Detection of testicular endocrine abnormalities and their correlation with serum antisperm antibodies in men following vasectomy

We measured the serum gonadotropin response to gonadotropin-releasing hormone in 25 men who underwent vasectomy 2 to 64 months before the study. Ten age-matched fertile men were used as controls. Baseline serum follicle-stimulating hormone, luteinizing hormone and testosterone levels were not significantly different between vasectomized men and controls. However, mean serum follicle-stimulating and luteinizing hormone responses to an intravenous bolus injection of 100 mcg. gonadotropin-releasing hormone were significantly greater in the vasectomy group (p equals 0.008 and 0.003, respectively). There was no correlation between these responses and the interval after vasectomy. Serum antisperm antibodies were present in 13 vasectomized men (52 per cent) using enzyme-linked immunosorbent assay and microagglutination techniques. A significant correlation (p equals 0.003) was found between the presence of serum antisperm antibodies and a normal follicle-stimulating hormone response to gonadotropin-releasing hormone stimulation. Of 13 patients with demonstrable antisperm antibody titers 9 (69 per cent) had normal follicle-stimulating hormone responses, compared to only 1 of 12 (8 per cent) without identifiable antisperm antibody titers. Our data suggest that certain men following vasectomy have abnormalities in seminiferous tubule and Leydig cell functions of the testes. These abnormalities are unrelated to the interval after vasectomy and are not identifiable with routine static hormonal measurements. In addition, serum antisperm antibodies are most likely to be present in men who demonstrate normal seminiferous tubular activity after vasectomy.     

Effect of vasectomy via inguinal canal on spermatogenesis in rabbits

Aim： To determine whether vasectomy away from the epididymal tail （via the inguinal canal） in rabbits can reduce the early postoperative effects on spermatogenesis. Methods： Twenty-nine normal male Japanese white rabbits （aged 4- 6 months） were subjected to unilateral close-ended （conventional） or open-ended （the cut end of the juxta-epididymal vas deferens not ligated） vasectomy via the inguinal canal. Ten days and 3 months after operation, testes, epididymides and vasa deferentia were removed and methacrylate resin-embedded sections prepared. The histology of the testis, epididymis and vas deferens was examined under light microscope, and the volume and diameter of the seminiferous tubules were quantitatively studied using stereological methods. Results： Neither of the methods of vasectomy led to apparent damage to spermatogenesis on the vasectomized side in comparison with the contralateral shamoperated side, but the juxta-epididymal vas deferens on the vasectomized side was highly distended and contained numerous sperm 3 months after operation. Conclusion： Vasectomy away from the cauda epididymis has no significant early postoperative effects on spermatogenesis in rabbits.     

A study on the mechanism of the spermatogenic damage after vasectomy in rats

PURPOSE: Vasectomy may result in damage to spermatogenesis. There are several explanations for this damage, including an increase of pressure in the seminiferous tubules and an autoimmune reaction. Recently, vasectomy has been reported to induce germ cell death by apoptosis. However, the exact mechanism of this vasectomy-induced germ cell apoptosis is unclear. To elucidate this mechanism, we designed a vasectomized rat model and examined the testiscular alterations and apoptotic degeneration biochemically and microhistopathologically. Particularly, we analyzed the expression of nitric oxide synthase (NOS) and nuclear factor kappa B (NF kappa B), which plays a critical role in the induction of the iNOS gene, in the testis after vasectomy to gain insight into the association between germ cell apoptosis and these factors. MATERIALS AND METHODS: The testes of 40 Wistar rats (10-weeks old) were studied at 1, 2, 5 and 10 weeks after unilateral (left) vasectomy. Wistar rats weighting 290 to 310 g were divided into 2 groups and subjected to underwent either unilateral vasectomy or sham surgery under ether anesthesia. Bilateral testes were carefully observed biochemically and histopathologically. Apoptosis was detected by an in situ end-labeling technique (detection of cellular DNA fragmentation) and electron microscopy. Neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) protein was detected by Western blotting and immunohistochemical studies using each NOS monoclonal antibody. To confirm the co-localization of cellular DNA fragmentation in germ cells and each NOS, each set of consecutive testis sections (one stained for cellular DNA fragmentation and the others for each NOS) were examined. Expression of NF kappa B proteins was examined immunohistochemically using a NF kappa B p65 polyclonal antibody. RESULTS: At 5 and 10 weeks after vasectomy, the vasectomized left testis was significantly lighter than the unvasectomized right testis and sham-operated testis. At that time, the seminiferous tubules of vasectomized testes were highly damaged, presenting narrow tubular diameter, disorder of cellular arrangement, depletion of the germ cells, and local interstitial fibrosis. Vasectomized testes demonstrated a significantly increased number of apoptotic germ cells per cross-sectional area compared with sham-operated testes at 5 and 10 weeks after operation (p < 0.01). Electron microscopy revealed apoptotic germ cells each with a darkly stained nucleus. Western blotting and immunohistochemical studies demonstrated that iNOS proteins were more strongly expressed on vasectomized testes as time passed after vasectomy. Examination of consecutive sections from the vasectomized testis revealed that visibly apoptotic germ cells that exhibited positive staining for cellular DNA fragmentation were also intensely stained for eNOS and iNOS. NF kappa B p65 proteins were more strongly expressed in the nucleus of germ cells in the vasectomized testis than in the sham-operated testis. CONCLUSIONS: We found that vasectomy results in damage to spermatogenesis in adult rats, that may induce germ cell apoptosis, and that iNOS and NF kappa B may play a critical role in the germ cell apoptosis after vasectomy.     

