| Background and Objective:As a safe and reliable method for male contraception, vasectomy has been widely used for over 30 years. About 100 million men worldwide[1] (including 50 million men in China[2] ) recieved vasectomy. Unfortunately, since 1990's, the use of vasectomy in China has been decreasing[3], mainly due to two factors: complications and irreversibility of contraception. The main complications after vasectomy include scrotal hematoma, epididymal stasis and spermatozoal granuloma. With the development of microsurgery and in vitro fertilization techniques, the effect of vasectomy on spermatogenesis has increasingly become a focus of research. Sperm counts and quality have a direct influence on the reversal of fertility and the chance of success in artificial fertilization.In our earlier work with vasectomy via scrotum on rabbits, both close-ended and open-ended vasectomy resulted in damages to spermatogenesis[4, 5]. We speculated that the effects of vasectomy on spermatogenesis might be related to the distance between the site of vasectomy and the cauda of epididymis and operational trauma. The current research utilized a new technique of vasectomy via inguinal canal in rabbits, in which the vasal ligation is farther away from the epididymal cauda and has little influence around testis, and compared with previous vasectomy via scrotum so as to determine whether it could indeed reduce surgical trauma and early post-operational complications.Methods1. Experimental Designs:Experiment 1: Thirty normal male Japanese white rabbits, aged approximately 4 months, were randomly divided into three groups:①The control group: no treatment was given.②The UCVI (unilateral close-ended vasectomy via inguinal canal) group: the vas deferens in the inguinal canal was exposed through a small ventral midline incision, and a segment of the left or right (alternately chosen) vas deferens was excised with both vasal ends being ligated (vasectomized side). A sham operation was performed on the contralateral side in the same way except that the vas was not cut or ligated (sham-operated side).③The UCVS (unilateral close-ended vasectomy via scrotum) group: the vas deferens, epididymis and testis were exposed through a scrotal incision, and a segment of the left or right (alternately chosen) vas deferens was excised and both vasal ends were ligated with the juxta-epididymal ligature being approximately 1 cm away from the cauda of epididymis. A sham operation was performed on the contralateral side.Ten days after vasectomy, organs– testis, epididymis and vas deferens– were removed on one side (randomly chosen) in the control group and on both sides in other groups.Experiment 2: Thirty-two normal male Japanese white rabbits, aged approximately 6 months, were randomly divided into two groups:①The UCVI (unilateral close-ended vasectomy via inguinal canal) group: left or right (alternately chosen) vas was ligated and a sham operation was performed on the contralateral side.②The UOVI (unilateral open-ended vasectomy via inguinal canal) group: left or right (alternately chosen) vas was ligated and a sham operation was performed on the contralateral side. The UOVI was the same as UCVI except that the juxta-epididymal end of the vas was left open and only the other end was ligated.Ten days after operation, bilateral testes, epididymides and vasa deferentia were removed from five animals randomly chosen. The organs in other animals were removed three months after operation.2. Tissue ProcessingAfter perfusion fixation in Bouin's solution for approximately 48 hours, 1~3 blocks were obtained randomly frome each testis, epididymis or vas deferens and embedded in hydroxyethyl methacrylate resin, and sections were cut at 10μm in thickness and stained with periodic acid-Schiff's reagent and hematoxylin (testis) or hematoxylin alone (epididymis and vas).3. Observation and MeasurementIn removing the organs, the shape of the testis and epididymis and the adhesion with surrounding tissues were noted. The histology of the testis, epididymis and vas deferens was examined under light microscope, and the following parameters were abtained using stereological methods:①diameter and volume of the seminiferous tubules in the testis;②volumes of the epididymal ducts, spermatozoal mass and others in the epididymis;③volume fraction of the spermatozoal mass and others in the vas deferens.4. Statistical TestComparision of mophometric parameters between the vasectomized side and the (contralateral) sham-operated side in the same group was performed using paired t-test, and significant difference was set at P≤0.05.Results1. Anatomical ObservationTen days after vasectomy, the testis, epididymis and vas in the UCVI or UOVI group had normal shape and no adhesion with surrounding tissue. The distance from the cauda of epididymis to the juxta-epididymal ligated end of the vas or the juxta-epididymal open end of the vas on the vasectomized side was about 5~8 cm. The testis and epididymis in the UCVS group had adhesion with surrounding tissue and some organs were distorted in shape.Three months after vasectomy, the cauda of epididymis and the vas deferens in the UCVI and UOVI groups were severely distended; the distended vasal part looked milky white and accounted for approximately 3/5 of the length of the vas between the cauda of epididymis and the vasal ligature or open end.2. Histology of the TestisThe primary characteristics of spermatogenic damage include atrophy of the seminiferous tubules with reduced tubule diameter and thinned seminiferous epithelium, sloughing and reduction in number of spermatogenic cells, and presence of multi-nucleated or degenerated cells.Ten days after vasectomy, severe