经腹股沟管输精管结扎对兔睾丸、附睾和输精管的组织学影响
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摘要
研究背景与目的:
输精管结扎是一种安全、可靠的男性节育方法,已在全世界开展30多年,实施了约1亿例[1],在我国已实施近5千万例[2],但自上世纪90年代以来,我国男性绝育术开展呈明显下降趋势[3]。有两个主要因素影响其开展,一是并发症,二是绝育的不可逆性。输精管结扎的并发症主要是阴囊血肿、附睾郁积、痛性精子肉芽肿等。不过,随着显微外科技术以及试管婴儿技术的发展,人们越来越关心输精管结扎对睾丸内精子发生的影响,精子的数量和精液的质量直接关系到输精管吻合术后生育能力的恢复和人工授精的成功率。
在既往研究中,我们采用经阴囊进行兔输精管结扎的手术方式,无论开放性还是闭合性输精管结扎均引起了较大程度的精子发生损害[4,5]。我们推测,输精管结扎术后对精子发生的影响,可能与输精管结扎的部位——结扎处离输精管附睾端的距离以及结扎术局部的手术创伤有关。因此,本研究采用一种离输精管附睾端较远因此对睾丸局部影响小的输精管结扎术——经腹股沟管输精管结扎术,通过与以前的经阴囊输精管结扎术进行对比,探讨这种手术方式是否可以减轻手术局部的创伤和术后早期(10天、3月)对精子发生等的影响。
方法:
1、实验方案:
实验1:正常雄性日本大耳白兔30只,4月龄,随机分为3组:①对照组——不作任何处理。②经腹股沟管单侧闭合性输精管结扎组(管-单-闭)——经腹正中线切口,打开两侧腹股沟管,挑出输精管,左侧或右侧(交替确定)输精管结扎——切除一段输精管并结扎两侧断端;对侧作假手术(除没剪断并结扎输精管外,其余步骤均同结扎侧)。③经阴囊单侧闭合性输精管结扎组(囊-单-闭)——经阴囊切口暴露睾丸、附睾、输精管,左侧或右侧(交替确定)输精管结扎——切除一段输精管并结扎两侧断端,近附睾端结扎处距离附睾尾约1cm;对侧作假手术。术后10天取材:对照组的动物选一侧(左侧或右侧,交替确定)取材,其余组的动物均取两侧的睾丸、附睾和输精管。
实验2:6月龄正常雄性日本大耳白兔32只,随机分为2组:①经腹股沟管单侧闭合性输精管结扎组(管-单-闭)——左侧或右侧(交替确定)结扎,对侧假手术;②经腹股沟管单侧近端开放性输精管结扎组(管-单-开)——左侧或右侧(交替确定)结扎,对侧假手术。管-单-开组的结扎术同管-单-闭组,但近附睾端输精管断端保持开放,另一断端的输精管才结扎。术后10天从各组随机抽选5只动物取材,其余的动物术后3月取材;取材包括双侧的睾丸、附睾和输精管。
2.组织处理
取下的睾丸、附睾和输精管用Bouin液浸润固定约48小时,然后从睾丸、附睾和输精管各随机切取1～3个组织块,用羟乙基甲基丙烯酸树脂包埋,切25μm厚的树脂切片,睾丸用PAS和苏木精染色,附睾、输精管用苏木精染色。
3.观察与测量
术后取材时肉眼观察睾丸、附睾的形态及其与周围组织的粘连情况;光镜下观察睾丸、附睾和输精管的组织学结构;用体视学方法测量睾丸内生精小管的直径和体积,附睾内的附睾管和精子团等的体积,输精管内精子团等的体积分数(比例)。
4.统计检验
同组动物结扎侧与(对侧)假手术侧的形态定量指标的比较,采用配对t检验,以P≤0.05表示有显著性差异。
结果:
1.解剖观察:
术后10天,管-单-闭或管-单-开组的睾丸、附睾形态正常,与周围组织无粘连,近附睾端输精管结扎处或近附睾端输精管断口处距附睾尾约5～8cm。囊-单-闭组的睾丸、附睾与周围组织粘连,且部分器官变形。
术后3月,管-单-闭组或管-单-开组结扎侧的附睾尾和输精管高度肿胀,肿胀的输精管呈乳白色,约占附睾尾至结扎处或开放处的输精管长度的3/5。
2.睾丸组织学:
精子发生损害的主要特征是:生精小管萎缩,直径变小,生精上皮变薄,生精细胞脱落、数量减少且出现退化细胞和多核细胞。
术后10天,管-单-闭组结扎侧和假手术侧各有1/7个睾丸的精子发生受到严重损害,两侧的睾丸体积或生精小管平均直径无显著性差异。囊-单-闭组结扎侧和假手术侧各有3/8个睾丸的精子发生受到严重损害,另2/8个睾丸的精子发生受到轻度损害,两侧的睾丸体积或生精小管平均直径无显著性差异。管-单-开组结扎侧(所有4个睾丸)和假手术侧(所有5个睾丸)精子发生均未受到严重损害,两侧的睾丸体积或生精小管平均直径无显著性差异。