Testicular reaction to prolonged exposure to nitrous oxide.

Male LEW/f Mai rats were exposed to an atmosphere of 20 per cent N2O, 20 per cent O2, and 60 per cent N2 for a maximum of 35 days. Evidence of injury to the seminiferous tubules was found in some animals by the second day. By 14 days, such damage was found in all animals. The toxic effect was confined to the spermatogenic cells, with consequent reduction in mature spermatozoa and appearance of multinucleated forms. Other cells within the testes were resistant to damage. Recovery of spermatogenesis occurred after return to room air for more than three days. Serum testosterone levels were not significantly affected during the prolonged exposure.     

Sperm antibodies after vasectomy with fulguration.

Whether antisperm antibodies develop after vasectomy probably depends on several variables, 1 of which may be the surgical technique. The levels of serum antisperm antibodies were compared in men vasectomized by 1 techniques: vasoligation and fulguration. No difference in the incidence of spermagglutinating antibody was found in the 2 groups. However, immobilizing antibodies were observed in 43 per cent of the men undergoing vasoligation but in only 29 per cent of the men vasectomized by fulguration.     

Effect of vasectomy on sperm nuclear chromatin condensation in the rabbit

Histone-to-protamine exchange in haploid spermatids is known to play a central role for male fertility. The present study investigates, for the first time, the effects of vasectomy on the expression of protamines in the rabbit. During normal spermatogenesis, protamine-1 and protamine-2 mRNA were expressed from step 5 round spermatids to step 11 elongated spermatids. In unilaterally vasectomized animals, control testes revealed normal spermatogenesis with normal protamine expression, while vasectomized testes exhibited both normal spermatogenesis and spermatogenic arrest. Some testes with normal spermatogenesis revealed delayed expression of both protamine-1 and protamine-2. Furthermore, multinucleated round spermatids were a regular finding in these testes. In both treated and untreated animals, a higher percentage of spermatozoa from the cauda epididymis had highly condensed chromatin when compared with those from the testis. The percentage of spermatozoa with highly condensed chromatin from testes and epididymides from the vasectomized side of treated animals remained unchanged from controls. As the integrity of nuclear chromatin is important for oocyte fertilization, especially in intracytoplasmic sperm injection (ICSI), where most of the natural selection mechanisms are bypassed, our data add valuable information for the treatment of infertility by ICSI, showing that vasectomy may affect nuclear chromatin integrity of testicular spermatids but not epididymal spermatozoa. Microsurgical epididymal sperm aspiration (MESA), therefore, may be superior to testicular sperm extraction (TESE) in vasectomized patients.     

