:: Asian Journal of Andrology ::

Histological study of vas deferens following intravasal laser irradiation

Xiao-Hong Wen¹, Xin-Min Xiao², Peng Huang², Xian-Yong Xie³, Zheng-Wei Yang¹

¹Morphometric Research Laboratory, 3Department of Pathology, North Sichuan Medical College, Nanchong 637007, China
²Department of Urology, Chinese PLA 452nd Hospital, Chengdu 610021, China

Asian J Androl 2003 Dec; 5: 287-294

Keywords: histology; laser; male contraception; photocoagulation; vas deferens; vas occlusion

Abstract

Aim: To study the histologic changes of the vas deferens following Nd: YAG laser irradiation. Methods: Intravasal laser irradiation was given to (i) 52 segments of rabbit (laser dosage: 2 seconds at 40 W ~ 50 W) and 16 segments of human (3 seconds at 45 W ~ 55 W) vas deferens in vitro, (ii) 25 rabbit vasa (2 seconds ~ 2.5 seconds at 40 W ~ 45 W) in vivo and (iii) 2 human vasa (3 seconds at 55W) in vivo. Segments of vasa were removed from the in vivo irradiated vasa deferentia 15 days ~ 180 days (rabbit) or 15 days (man) after the exposure. All vas segments were embedded in methacrylate resin. Serial sections (thickness 25 mm ~ 30 mm) were obtained and observed under a light microscope. Results: (i) Laser-induced damage reached the muscularis layer in 27 % and 94 % of the rabbit and human vas segments in vitro, respectively. (ii) Fourteen of the 25 in vivo rabbit vasa were completely occluded by fibrous tissue and the longer the time interval after treatment, the more likely was the vas occluded. Those unoccluded vasa had either a normal histology or a mucosal damage. (iii) One in vivo human vas was almost completely occluded by the fibrous tissue but the other had a relatively large lumen packed with sperm granulomatous tissue and partial destruction of the smooth muscle layer. Conclusion: Laser irradiation can induce long-term vas occlusion; for rapid occlusion, laser doses just completely destroying the mucosal layer will be advisable.

1 Introduction

The vas deferens has been the primary target for male contraception [1]. It can be transected (through vasectomy), diverted (vasocystostomy) or occluded. Vas occlusion may be achieved by intravasal placement of different materials, such as formed-in-place silicone rubber or styrene maleic anhydride, vasal clipping or pathological growth of vasal and surrounding tissues [1]. The latter can be induced through tissue damage by means of, for example, sclerosing agents [1-2], cautery [3], high intensity focused ultrasound [4] or high power laser.

Vasectomy remains the only male contraceptive that is most reliable, widely accepted and generally safe in terms of the morphological effects on the reproductive organs or the long-term complications [5-7]. Since 1985, the no-scalpel vasectomy technique has been widely used [8]. However, studies have been continuing to establish even less invasive non-surgical methods. By means of the CO2 laser, Shi et al transected (burned off) the vas deferens in dogs and men [9] and Zhang tried to occlude the vas in rabbits and dogs [10]; similar procedure was employed in rabbits and rats using the Ar+ and Nd: YAG lasers [11, 12]. Xiao et al tried further to perform transcutaneous vas occlusion in men using YAG laser in 1997 [13] and have continued the work since then [14].

Transcutaneous vas occlusion by photocoagulation appears to be potentially more advantageous than vasectomy in that (i) a cutaneous incision is not involved and (ii) the vas reversal (via, e.g., reopening of the vas by transcutaneous placement of a thread running through the occluded node of the vas as shown by He and Tian [15] and Xiao et al [14]) would be easier than microsurgical vasovasostomy following vasectomy. Preliminary histological studies using paraffin sections indicated that vas occlusion could be achieved after photocoagulation by the growth of fibrous tissue [14]. However, careful histological study is lacking and the question when, by what tissue and to what degree the vas will be occluded following photocoagulation remains to be further clarified. This study was therefore undertaken to investigate the histological basis of photocoagulation, using methacrylate resin embedded serial sections.

2 Materials and methods

Animals were obtained from and cared for at the Animal Center of Sichuan University. Experiment protocols were approved by the relevant departments of North Sichuan Medical College and Chinese PLA 452nd Hospital and ethical guidelines were followed during experiments.

2.1 Laser irradiation

Laser generating machine: computer controlled coagulation system, Dornier Medilas Fibertom 6040, Germany. Laser delivering tool: optical fiber, TYP/TYPE:E-6100-B, fiber core diameter 0.6 mm. Laser type: Nd:YAG, wave length 1060 nm.