术后3月,管-单-闭组或管-单-开组结扎侧和假手术侧睾丸的精子发生均未受到严重损害,两侧的睾丸体积或生精小管平均直径无显著性差异。
3.附睾组织学:
结扎对附睾的影响主要表现在:结扎侧附睾管的扩张和精子在附睾管内郁积。
术后10天,管-单-闭组结扎侧附睾内精子团的总体积、尾部附睾管或尾部精子团的体积与假手术侧相比有增加趋势,增加43%-62%,但差异无显著性。囊-单-闭组结扎侧尾部附睾管或尾部附睾管腔的体积与假手术侧相比均有显著性增加,增加38%-73%;附睾总体积或附睾内精子团的总体积与假手术侧相比有增加趋势,增加13%-95%,但差异无显著性。管-单-开组结扎侧附睾尾的体积或尾部附睾管的体积与假手术侧相比似有减小趋势(减少21%-24%),而精子团的总体积或尾部精子团的体积似有增加趋势(增加14%-17%),但差异均无显著性。
术后3月,管-单-闭组和管-单-开组的结扎侧均出现附睾管的高度扩张和精子郁积。结扎侧附睾或附睾尾的体积、附睾内精子团的总体积以及附睾尾内附睾管、附睾管管腔、精子团的体积均显著增加到假手术侧1.64～3.87倍(管-单-闭组)或1.43~2.61倍(管-单-开组)。
4、输精管组织学:
输精管结扎对输精管的影响主要表现在结扎侧输精管的扩张和精子郁积。
术后10天,管-单-闭组或管-单-开组结扎侧输精管的管径,输精管内上皮层及管腔的体积分数或输精管内精子团的体积分数,与假手术侧相比均无显著性差异。
术后3月,管-单-闭组或管-单-开组均有结扎近端输精管的高度扩张和精子郁积。结扎侧的输精管内上皮层及管腔的体积分数和精子团的体积分数分别显著增加到假手术侧的2.52、4.28倍(管-单-闭组)或2.06、3.60倍(管-单-开组)。
结论:
1.该研究显示,①经腹股沟管的输精管结扎,不引起睾丸、附睾、输精管与周围组织的粘连。②经腹股沟管的开放性(共4个睾丸)及闭合性(6/7个睾丸)输精管结扎术后10天,睾丸的精子发生未见明显损害。本文研究和我们既往的研究显示,经阴囊的近附睾端输精管结扎术后10天均引起睾丸与周围组织的粘连,闭合性(见于4/12[5]、3/8个睾丸)及开放性(所有12个睾丸[5])结扎术后10天引起了严重的精子发生损害。这有力的说明,输精管结扎术后早期对精子发生的损害与手术创伤或睾丸周围的炎症刺激有关。
2.该研究显示,经腹股沟管的闭合性及开放性输精管结扎术后3月,附睾尾、输精管肿胀,附睾管及输精管内精子郁积,但睾丸内的精子发生未见明显损害。我们既往的研究[5,6]显示,经阴囊的近附睾端输精管闭合性结扎术后3月,约有一半的睾丸有严重的精子发生损害。经阴囊的近附睾端闭合性输精管结扎后,睾丸产生的精子不能贮存于扩张性好的兔输精管,附睾内贮存的精子也很有限[5,6];而经腹股沟管的远离附睾的闭合性或开放性输精管结扎术后,睾丸产生的精子可继续输送出来,贮存在较长的输精管内,因而可缓解睾丸内压。这说明,输精管结扎对精子发生的损害与睾丸产生的精子不能排出有关。睾丸产生的精子不能排出,就势必导致睾丸内压升高,进而影响精子发生。该研究为输精管结扎致精子发生的损害主要是由于睾丸内压所致的假说[4,7]提供了有力的证据。
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.
Methods
1. 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 Processing
After 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 Measurement
In 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 Test
Comparision 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.