Quantitative testicular biopsy in congenital and acquired genital obstruction

To determine if congenital obstruction of the genital tract is associated with significant testicular histopathological conditions compared to acquired forms of obstruction we performed testicular biopsy in 8 vasectomized men and 5 men with vasal agenesis. Quantitative analysis of the seminiferous tubular and epithelial parameters demonstrated a statistically significant increase in tubular wall thickness in the vasectomized group. There was no significant difference among the groups with reference to the mean number of late spermatids per seminiferous tubules, mean number of Sertoli cells per seminiferous tubules, mean number of seminiferous tubules per field (100 times) or mean seminiferous tubular diameter. We conclude that despite a lifelong duration of obstruction, men with vasal agenesis demonstrate a more favorable testicular histological status compared to men after vasectomy. This finding may have therapeutic implications when considering assisted pregnancy techniques as a method of treatment of male genital tract atresia.     

Effect of Freund's complete adjuvant on the immune response of vasectomized rats.

Rats were vasectomized, vasectomized and injected in the epididymis with Freund's complete adjuvant (FCA), or only injected with FCA without vasectomy. All were positive for sperm agglutinins, in contrast to sham-operated or untreated controls. Generally, the highest titers were found in vasectomized or unoperated animals given FCA, indicating that local granuloma formation promotes the production of sperm autoantibody, even in nonvasectomized rats.     

Effect of hydrocele on testis and spermatogenesis

One hundred and twenty cases of big unilateral hydrocele of the tunica vaginalis testis have been studied to ascertain the effect on the structure and function of the testis, taking the normal side as control. There was no pressure effect from the hydrocele on the structure of the testis in 70 per cent, a flattening of testis in 22 per cent, and atrophy of testis in 8 per cent of cases. There was partial arrest of spermatogenesis in 10 per cent and total arrest of spermatogenesis in 8 per cent of cases. The remaining 82 per cent showed normal spermatogenesis.     

Dibromoacetic acid, a prevalent by-product of drinking water disinfection, compromises the synthesis of specific seminiferous tubule proteins following both in vivo and in vitro exposures.

Dibromoacetic acid (DBA) is a by-product of drinking water disinfection that alters spermatogenesis in adult male rats. To identify a mechanism by which DBA alters spermatogenesis, seminiferous tubules representing specific groups of spermatogenic stages were exposed either in vivo or in vitro, and structural and functional consequences were evaluated. Seminiferous tubules representing stages I-V, VI-VIII, and IX-XIV were isolated from testes of adult rats and cultured overnight in conditions of reduced oxygen and temperature. For in vivo exposures, seminiferous tubules were recovered from animals that had received 250 mg/kg DBA via gavage for 5 days. For in vitro exposures, 180 and 600 microM concentrations were tested; these concentrations bracket the concentration of DBA observed within the testis following in vivo exposure. Protein synthesis was evaluated by 35S-methionine labeling overnight and quantitative analysis of radiolabeled proteins in mini, 2-dimensional (2D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Radio-inert cultures were processed for light and electron microscopy. Morphologicaf evaluation indicated that all spermatogenic stages of the seminiferous tubules from control animals were well maintained during the isolation and culture period. Although no treatment-related lesions were observed following in vivo exposure, histological alterations were observed at the lowest in vitro exposure. There was a significant diminution (P < .05) in the synthesis of 4 cytosolic proteins following both in vivo and in vitro exposures. Diminution in these proteins was restricted to stages I-V and IX-XIV of spermatogenesis, suggesting that proteins involved in the early stages of spermiogenesis are uniquely sensitive to DBA exposure. Because histology and protein synthesis were affected by relevant in vitro exposures, this indicates that DBA is capable of altering spermatogenesis directly.     

Histologic changes in the mouse testis after bilateral vasectomy

Aim: To study the effect of vasectomy on histological appearance of the testis. Methods: Parkes strain mice were used as the animal model; they were bilaterally vasectomized (Vx) or sham-operated (So) and killed at intervals of 4, 6, 9, and 12 months after the operation. Testes were excised from 5 Vx and 5 So mice at each interval and processed for histological examination. Results: Testes of So mice showed normal histological features. By contrast, marked alterations were observed in the seminiferous tubules in testes of Vx mice, except in those killed 4 months after the operation. The seminiferous epithelium in the tubules was only 2 - 3 layers thick and showed much depletion of germ cells; in severe cases, the epithelium consisted of only a thin layer of Sertoli cells, spermatogonia and a few spermatocytes. Exfoliation of germ cells, occurrence of multinucleated giant cells and vacuolated appearance of the epithelium were of common features in the tubules. Furthermore, lumen of the rete testis in Vx mice was greatly dilated and showed accumulation of spermatozoa with immature germ cells; in mice vasectomized for 6 - 12 months, several macrophages ingesting spermatozoa were often observed in the lumen of the rete testis. Spermatic granuloma was also sometimes noticed in corpus or in cauda regions of the epididymis in mice vasectomized for 6 - 12 months. Conclusion: We suggest that consequences of vasectomy should be thoroughly understood in order to make this method rather more popular as a reversible method of male contraception.     

