesis in vasectomized guinea pigs. She noted degeneration of seminiferous tubules in both testes of unilaterally vasectomized animals but made no mention of the presence of lymphocytes. A later paper, Tung and Alexander (1977), made clear that the attribution of this testicular damage to immunological mechanisms had been premature. This paper also clarified that the bilateral atrophy following unilateral vasectomy was found only in the longer term after vasectomy. Muir et al. (1976) noted changes similar to those described by Alexander

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(1973) in their guinea pigs 8 months after bilateral vasectomy. In addition, however, they noted that some of the testes appeared swollen and that some seminiferous tubular profiles were distended, consistent with raised intraluminal pressure. Hutson et al. (1976) reported degeneration of the seminiferous tubules in almost half of their bilaterally vasectomized guinea pigs in the first 6 months after operation. Of the animals, 93% also showed serum antisperm antibodies. Although these authors thought the mechanism to be autoimmune, there was no clear evidence of this and, again, there was no mention of lymphocytic infiltration. Tung (1979), in a report which furthered a study started with Alexander (Tung and Alexander, 1977), described lymphocyte and macrophage invasion of testes of guinea pigs 16 months after vasectomy. This was seen following bilateral vasectomy and in both testes following unilateral vasectomy. Cell-mediated orchitis is also reported in other species. Tung and Alexander (1980) saw infiltrates of lymphocytes, plasma cells, and macrophages in the interstitium adjacent to seminiferous tubules and the rete testis in rhesus monkeys vasectomized for several years. My own group has investigated the long-term effects of vasectomy on the guinea pig testis. Three years after vasectomy and control sham operation, the guinea pig testis showed a number of degenerative features (Aitken et al., 1999). Most of these occurred in the controls as well as in the experimental animals. Both groups exhibited tubular atrophy with sloughing of germ cells. Spermatids were most frequently lost, but some tubular profiles were devoid of all sperm precursors. Some profiles in all groups contained clusters of macrophages showing intracytoplasmic sperm heads. The changes seemed to be more marked in the material from vasectomized guinea pigs, but evaluation of the percentage of atrophic tubular profiles and of tubular dimensions failed to reveal any statistically significant differences. These changes are likely to be explained by the age of the guinea pigs and appear to be nonspecific phenomena. Vesicles were frequently seen in cells of the seminiferous epithelium in both the experimental and control testes and were also likely to be agerelated (Fig. 18). They were particularly associated with the spermatids. Determination of the percentage of tubular profiles showing vesicles in our wax sections showed significantly higher values on the ipsilateral side following unilateral vasectomy. We suggest that vasectomy may have exacerbated an age-related phenomenon. We found, however, that lymphocyte invasion of the testicular interstitium and seminiferous tubules was confined to vasectomized animals (Fig. 18). Infiltration of the interstitium was observed in five of the eight left testes following left unilateral vasectomy. In three of these there was also invasion of the seminiferous tubules. Two right testes following left-sided

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FIG. 18 Two profiles of seminiferous tubules from a left testis 3 years after left unilateral vasectomy in the guinea pig. One contains and is surrounded by small lymphocytes. The other shows vesicle formation in otherwise healthy-looking epithelium. Resin; Azur II. ⫻ 250. Reproduced from Aitken et al. (1999) by permission of the John Wiley & Sons, Inc.

vasectomy also showed very occasional small foci of interstitial lymphocytes (Fig. 19). Even though the number of animals was small, we tentatively suggested that lymphocyte infiltration may be specific for the vasectomized group. Conclusions about the mechanism of testicular degeneration following vasectomy must, however, be guarded because some vasectomized guinea pigs showed atrophy which was not accompanied by lymphocyte invasion. It is possible that, following vasectomy, mechanical factors may be the primary cause of degeneration and that a lymphocyte response supervenes on an already damaged testis. In my group’s study, the absence

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FIG. 19 Profile of a degenerated tubule (D) containing macrophages (M) in a right testis 3 years after left unilateral vasectomy in the guinea pig. Numerous small lymphocytes (L) have infiltrated the adjacent interstitium. Wax; PAS & H. ⫻ 300. Reproduced from Aitken et al. (1999) by permission of the John Wiley & Sons, Inc.

of tubular distension in the left testes following ipsilateral vasectomy does not rule out mechanical damage. Initial mechanical damage may have been followed by degeneration and shrinkage of previously distended tubules. We can, therefore, confirm Tung’s (1979) observation of lymphocyte invasion of the guinea pig testis following vasectomy but are unable to exclude Muir et al.’s (1976) hypothesis that the testicular degeneration is primarily mechanical in aetiology.