Results
1. Anatomical Observation
Ten 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 Testis
The 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 Epididymis
The 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 Deferens
The 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.
Conclusions
1. 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].
输精管结扎是一种安全、可靠的男性节育方法,已在全世界开展30多年,实施了约1亿例[1],在我国已实施近5千万例[2],但自上世纪90年代以来,我国男性绝育术开展呈明显下降趋势[3]。有两个主要因素影响其开展,一是并发症,二是绝育的不可逆性。输精管结扎的并发症主要是阴囊血肿、附睾郁积、痛性精子肉芽肿等。不过,随着显微外科技术以及试管婴儿技术的发展,人们越来越关心输精管结扎对睾丸内精子发生的影响,精子的数量和精液的质量直接关系到输精管吻合术后生育能力的恢复和人工授精的成功率。
在既往研究中,我们采用经阴囊进行兔输精管结扎的手术方式,无论开放性还是闭合性输精管结扎均引起了较大程度的精子发生损害[4,5]。我们推测,输精管结扎术后对精子发生的影响,可能与输精管结扎的部位——结扎处离输精管附睾端的距离以及结扎术局部的手术创伤有关。因此,本研究采用一种离输精管附睾端较远因此对睾丸局部影响小的输精管结扎术——经腹股沟管输精管结扎术,通过与以前的经阴囊输精管结扎术进行对比,探讨这种手术方式是否可以减轻手术局部的创伤和术后早期(10天、3月)对精子发生等的影响。
方法:
1、实验方案:
实验1:正常雄性日本大耳白兔30只,4月龄,随机分为3组:①对照组——不作任何处理。②经腹股沟管单侧闭合性输精管结扎组(管-单-闭)——经腹正中线切口,打开两侧腹股沟管,挑出输精管,左侧或右侧(交替确定)输精管结扎——切除一段输精管并结扎两侧断端;对侧作假手术(除没剪断并结扎输精管外,其余步骤均同结扎侧)。③经阴囊单侧闭合性输精管结扎组(囊-单-闭)——经阴囊切口暴露睾丸、附睾、输精管,左侧或右侧(交替确定)输精管结扎——切除一段输精管并结扎两侧断端,近附睾端结扎处距离附睾尾约1cm;对侧作假手术。术后10天取材:对照组的动物选一侧(左侧或右侧,交替确定)取材,其余组的动物均取两侧的睾丸、附睾和输精管。
实验2:6月龄正常雄性日本大耳白兔32只,随机分为2组:①经腹股沟管单侧闭合性输精管结扎组(管-单-闭)——左侧或右侧(交替确定)结扎,对侧假手术;②经腹股沟管单侧近端开放性输精管结扎组(管-单-开)——左侧或右侧(交替确定)结扎,对侧假手术。管-单-开组的结扎术同管-单-闭组,但近附睾端输精管断端保持开放,另一断端的输精管才结扎。术后10天从各组随机抽选5只动物取材,其余的动物术后3月取材;取材包括双侧的睾丸、附睾和输精管。
2.组织处理
取下的睾丸、附睾和输精管用Bouin液浸润固定约48小时,然后从睾丸、附睾和输精管各随机切取1～3个组织块,用羟乙基甲基丙烯酸树脂包埋,切25μm厚的树脂切片,睾丸用PAS和苏木精染色,附睾、输精管用苏木精染色。
3.观察与测量
术后取材时肉眼观察睾丸、附睾的形态及其与周围组织的粘连情况;光镜下观察睾丸、附睾和输精管的组织学结构;用体视学方法测量睾丸内生精小管的直径和体积,附睾内的附睾管和精子团等的体积,输精管内精子团等的体积分数(比例)。
4.统计检验
同组动物结扎侧与(对侧)假手术侧的形态定量指标的比较,采用配对t检验,以P≤0.05表示有显著性差异。
结果:
1.解剖观察:
术后10天,管-单-闭或管-单-开组的睾丸、附睾形态正常,与周围组织无粘连,近附睾端输精管结扎处或近附睾端输精管断口处距附睾尾约5～8cm。囊-单-闭组的睾丸、附睾与周围组织粘连,且部分器官变形。
术后3月,管-单-闭组或管-单-开组结扎侧的附睾尾和输精管高度肿胀,肿胀的输精管呈乳白色,约占附睾尾至结扎处或开放处的输精管长度的3/5。
2.睾丸组织学:
精子发生损害的主要特征是:生精小管萎缩,直径变小,生精上皮变薄,生精细胞脱落、数量减少且出现退化细胞和多核细胞。