The number of spermatogonia in various congenital testicular disorders

Various congenital testicular disorders, including monorchism, retractile testis, cryptorchidism and male intersex, were investigated by counting the number of spermatogonia per seminiferous tubule. The results showed that all 7 cases of monorchism had normal numbers of spermatogonia per seminiferous tubule. However, in 29 cases of a retractile testis a normal testis was observed in 13 (44.8 per cent). Therefore testicular dysgenesis is suggested to exist in more than half of cases of the retractile testis. Of 150 cases of cryptorchidism 82 were bilateral and 68 were unilateral. There was no significant difference in the number of spermatogonia per seminiferous tubule between these 2 groups. The higher the testes were located the worse the ratio of spermatogonia per seminiferous tubule. Fewer or absent spermatogonia were observed in 2 patients less than 2 years old. Of 28 contralateral scrotal testes in patients with unilateral cryptorchidism 4 (14.3 per cent) had no spermatogonia per seminiferous tubule and 8 (28.0 per cent) had a decreased number of spermatogonia per seminiferous tubule. The male intersex patients had much damage even in the scrotal testes. From these results it is suggested that these congenital testicular disorders, except monorchism, have similar histological features. Moreover, these conditions are possibly related in etiology to the phenomenon of deficient androgen stimulation.     

Mast cells in testicular biopsies of infertile men with 'mixed atrophy' of seminiferous tubules

Mast cells in the bilateral testicular biopsies of 30 patients with a 'mixed atrophy' of seminiferous tubules were analysed. Seven biopsies from vasectomized patients served as controls. With regard to their characteristic location within testicular tissue, two groups of mast cells could be distinguished, in both control and infertile patients: 'interstitial' mast cells (located between Leydig and other interstitial cells as well as in the vicinity of blood vessels) and 'peritubular' mast cells (located in the close proximity of the tubular lamina propria or incorporated in the lamina propria itself). Morphometric data indicated a significant increase in the number and volume of mast cells in infertile patients when compared with controls. In the biopsies of infertile patients that were analysed both 'interstitial' and 'peritubular' mast cells showed a significant increase in their number and volume, although it appeared that 'peritubular' mast cells increased at a higher rate than 'interstitial' mast cells. A significant negative correlation was found between the following variables: volume and number of mast cells, testis volume and the status of spermatogenesis evaluated by Johnsen's scoring. It was concluded that the increased presence of mast cells is closely associated with an impairment of spermatogenesis.     

Presence and distribution of leucocyte subsets in the murine epididymis after vasectomy

Male mice were vasectomized by 'open-ended' or 'closed' techniques. After 4 weeks the cell-mediated immune reactions were compared with those of sham-operated animals by immunohistochemical localization of leucocytes, using specific monoclonal antibodies. Macrophages and MHC class II antigen-positive cells were the major cell types to appear in all regions of the epididymis after both types of operation. There was recruitment of T-helper/inducer leucocytes but not of T-suppressor-cytotoxic cells. An increased presentation of macrophage-migration inhibiting factor antigen appeared in interstitial and peritubular locations. After 'closed' and 'open-ended' vasectomy granulomata developed in the epididymis. The sperm-containing lumen of these granulomata was invaded by macrophages, MHC class II-positive cells and T-helper/inducer lymphocytes. This mouse model thus reveals a significant epididymal inflammatory response of the epididymis to vasectomy.     