damages to spermatogenesis were observed in 1/7 testes on both vasectomized and sham-operated side in the UCVI group, and no significant difference was found between the two sides in the volume of testis or the mean diameter of serminiferous tubules. In the UCVS group, severe damages to spermatogenesis were observed in 3/8 testes and slight damages in another 2/8 testes on the vasectomized side or the sham-operated side. The testicular volume and mean tubular diameter had no significant difference between the two sides. No severe spermatogenic damage was observed on the vasectomized (all 4 testes) or sham-operated (all 5 testes) side in the UOVI group, and there was no significant difference between the two sides in the testicular volume or mean tubular diameter.Three months after operation, no severe spermatogenic damage was observed on the vasectomized or sham-operated side in the UCVI or UOVI group, and no significant difference was found between the two sides in either group in the testicular volume or mean tubular diameter.3. Histology of EpididymisThe main effects of vasectomy on epididymis are distension of the epididymal duct and spermatozoal stasis in the epididymal duct on the vasectomized side.Ten days after operation, in the UCVI group, the total volume of spermatozoal mass in the epididymis and the volume of epididymal duct or spermatozoal mass in the cauda of epididymis had a trend of increase on the vasectomized side, about 1.43~1.62 times that on the sham side but without statistical significance. In the UCVS group, the volume of the epididymal duct or the duct lumen in the cauda epididymidis significantly increased on the vasectomized side about 1.38~1.73 folds, and a trend of increase (1.13~1.95 folds) was observed in the volume of epididymis and the volume of spermatozoal mass in the epididymis but without statistical significance. In the UOVI group, there seemed a slight decrease in the volume of the epididymal cauda or the epididymal duct in the epididymal cauda on the vasectomized side, and a slight increase in the volume of spermatozoal mass in the epididymis or the epididymal cauda, but the differences were of no statistical significance.Three months after operation, marked distension of epididymal duct and spermatozoal stasis were observed on the vasectomized side in both the UCVI and UOVI groups. The volumes of epididymis or epididymal cauda and the volumes of spermatozoal mass in the epididymis and the epididymal duct or duct lumen in the epididymal cauda on the vasectomized side significantly increased 1.64~3.87 folds (UCVI group) and 1.43~2.61 folds (UOVI group) compared with that on the sham-operated side.4. Histology of Vas DeferensThe primary effects of vasectomy on the vas deferens are vasal distension and spermatozoal stasis.Ten days after operation, the vassal diameter, the volume fraction of the epithelial layer and lumen or the volume fraction of the spermatozoal mass in the vas deferens had no significant difference between the vasectomized and sham-operated sides in the UCVI and UOVI groups.Three months after vasectomy, marked vasal distension and spermatozoal stasis were observed in both the UCVI and the UOVI groups. The volume fraction of the epithelial layer and lumen and the volume fraction of the spermatozoal mass in the vas deferens on the vasectomized side significantly increased 2.52 and 4.28 folds (UCVI group) and 2.06 and 3.60 folds (UOVI group), respectively, compared with that on the sham-operated side.Conclusions1. The present research showed that vasectomy via the inguinal canal did not lead to testicular, epididymal and vasal adhesion with surrounding tissue, and ten days after operation, no obvious spermatogenic damage was observed in all 4 testes (open-ended vasectomy via inguinal canal) and 6/7 testes (close-ended vasectomy via inguinal canal). In this and our previous studies, however, 10 days after vasectomy via scrotum, adhesion of testis with surrounding tissue was induced, and severe spermatogenic damage was observed in 4/12 testes[5] and 3/8 testes (close-ended operation) and 12/12 testes[5] (open-ended operation). This strongly suggest that spermatogenic damages induced shortly after vasectomy was related to operational trauma or inflammatory irritation around the testis.2. The present research showed that there were distension of the epididymal cauda and the vas deferens, and spermatozoal stasis in the epididymal duct and the vas deferens three months after the close-ended and open-ended vasectomy via the inguinal canal, with no spermatogenic damage to the testis. We previously showed[5, 6], however, that severe spermatogenic damages were induced in about half of testes three months after the close-ended vasectomy (near the epididymal cauda) via scrotum. After close-ended vasectomy (near the epididymal cauda) via scrotum, spermatozoa produced by the testis were unable to be stored in the distensible vas deferens, and only a limited amount of sperm were stored in the epididymis [5, 6]. However, after vasectomy (distant from the epididymal cauda) via the inguinal canal, spermatozoa produced by the testis could be transported to and stored in the longer vas deferens, thus being able to relieve intra-testicular pressure. This suggested that the spermatogenic damages after vasectomy are associated with the incapability of sperm transport out of the testis. Retaining of sperm in the testis would increase the intra-testicular pressure and, in turn, affect spermatogenesis. The present research provided strong evidence for the hypothesis that spermatogenic damage induced after vasectomy was mainly intra-testicular pressure-mediated[4, 7].
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