术后10天,管-单-闭组结扎侧和假手术侧各有1/7个睾丸的精子发生受到严重损害,两侧的睾丸体积或生精小管平均直径无显著性差异。囊-单-闭组结扎侧和假手术侧各有3/8个睾丸的精子发生受到严重损害,另2/8个睾丸的精子发生受到轻度损害,两侧的睾丸体积或生精小管平均直径无显著性差异。管-单-开组结扎侧(所有4个睾丸)和假手术侧(所有5个睾丸)精子发生均未受到严重损害,两侧的睾丸体积或生精小管平均直径无显著性差异。
术后3月,管-单-闭组或管-单-开组结扎侧和假手术侧睾丸的精子发生均未受到严重损害,两侧的睾丸体积或生精小管平均直径无显著性差异。
3.附睾组织学:
结扎对附睾的影响主要表现在:结扎侧附睾管的扩张和精子在附睾管内郁积。
术后10天,管-单-闭组结扎侧附睾内精子团的总体积、尾部附睾管或尾部精子团的体积与假手术侧相比有增加趋势,增加43%-62%,但差异无显著性。囊-单-闭组结扎侧尾部附睾管或尾部附睾管腔的体积与假手术侧相比均有显著性增加,增加38%-73%;附睾总体积或附睾内精子团的总体积与假手术侧相比有增加趋势,增加13%-95%,但差异无显著性。管-单-开组结扎侧附睾尾的体积或尾部附睾管的体积与假手术侧相比似有减小趋势(减少21%-24%),而精子团的总体积或尾部精子团的体积似有增加趋势(增加14%-17%),但差异均无显著性。
术后3月,管-单-闭组和管-单-开组的结扎侧均出现附睾管的高度扩张和精子郁积。结扎侧附睾或附睾尾的体积、附睾内精子团的总体积以及附睾尾内附睾管、附睾管管腔、精子团的体积均显著增加到假手术侧1.64～3.87倍(管-单-闭组)或1.43~2.61倍(管-单-开组)。
4、输精管组织学:
输精管结扎对输精管的影响主要表现在结扎侧输精管的扩张和精子郁积。
术后10天,管-单-闭组或管-单-开组结扎侧输精管的管径,输精管内上皮层及管腔的体积分数或输精管内精子团的体积分数,与假手术侧相比均无显著性差异。
术后3月,管-单-闭组或管-单-开组均有结扎近端输精管的高度扩张和精子郁积。结扎侧的输精管内上皮层及管腔的体积分数和精子团的体积分数分别显著增加到假手术侧的2.52、4.28倍(管-单-闭组)或2.06、3.60倍(管-单-开组)。
结论:
1.该研究显示,①经腹股沟管的输精管结扎,不引起睾丸、附睾、输精管与周围组织的粘连。②经腹股沟管的开放性(共4个睾丸)及闭合性(6/7个睾丸)输精管结扎术后10天,睾丸的精子发生未见明显损害。本文研究和我们既往的研究显示,经阴囊的近附睾端输精管结扎术后10天均引起睾丸与周围组织的粘连,闭合性(见于4/12[5]、3/8个睾丸)及开放性(所有12个睾丸[5])结扎术后10天引起了严重的精子发生损害。这有力的说明,输精管结扎术后早期对精子发生的损害与手术创伤或睾丸周围的炎症刺激有关。
2.该研究显示,经腹股沟管的闭合性及开放性输精管结扎术后3月,附睾尾、输精管肿胀,附睾管及输精管内精子郁积,但睾丸内的精子发生未见明显损害。我们既往的研究[5,6]显示,经阴囊的近附睾端输精管闭合性结扎术后3月,约有一半的睾丸有严重的精子发生损害。经阴囊的近附睾端闭合性输精管结扎后,睾丸产生的精子不能贮存于扩张性好的兔输精管,附睾内贮存的精子也很有限[5,6];而经腹股沟管的远离附睾的闭合性或开放性输精管结扎术后,睾丸产生的精子可继续输送出来,贮存在较长的输精管内,因而可缓解睾丸内压。这说明,输精管结扎对精子发生的损害与睾丸产生的精子不能排出有关。睾丸产生的精子不能排出,就势必导致睾丸内压升高,进而影响精子发生。该研究为输精管结扎致精子发生的损害主要是由于睾丸内压所致的假说[4,7]提供了有力的证据。
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.
Methods
1. 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 Processing
After 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 Measurement
In 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 Test
Comparision 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.