Secondary changes in the scrotal testis in experimental unilateral cryptorchidism

Unilateral cryptorchidism was induced in under 2-day-old Wistar rat pups. A control group of rats underwent sham operation at the same age. The animals were killed at intervals from 5 to 120 days, both testes were excised, weighted, and processed for histological examination, morphometric measurement of the seminiferous tubules, and DNA flow cytometry. There was no difference in weight, Johnsen score, and tubular size between the scrotal testis of cryptorchid animals and control testes at any age. Significant decreases in all of these parameters occurred in the undescended testis from 30 days when compared with the scrotal testis in cryptorchid animals and controls. Using flow cytometry to measure changes in the DNA ploidy of the cells of the seminiferous epithelium during spermatogenesis, a significant decrease in the haploid population of cells occurred in the scrotal testis of cryptorchid animals at 40 days. This difference continued into adult life (P less than .005). Flow cytometry demonstrates a secondary decrease in spermatogenesis in the scrotal testis in experimental unilateral cryptorchidism.     

A stereological study of the effects of experimental inguinal cryptorchidism and subsequent orchiopexy on spermatogenesis in adult monkeys

Our previous study demonstrated that experimental intra-abdominal cryptorchidism in adult rabbits for 13 weeks resulted in severe spermatogenic arrest: type A spermatogonia was the only germ cell type seen in the seminiferous epithelium and its number per testis was reduced by 84%. Seven weeks following orchiopexy, the type A spermatogonial number returned to the near-normal range in most animals and spermatogenesis partially recovered (Reproduction 2002, 124, 95-105). This study aimed to determine whether inguinal cryptorchidism would produce less-severe damage to spermatogenesis and whether subsequent orchiopexy would better restore spermatogenesis. Five normal adult male rhesus monkeys (Macaca mulatta) underwent bilateral artificial inguinal cryptorchidism. Half a year later, one testis together with the ipsilateral epididymis were removed from each animal and then unilateral orchiopexy was performed on the contralateral side, with the remaining testis and epididymis being removed another half a year later. A contemporary unbiased and efficient stereological tool, the optical disector, was used to estimate numbers of all types of spermatogenic cells in the testis and spermatozoa in the epididymis. Spermatogenic arrest was induced by cryptorchidism at the stage of spermatogonia (n = 1), spermatocytes (n = 2) or early spermatids (n = 1), with the type A spermatogonial numbers per testis being reduced to 14.8-57.2% of the control average; in one of the five cryptorchid animals, however, spermatogenesis remained normal. Subsequent orchiopexy, which was successfully performed on two animals with cryptorchidism-induced spermatogenic arrest, brought on a full or partial recovery of spermatogenesis. In conclusion, inguinal cryptorchidism induces less severe (in comparison with an intra-abdominal one) and variable damage to spermatogenesis, which is restored, at least in part, by subsequent orchiopexy.     

Inhibition of Canalization in Rat Seminiferous Cords by Medroxyprogesterone Acetate

Due to interest in progestins as male contraceptives, Provera was used to study lumen formation in seminiferous cords. Seven day old rats received daily subcutaneous Depo Provera injections (500 μg/100 g b. w.). At 23 and 28 days of age testes from treated and control animals were prepared for 1 μ plastic sections. At least 100 cross sections of seminiferous cords and tubules were studied in each animal and the per cent of tubules with lumens was calculated. Lumen formation was significantly retarded in the experimental animals. At 23 days tubules in the treated and control animals comprise 7.5 ± 4.1 and 33.3 ± 13.6 % respectively, and at 28 days 15.8 ± 8.3 and 47.5 ± 16.0%. Another series of animals received daily doses of FSH (12.5 μg/100 g b. w.) and LH (50 μg/100 g b. w.) with Depo Provera. Lumen formation in this series was similar to that in animals receiving Depo Provera alone.     

Vasectomy in rhesus monkeys II. Failure to demonstrate humoral and cellular immune responses specific for sperm