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A number of investigators have reported abnormalities in testicular biopsies of vasectomized men but make no mention of lymphocyte infiltration (Derrick et al., 1974; Gupta et al., 1975; Fallon et al., 1978; Jenkins et al., 1979; Bigazzi et al., 1979; Choi and Reiner, 1983; Jarow et al., 1985; Mehrotra et al., 1985; Jarow et al., 1994). e. Antibody Induced Degeneration Several papers have described immune complex deposition around seminiferous tubules and other parts of the reproductive tract after vasectomy. In the testis, there is a general assumption that these antibodies have caused accompanying degeneration of the seminiferous epithelium (see Salomon et al., 1982). There seems to be no reason for this premise; the immune complexes could reflect antigen escape following some other insult. Bigazzi et al. (1976) were the leaders in detecting immune complexes in the seminiferous tubular basement membrane. In long-term vasectomized rabbits, they found immune complex deposition around seminiferous tubules which frequently showed degenerative changes. Alexander and Tung (1977) found degenerative changes in seminiferous tubules of long-term vasectomized rabbits and later, like Bigazzi et al. (1976), confirmed immune complex deposition in the basement membrane (Alexander and Tung, 1979). Immune complex deposition has also been observed in the basement membrane of efferent ductules of long-term vasectomized monkeys (Alexander, 1972; Marsh and Alexander, 1982). Tung and Alexander (1980) found immunoglobulin deposits in the efferent ductules and epididymal head in 43% of control monkeys and in 91% of monkeys after long-term vasectomy. The deposits were more widespread in the vasectomized group. They also found no particular association between the presence of the immunoglobulins in the basal lamina and the presence of serum antibodies or testicular or epididymal damage. Many questions remain unanswered about whether antisperm antibodies can damage the testis. There also seem to be marked species differences in the occurrence and distribution of immune complex deposition following vasectomy. Bagshaw et al. (1980) and Mehrotra et al. (1985) looked for immune complex deposition in the human testis with negative results. Another controversial topic is whether degenerative changes in the testis are associated with serum antisperm antibodies. Bigazzi et al. (1976) and Alexander and Tung (1977) found orchitis in rabbits to be associated with the presence of serum antisperm antibodies. Similarly, Flickinger et al. (1990) found that, in their Lewis rats, inflammatory changes in the epididymis were associated with degenerative changes in the seminiferous tubules and with the presence of serum antisperm antibodies. However, Jarow et

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al. (1994) found no relationship between the presence of testicular changes and serum antisperm antibodies in human vasectomy patients. f. Testosterone Production The effect of vasectomy on interstitial cell function is poorly documented and has received little attention in recent years. Evidence on hormone levels has been conflicting but there is a general consensus that they are within the normal range following vasectomy (Richards et al., 1981). Wild claims have been made that vasectomy lowers testosterone production and leads to premature aging (Petty, 1994) or erectile dysfunction (Carruthers, 1991); these are without sound scientific foundation. Psychological problems following vasectomy may, however, lead to erectile dysfunction (Buchholz et al., 1994). An interesting study by Geierhaas et al. (1991) described reduced testosterone and elevated FSH and LH after vasectomy in rats. They also observed hypertrophy of the Leydig cells and of adjacent macrophages, which appeared in close contact with the Leydig cells. Unfortunately, the authors did not report on the appearance and integrity of the seminiferous tubules.