Results
1. Anatomical Observation
Ten 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 Testis
The 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 Epididymis
The 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 Deferens
The 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.
Conclusions
1. 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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[29]Ren-Dong Zhang, Xian-Zhong Deng, Ya-ping Wang, Zheng-Wei Yang, et al. Spermatogenic damage induced shortly after vasectomy was related to operational truma? Asian J Androl. 2006(suppl.).
[30]陈胜民, 向前, 邹学正, 等. 输精管组织学观察及输精管结扎术后并发症的探讨 [J].生殖与避孕.1998, 18(3): 185-186
[31]徐冰, 黄卫东. 直视钳穿法在国外开展状况 [J].中国计划生育学杂志. 2000,8(7): 332-334
[32]徐传良,范强,齐威琴等. 家兔高位输精管结扎后期近端结构变化的观察[J]. 解剖学报,1995,26(1):105-111
[33]Matsuda T, Hiura Y, Muguruma K, et al. Quantitative analysis of testicular histology in patients with vas deferens obstruction caused by childhood inguinal herniorrhaphy :comparison to vasectomized men[J]. J Urol.1996,155(2):564-567
[34]Bedford JM. Unilateral vasectomy in rabbits. J Anat. 2004,205(5):413
[35]郑天贵、张玉兰等. 腹股沟管入路法输精管结扎术后 186 例临床观察. 中国男科学杂志.2006,20(4):58-59
[36]杜卓明主编,实用组织学技术[M]. 人民卫生出版社.1998,p8-21,36-57,282-285
[37]Zhang RD,Wen XH, Kong LS, et al. A quantitative (stereological) study of the effect of experimental unilateral cryptorchidism and subsequent orchiopexy on spermatogenesis in adult rabbit testis[J].Reproduction. 2002,124:95-105
[38]Wen XH, Xiao XM, Huang P, Xie XY, Yang ZW. Histological study of vas deferens following intravasal laser irradiation[J]. Asian J Androl.2003, 5(4):287-94
[39]赵圆宇,王亚平,杨正伟. 固定与脱水对睾丸、附睾尾和眼球的密度与体积的影响[J].川北医学院学报.2006,21(1):4-7
[40]Weast RC, Astle MJ, Beyer WH. CRC Handbook of Chemistry and Physics[M]. 66th edition. Florida: CRC Press, Inc.,1985, D-228,D-262
[41]杨正伟. 形态定量. 见:现代组织学[M].主编:成令忠,钟翠平,蔡文琴. 上海:上海科学技术文献出版社 2003 年 1 月第一版,p34-39,58
[42]杨正伟. 睾丸组织结构的若干定量方法及其应用. 见:临床生殖内分泌学:女性与男性[M]. 主编:葛秦生. 北京:科学技术文献出版社 2001 年 11 月第一版,p1010-1022
[43]Reddy NM, Gerscovieh EO, Jain KA,et al. Vasectomy-Related Changes on Sonographic Examination of the Scrotum[J].J Clin Ultrasound, 2004,32 (8): 394-398
[1]Weiske WH. Vasectomy[J]. Andrologia. 2001, 33(3): 125-34
[2]刘云嵘. 90 年代我国男性绝育术开展的趋势分析 [J]. 中国计划生育学杂志. 1997, 5(3): 152-159
[3]刘云嵘,我国男性绝育使用者人数锐减值得引起高度重视[J]. 中国计划生育学杂志 2000. 61(5):195-197
[4]宋黎明等。男性节育技术回顾与展望[J]. 国外医学计划生育分册.2005,24(6):321-324
[5]刘小章. 输精管绝育术的研究进展[J]. 国外医学计划生育分册. 1992,11(3):130-133
[6]刘云嵘. 90 年代以来我国男性绝育术开展呈明显下降趋势[J]. 生殖医学杂志. l996, 3(4): 198
[7]Bing XU, Wei-Dong Huang. No-scaipel vasectomy outside China[J]. Asian J Androl. 2000, 2: 21-24
[8]Grimes DA, Satterthwaite AP, Rochat RW et al. Deaths from contraceptivesterilization in Bangladesh: rates, causes and prevention[J]. Obstet Gynecol.1982, 60: 635-640