A study designed to determine whether humoral antibodies and a cell-mediated immune response to sperm antigen(s) in rhesus monkeys after unilateral and bilateral vas ligations has revealed negative findings. Kibrick agglutination, indirect hemagglutination, and sperm immobilization tests on pre- and postvasectomy sera obtained at timed intervals of one to one hundred and two weeks indicated that significant sperm agglutinating, hemagglutinating, and immobilizing antibodies did not develop in these animals. Also, lymphocyte transformation studies showed that a significant cellular antibody response to sperm antigen(s) did not develop in vasectomized animals. Furthermore, sera from rhesus monkeys injected with autologous and homologous washed sperm in Freund's adjuvant failed to demonstrate a positive reaction in agglutination and immobilization tests. On the other hand, immunization of a female rhesus monkey with washed sperm in Freund's adjuvant produced high titer sperm agglutinating and immobilizing antibodies. Therefore, it has not been possible to repeat or contribute supporting data to literature reports concerning the presence of antibodies to sperm in vasectomized rhesus monkeys. It is further suggested that a number of antigens unrelated to sperm might react with sera obtained from vasectomized animals and lead to positive findings in sperm agglutination and immobilization tests. As a result, erroneous conclusions may be drawn about the development of antibodies specific for sperm in vasectomized rhesus monkeys. Definitive conclusions concerning the development of specific antibodies to sperm antigens after vasectomy will be possible only when sperm antigens have been isolated, purified, and chemically and immunologically characterized for use in serologic tests.     

The Ligation of the Male Reproductive Organs and the Role of the Spermatic Cyst

The effects of ligation of the vas deferens, the corpus epididymis and the vasa efferentia on spermatozoa and testicular morphology were studied in sexually mature rats. Following the ligation of the vas deferens, headless and immotile spermatozoa were observed on the second day in the vas deferens. Decapitation occured in more than ninty per cent of the spermatozoa on the sixth day and the motility became almost zero. On the contrary, in the epididymis normal spermatozoa were observed for a relatively long period. Even three weeks after the vas ligation, more than ninty per cent of spermatozoa showed normal morphology. Spermatic cyst formation was observed so early as four days following ligation of the vas. By the third week cysts were observed in most rats, either unilaterally or bilaterally. In addition, ligation of the corpus epididymis resulted also in the formation of a spermatic cyst on the proximal site of the ligature. A strong correlation was observed between spermatic cyst formation and the occurrence of morphological changes in the testis, as well as the motility and the normality of spermatozoa. When the spermatic cyst was formed, the testis showed almost normal morphology for a long period as well as spermatozoa in the ductal system. When a spermatic cyst was not formed, degenerative changes took place promptly and abnormal spermatozoa were observed in the ductal system. These observations suggest that the seminiferous tubules may be very sensitivie to the increase in intratubular pressure and in such instances the spermatic cyst acts as a "shock absorber" to prevent the abnormal increase of pressure within the ductal system, especially the seminiferous tubules. In addition, the result suggests further that a need for caution and careful follow-up are necessary in the vasectomized man.     

Does ethane 1,2-dimethanesulphonate (EDS) have a direct cytotoxic effect on the seminiferous epithelium of the rat testis?

The authors examined the possibility that ethane 1,2-dimethanesulphonate (EDS) has a cytotoxic effect on spermatogenesis that is not secondary to androgen withdrawal resulting from the well known cytotoxic effect of EDS on Leydig cells. Adult male rats were implanted with polydimethylsiloxane (PDS) capsules containing testosterone (T) and estradiol (E), and were simultaneously injected with EDS. The PDS-TE implants, by inhibiting luteinizing hormone (LH) production, prevented Leydig cells from repopulating the testis and clamped testosterone within the seminiferous tubules at increasing concentrations relative to implant size. In rats that received EDS alone, the number of advanced spermatids per testis was significantly reduced by 2 weeks, but within 8 weeks returned to the numbers maintained in vehicle-injected control rats or in vehicle-injected rats that received testosterone- and estradiol-filled capsules of 24 cm and 0.1 cm, respectively (PDS-24TE). Surprisingly, in rats that received an EDS injection plus PDS-24TE implants, the number of advanced spermatids per testis was significantly reduced at 8 weeks and severe seminiferous tubule atrophy occurred despite the fact that the testosterone concentration was sufficient to quantitatively maintain spermatogenesis in vehicle-injected rats. In rats injected with EDS and implanted with 24 cm testosterone but not estradiol-filled capsules (PDS-24T), the advanced spermatid number per testis was significantly higher than that in the EDS plus PDS-24TE rats, but significantly lower than that in control rats. These results suggest that EDS may have a cytotoxic effect on the seminiferous epithelium that is independent of the elimination of Leydig cells, and the EDS and estradiol act synergistically to exert a profound toxic effect on spermatogenesis.