B. Effect on the Cycle of the Seminiferous Epithelium McDonald and Scothorne (1988) carried out a detailed study to determine whether seminiferous tubules which appeared normal following vasectomy in the Albino Swiss rat were truly healthy and not subject to some subtle disturbance of spermatogenesis. A quantitative analysis of the apparently healthy seminiferous tubules seen in wax sections of testes harvested 6 months after vasectomy was made and the data were compared with that from sham-operated control rats. If the rate of spermatogenesis were to be reduced as a result of vasectomy, it might have been expected to produce any or all of the following changes in each tubular profile: (i) A decrease in the total area; (ii) A reduction in the area of seminiferous epithelium; (iii) A reduction in the absolute number of pachytene spermatocyte nuclei; (iv) A reduction in the number of pachytene spermatocyte nuclei per unit length of perimeter. On the other hand, damming back of sperm into the testis as a result of blockage might have been expected to produce any of the following changes in the tubular profiles:

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(i) An increase in the total area as a result of tubular distension; (ii) A change in the area of the lumen; (iii) A reduction in the number of pachytene spermatocyte nuclei per unit length of perimeter due to thinning of the epithelium. That there was no significant difference in any of the parameters studied after vasectomy compared with sham operation suggested strongly that there was neither alteration in the rate of spermatogenesis nor damming back of sperm in the testicular tubules as a result of vasectomy. The study also examined the effect of vasectomy on the cycle of the seminiferous epithelium. Again, only testes which appeared grossly healthy were assessed. The staging of the seminiferous tubules was carried out according to the classification of Leblond and Clermont (1952) which defines 14 stages of the seminiferous cycle, based on the development of the acrosome in the spermatids. Our results showed that there was no important alteration in the seminiferous cycle after left unilateral vasectomy. This was supported by the observation that there was no change in the relationship of the spermatids, on which the staging was primarily based, with the spermatogonia and spermatocytes; the spermatogonia and spermatocytes were always at the stage of development described by Leblond and Clermont (1952) for the corresponding stage of spermatid development. If there were to be an alteration in the rate of some step in spermatogenesis, it would have been expected to put the development of the spermatogonia, spermatocytes, and spermatids out of phase with each other. Other workers (Lamano-Carvalho et al., 1984), using a simplified version of Leblond and Clermont’s classification, reported a higher frequency of Stages VII and VIII, the stages with the most mature spermatids. They thought they were seeing sperm accumulation within the testes as a result of vasectomy and speculated that this might have been due to partial denervation of the myoepithelial cells, with reduced peristaltic tubular contraction. The results of my own study (McDonald and Scothorne, 1988) indicated that, at least in Albino Swiss rats 6 months after vasectomy, there was no retention of mature spermatozoa in the seminiferous tubules. Sections of tubules at Stage IX were consistently devoid of mature spermatozoa, after both vasectomy and sham operation, as they were, at that stage, in the original description of Leblond and Clermont (1952). C. Effect on the Boundary Zone of the Seminiferous Epithelium Dobson et al. (2000) in an ultrastructural study on rat testes following vasectomy found that the boundary zone of healthy seminiferous tubules

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resembled that of controls but that there were marked changes when the tubules degenerated. Similar studies on vasectomized rabbits documented deposition of immune complexes (Alexander and Tung, 1979; Bigazzi, 1979), but no morphological evidence of this was observed in the rat. In the healthy rat testis, several layers in the periphery of the seminiferous tubules compose the boundary zone: the basal lamina of the seminiferous epithelium, fine collagen fibers of the basement membrane, the inner basal lamina of the myoid cells, the myoid cells themselves, and their outer basal lamina (Hadley and Dym, 1987; Christl, 1990). Dobson et al. (2000) found that when testes degenerated following vasectomy, changes occurred in all layers of the boundary zone in affected seminiferous tubules (Fig. 20). The myoid cells persisted as a single layer of cells but the nucleus and the region of the cell in which it lay assumed a triangular profile. All three basal laminae, that of the seminiferous epithelium and the two layers belonging to the myoid cells, became more undulating. This was particularly marked

FIG. 20 Transmission electron micrograph of boundary zone of left testis of a rat showing bilateral testicular degeneration following left-sided vasectomy. A Sertoli cell nucleus (N) is euchromatic and deeply indented. Its cytoplasm shows few organelles. The basal lamina of the seminiferous epithelium (s) and the inner (i) and outer (o) basal laminae of the elongated myoid cell (M) are all folded. Fine collagen fibers (f ) separate the basal lamina of the seminiferous epithelium and the inner basal lamina of the myoid cell. Lymphatic endothelium (E) lies peripheral to the outer myoid cell basal lamina. A small lymphocyte (L) lies between the myoid cell and its outer basal lamina. Bar ⫽ 10 애m. Reproduced from Dobson et al. (2000) by permission of the John Wiley & Sons, Inc.