[9]丁玉庆。7248 例输精管结扎术后并发症随访调查报告[J]。男性学杂志.1996,10(1):44-45
[10]STUART W. McDONALI .Vasectomy Review: Sequelae in the Human Epididymis and Ductus Deferens[J]. Clinical Anatomy. 1996,9:337-342
[11]Massey FJ et al. JAMA. 1984, 252: 1023-102
[12]Peterson HD et al. Obstet Gynecol. 1990, 76 (3): 568-571
[13]Flickinger CJ et al. J Androl. 1986, 7 (6): 285
[14]Michael McCormack, MD, Steven Lapointe, MD, CSPQ, FRCSC. Physiologic consequences and complications of vasectomy[J]. CMAJ. 1988, 138 (1): 223-225
[15]Raleigh D, Hons BS., O’Donnell L, Southwick GJ, Kretser DM, and McLachlan RI. Stereological analysis of the human after vasectomy indicates impairment spermatogenic efficiency with increasing obstructive interval[J]. Fertil Steril. 2004; 81: 1595–603
[16]Peng B, Zhang RD, Dai XS, et al. Quantitative (stereological) study of the effect of vasectomy on spermatogenesis in rhesus monkys (Macaca mulatta) [ J ] . Reproduction. 2002, 124(6): 847-856
[17]刘云嵘,吴世仲. 影响男性参与计划生育的因素:中国定性研究的发现[J]. 中国计划生育学杂志. 1995,1:8-17
[18]Hair WM, Wu FC. Male contraception: prospects for the new millennium[J]. Asian J Androl .2000, 2: 3-12
[19]张仁东,王亚平,杨正伟. 雄孕激素男性避孕研究进展[J]. 中国计划生育杂志. 2004. 12: 758-61
[20]谷翊群,王兴海,徐铎,彭林,程立法,黄明孔,黄真嘉,张桂元. 十一酸睾酮酯注射液避孕有效性的多中心 II 期临床研究[J]. 生殖医学杂志. 1997,5:50-51
[21]吴伟雄,谷翊群,张伟铭,王学本,陈振文. 非阻塞性输精管滤过装置节育术远期有效性和安全性的临床研究[J]. 中国计划生育杂志. 2001, 5:269-278
[22]欧汝强,姜彦嘉,陈巧儿,杨宁等. 非阻塞性输精管滤过装置节育术后抗精子抗体的分析[J]. 生殖与避孕.1999, 18(1):53-55
[23]Shapiro EI, Silber SJ. Open-ended vasectomy, sperm granuloma, and postvasectomy orchialgia[J]. fertile Steril. 1979,32(5): 546-550
[24]Errey B, Edwards I. Open-ended vasectomy: an assessment[J]. Fertil Steril .1986, 45:843–846
[25]Matthews GJ, Schlegel PN &Goldstein M. Patency following microsurgical vasoepididymostomy and vasovasotomy: temporal considerations[J]. J Urol. 1995, 154: 2078-2079
[2]刘云嵘. 90 年代我国男性绝育术开展的趋势分析[J].中国计划生育学杂志, 1997, 5(3): 152-159
[3]刘云嵘. 90年代以来我国男性绝育术开展呈明显下降趋势[J].生殖医学杂志l996, 3(4): 198
[4]Kong LS, Huang AP, Deng XZ, et al. Quantitative (stereological) study of the effect of vasectomy on spermatogenesis in Rabbits[J].J Anat. 2004,205:147-156
[5]张仁东. 常规与近端开放输精管结扎对兔睾丸和附睾结构的影响[D]. 重庆:重庆医科大学组织胚胎学教研室,2006
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[7]McDonald SW. Cellular responses to vasectomy[J].Int Rev Cytol. 2000;199:295-339
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[9]刘云嵘. 我国男性绝育使用者人数逐年锐减值得高度重视[J].中国计划生育杂志.2000(8):195-197
[10]Raleigh D, Hons BS., O’Donnell L, , Southwick GJ, Kretser DM, and McLachlan RI. Stereological analysis of the human after vasectomy indicates impairment spermatogenic efficiency with increasing obstructive interval[J].Fertil Steril. 2004; 81: 1595–603
[11]陈巧儿、文任乾、叶嘉玲、刘美意、黄健初、秦卫兵. 输精管吻合术后精子超微结构观察[J].中国计划生育学杂志.2004,9:543-546.
[12]Michael McCormack, MD, Steven Lapointe, MD, CSPQ, FRCSC. Physiologic consequences and complications of vasectomy[J].CMAJ.1988,138(1): 223-225
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[14]Silber SJ. Reversal of vasectomy and the treatment of male infertility: Role of microsurgery, vasoepididymostomy, and pressure-induced changes of vasectomy[J]. Urol Clin North Am.1981,8:53-62
[15]Bigazzi PE. Immunologic effects of vasectomy in men and experimental animals[J].Prog Clin Biol Res. 1981,70:461-476
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[17]赵雪俭,王忠山,林桦,刘复兴,王泰玲,王石林,赵静波,张菱. 输精管结扎术后睾丸组织学变化及其机制的实验研究[J].生殖与避孕.1989;9(3):20-24
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[19]Shiraishi K, Naito K, Yoshida K. Vasectomy impairs spermatogenesis through germ cell apoptosis mediated by the p53-Bax pathway in rats[J]. J Urol 2001.166:1565–1571.
[20]Whyte J, Sarrat R, Cisneros AI, Whyte A, Mazo R, Torres A and Lazaro J. The vasectomized testis[J].Int Surg 2000.85:167–174
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[22]邹雁宾,章志华,禹华月. 710 例输精管结扎术所致并发症分析[J].中国计划生育学杂志.1997,5:50-51
[23]顾益斌,陈国荣,杨兵. 男性绝育术后并发症的防治体会. 生殖与避孕 1997;17:183-4
[24]王毅,张燕,郭贤坤. 输精管结扎对男性生殖系统的影响[J].实用医学进修杂志.2002, 30:61-4
[25]Raleigh D, Hons BS., O’Donnell L, , Southwick GJ, Kretser DM, and McLachlan RI. Stereological analysis of the human after vasectomy indicates impairment spermatogenic efficiency with increasing obstructive interval [ J ] .FertilSteril.2004,81: 1595–603
[26]Shapiro EI, Silber SJ. Open-ended vasectomy, sperm granuloma, and postvasectomy orchialgia[J].Fertil Steril.1979, 32(5):546-550
[27]Moss WM. A comparison of open-ended versus closed-end vasectomies: a report on 6220 cases[J].Contraception,1992, 46( 6):521-525
[28]Errey B, Edwards I. Open-ended vasectomy: an assessmen[tJ].Fertil Steril.1986, 45: 843-846
[29]Ren-Dong Zhang, Xian-Zhong Deng, Ya-ping Wang, Zheng-Wei Yang, et al. Spermatogenic damage induced shortly after vasectomy was related to operational truma? Asian J Androl. 2006(suppl.).
[30]陈胜民, 向前, 邹学正, 等. 输精管组织学观察及输精管结扎术后并发症的探讨 [J].生殖与避孕.1998, 18(3): 185-186
[31]徐冰, 黄卫东. 直视钳穿法在国外开展状况 [J].中国计划生育学杂志. 2000,8(7): 332-334
[32]徐传良,范强,齐威琴等. 家兔高位输精管结扎后期近端结构变化的观察[J]. 解剖学报,1995,26(1):105-111
[33]Matsuda T, Hiura Y, Muguruma K, et al. Quantitative analysis of testicular histology in patients with vas deferens obstruction caused by childhood inguinal herniorrhaphy :comparison to vasectomized men[J]. J Urol.1996,155(2):564-567
[34]Bedford JM. Unilateral vasectomy in rabbits. J Anat. 2004,205(5):413
[35]郑天贵、张玉兰等. 腹股沟管入路法输精管结扎术后 186 例临床观察. 中国男科学杂志.2006,20(4):58-59
[36]杜卓明主编,实用组织学技术[M]. 人民卫生出版社.1998,p8-21,36-57,282-285
[37]Zhang RD,Wen XH, Kong LS, et al. A quantitative (stereological) study of the effect of experimental unilateral cryptorchidism and subsequent orchiopexy on spermatogenesis in adult rabbit testis[J].Reproduction. 2002,124:95-105
[38]Wen XH, Xiao XM, Huang P, Xie XY, Yang ZW. Histological study of vas deferens following intravasal laser irradiation[J]. Asian J Androl.2003, 5(4):287-94