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in the basal lamina of the seminiferous epithelium which became extensively folded. The myoid cell basal laminae were also folded and became separated from the cells in places. There was thickening of the layer of fine collagen fibers that forms part of the basement membrane between the Sertoli and myoid cells. The fibers seemed more numerous and irregular in their orientation. Occasional leukocytes, mostly lymphocytes, were observed among the layers of the boundary zone and seemed more numerous in degenerated tubules. This suggests possible immunological activity. Dym and Romrell (1975) did not find leukocytes internal to the myoid layer in healthy seminiferous tubules. Richardson et al. (1998) found evidence of increased basement membrane synthesis in testicular tubules with similar pathology to ours, but following efferent ductule ligation. Dobson et al. (2000) were unable to detect any structural differences between the tubules of unilaterally and bilaterally degenerated testes, and both groups had similar boundary zones. Altered boundary zones were only found in degenerated tubules suggesting that the boundary zone changes were a result, rather than a cause, of degeneration of the seminiferous tubules. The striking similarity of the boundary zone changes following vasectomy to those observed following administration of an antiandrogen, flutamide, and X-ray irradiation in the rat (Lacy and Rotblat, 1960) suggested that the boundary zone changes were nonspecific in nature, probably related to a decrease in tubular cross section. Bigazzi et al. (1976) and Alexander and Tung (1977, 1979) reported similar morphological changes in rabbit seminiferous tubules after vasectomy as did Marsh and Alexander (1982) in the efferent ductules of vasectomized rhesus macaques. Alexander and Tung (1977) found the basement membrane to be highly folded in rabbits, even in the contralateral testis after unilateral vasectomy. The thickened membranes contained immune complexes in all these studies. Few have looked for immune complexes in the rat boundary zone and early reports require confirmation (Aydos et al., 1998). The general topic of immune complex deposition has already been described.

IV. Conclusion Over the 30 years since vasectomy started to be carried out as a routine contraceptive measure, there has been a general realization that the resultant obstruction may have profound local effects on the reproductive tract and on health in general. This review is concerned with the cell biology of vasectomy. Although the epithelium of the obstructed epididymis and ductus deferens, in general, shows little alteration, it may have an important role in the uptake and processing of soluble material from the tract lumen.

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Spermatozoal phagocytosis by macrophages is important in all species, but its site of occurrence shows marked species variations. In all species, sperm granulomas are sites of high macrophage activity at sites of spermatozoal extravasation but occur early in some species and late in others. Most species also show much breakdown of spermatozoa by intraluminal macrophages. In some these are localized to the epididymal duct adjacent to granulomas, but in others they are widespread and by degrading spermatozoa may even prevent the vasectomized tract showing gross evidence of pathology in the testis or epididymis. Spermatozoal antigen escape from the reproductive tract stimulates an antisperm antibody response which varies between individuals, even in the same inbred strain. Following vasectomy, there is evidence of cell-mediated as well as humoral immunity against spermatozoa, but their roles in the aetiology of testicular damage remain poorly understood. Many men and animals retain normal testicular function after vasectomy. The cause and significance of reports of testicular damage in human testis following vasectomy in unclear, but laboratory animals may develop degeneration of the seminiferous tubules following sperm granuloma formation and obstruction at the head of the epididymis. Whether such pressure-induced degeneration occurs in man is uncertain. Sex hormones are probably within normal limits following vasectomy.

Acknowledgments I am grateful to the staff of the Life Sciences and Inter Library Loans Sections of the Glasgow University Library for all their kind help and to Mr. Iain Sim for assistance with photography.

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