[39]赵圆宇,王亚平,杨正伟. 固定与脱水对睾丸、附睾尾和眼球的密度与体积的影响[J].川北医学院学报.2006,21(1):4-7
[40]Weast RC, Astle MJ, Beyer WH. CRC Handbook of Chemistry and Physics[M]. 66th edition. Florida: CRC Press, Inc.,1985, D-228,D-262
[41]杨正伟. 形态定量. 见:现代组织学[M].主编:成令忠,钟翠平,蔡文琴. 上海:上海科学技术文献出版社 2003 年 1 月第一版,p34-39,58
[42]杨正伟. 睾丸组织结构的若干定量方法及其应用. 见:临床生殖内分泌学:女性与男性[M]. 主编:葛秦生. 北京:科学技术文献出版社 2001 年 11 月第一版,p1010-1022
[43]Reddy NM, Gerscovieh EO, Jain KA,et al. Vasectomy-Related Changes on Sonographic Examination of the Scrotum[J].J Clin Ultrasound, 2004,32 (8): 394-398
[1]Weiske WH. Vasectomy[J]. Andrologia. 2001, 33(3): 125-34
[2]刘云嵘. 90 年代我国男性绝育术开展的趋势分析 [J]. 中国计划生育学杂志. 1997, 5(3): 152-159
[3]刘云嵘,我国男性绝育使用者人数锐减值得引起高度重视[J]. 中国计划生育学杂志 2000. 61(5):195-197
[4]宋黎明等。男性节育技术回顾与展望[J]. 国外医学计划生育分册.2005,24(6):321-324
[5]刘小章. 输精管绝育术的研究进展[J]. 国外医学计划生育分册. 1992,11(3):130-133
[6]刘云嵘. 90 年代以来我国男性绝育术开展呈明显下降趋势[J]. 生殖医学杂志. l996, 3(4): 198
[7]Bing XU, Wei-Dong Huang. No-scaipel vasectomy outside China[J]. Asian J Androl. 2000, 2: 21-24
[8]Grimes DA, Satterthwaite AP, Rochat RW et al. Deaths from contraceptivesterilization in Bangladesh: rates, causes and prevention[J]. Obstet Gynecol.1982, 60: 635-640
[9]丁玉庆。7248 例输精管结扎术后并发症随访调查报告[J]。男性学杂志.1996,10(1):44-45
[10]STUART W. McDONALI .Vasectomy Review: Sequelae in the Human Epididymis and Ductus Deferens[J]. Clinical Anatomy. 1996,9:337-342
[11]Massey FJ et al. JAMA. 1984, 252: 1023-102
[12]Peterson HD et al. Obstet Gynecol. 1990, 76 (3): 568-571
[13]Flickinger CJ et al. J Androl. 1986, 7 (6): 285
[14]Michael McCormack, MD, Steven Lapointe, MD, CSPQ, FRCSC. Physiologic consequences and complications of vasectomy[J]. CMAJ. 1988, 138 (1): 223-225
[15]Raleigh D, Hons BS., O’Donnell L, Southwick GJ, Kretser DM, and McLachlan RI. Stereological analysis of the human after vasectomy indicates impairment spermatogenic efficiency with increasing obstructive interval[J]. Fertil Steril. 2004; 81: 1595–603
[16]Peng B, Zhang RD, Dai XS, et al. Quantitative (stereological) study of the effect of vasectomy on spermatogenesis in rhesus monkys (Macaca mulatta) [ J ] . Reproduction. 2002, 124(6): 847-856
[17]刘云嵘,吴世仲. 影响男性参与计划生育的因素:中国定性研究的发现[J]. 中国计划生育学杂志. 1995,1:8-17
[18]Hair WM, Wu FC. Male contraception: prospects for the new millennium[J]. Asian J Androl .2000, 2: 3-12
[19]张仁东,王亚平,杨正伟. 雄孕激素男性避孕研究进展[J]. 中国计划生育杂志. 2004. 12: 758-61
[20]谷翊群,王兴海,徐铎,彭林,程立法,黄明孔,黄真嘉,张桂元. 十一酸睾酮酯注射液避孕有效性的多中心 II 期临床研究[J]. 生殖医学杂志. 1997,5:50-51
[21]吴伟雄,谷翊群,张伟铭,王学本,陈振文. 非阻塞性输精管滤过装置节育术远期有效性和安全性的临床研究[J]. 中国计划生育杂志. 2001, 5:269-278
[22]欧汝强,姜彦嘉,陈巧儿,杨宁等. 非阻塞性输精管滤过装置节育术后抗精子抗体的分析[J]. 生殖与避孕.1999, 18(1):53-55
[23]Shapiro EI, Silber SJ. Open-ended vasectomy, sperm granuloma, and postvasectomy orchialgia[J]. fertile Steril. 1979,32(5): 546-550
[24]Errey B, Edwards I. Open-ended vasectomy: an assessment[J]. Fertil Steril .1986, 45:843–846
[25]Matthews GJ, Schlegel PN &Goldstein M. Patency following microsurgical vasoepididymostomy and vasovasotomy: temporal considerations[J]. J Urol. 1995, 154: 2078-2079