As previously described [14] (i) the laser delivering optical fiber was directly inserted into the vasal lumen for in vitro studies; (ii) for in vivo studies, the optical fiber was guided into the surgically exposed vas (rabbit) via a catheter needle or transcutaneously inserted into the vas (human).

2.2 Histology

Fresh vas segments were first immersion-fixed in Bouin's fluid for 24 h immediately after irradiation (in vitro groups) or after they were dissected (in vivo groups) and then stored in 70 % ethanol for a few weeks before being dehydrated in 100 % ethanol (3 changes, each 1 h) and butanol (2 changes, one 1 h and one 2.5 h). Then dehydrated blocks were embedded in methacrylate (hydroxyethylmethacrylate, HEMA) resin (Heraeus Kulzer GmbH, Germany) following the manufacturer's instruction. Serial sections were cut at 25 mm ~ 30 mm on a semi-automatic microtome (RM2145, Leica Instruments GmbH, Germany) and then stained with Harris hematoxylin. Sections were observed under a light microscope with special attention to trace the structural changes inside the vas in three dimensions.

2.3 Sample groups

2.3.1 In vitro rabbit group

Fifty-seven vas segments (each about 1 cm in length) were obtained from mature New Zealand white rabbits, of which 19, 17 and 16 were subjected to laser irradiation for 2 seconds at power 40 W, 45 W and 50 W, respectively, and 5 served as the controls. A few sections were longitudinally cut close to the middle of the vas.

2.3.2 In vitro human group

Sixteen vas segments (each about 1 cm in length) were obtained from adult men during vasectomy, of which 7, 4 and 5 were subjected to laser irradiation for 3 seconds at power 45W, 50 W and 55 W, respectively. Longitudinal sections were cut and a few sections around the middle of the vas were sampled and observed.

2.3.3 In vivo rabbit group

In 13 mature New Zealand white rabbits aged 4 months ~ 5 months, the vas deferens was exposed with a cutaneous incision and bilateral intravasal photocoagulation (for 2 s ~ 2.5 s at 40 W or 45 W depending on animal size) was performed under general anesthesia. A nylon thread was loosely tied around the vas to facilitate identification of the treated part when it was removed later. At day 15 ~ day 30, day 60 ~ day 90 and day 120 ~ day 180 after treatment, 4 rabbits, 7 rabbits and 2 rabbits were sacrificed, respectively, and the segments of vas subjected to laser irradiation removed from both sides. After embedding, the vas segments were longitudinally and serially sectioned and stained; the number of serial sections observed per segment was 20-75, with a mean of 4215 (SD).

2.3.4 In vivo human group

Bilateral transcutaneous intravasal photocoagulation (for 3 seconds at 55 W) was performed in an old man suffering from prostate cancer, on whom an operation was arranged 15 days later to remove the prostate and both vasa and testes. The two treated vas segments on both sides were removed at the operation and further cut into 2 pieces and 3 pieces. Serial sections were transversely cut and stained; the number of serial sections observed per block was 36 ~ 62, with mean 4910 (SD).

3 Results

3.1 In vitro rabbit group

The vas segments of the controls were normal in histology (Figure 1). Thirteen percent of the 52 treated vas segments had no apparent damage to the vasal histology, but 60 % had mucosa and 27 % muscularis damages; the higher the laser power, the deeper the vasal destruction was (Table 1, Figure 2).

Table 1. Laser-induced mucosal and muscular damages in in vitro rabbit vas segments.Laser dosage and histological alterations are significantly related (Chi-square test: c2 = 9.74, P<0.05).

Laser dose: power (W)/time(s)	Segments without damags	Segments with mucosal damge	Segments with muscular damage
40/2	4	14	1
45/2	3	8	6
50/2	0	9	7

Figure 1. (a) Longitudinal section of normal rabbit vas deferens; (b) and (c): higher magnification of part of (a). *: vasal lumen lined by pseudostratified columnar epithelium; A: adventitia (fibrosa). Lengths (left to right) of (a), (b) and (c): 3.73 mm, 0.38 mm and 0.76mm, respectively.

Figure 2. Longitudinal sections of two rabbit vasa deferentia immediately after intravasal laser irradiation for 2 seconds at power 40 W (a) or 50 W (b). h: normal epithelium (luminal side) of vas; m: mucosa destroyed; l: part of muscle destroyed; A: adventitia (fibrosa). Length (left to right) of either micrograph: 1.9 mm.

3.2 In vitro human group

In 15 of the 16 vas segments, laser induced destruction to the smooth muscle layer of the vas; only 1 segment appeared normal.

3.3 In vivo rabbit group

Twenty-five vas segments obtained from 13 rabbits were observed (one missing). Histological findings were shown in Table 2. Fourteen segments were completely occluded by fibrous tissue (Figure 3). The longer the time after exposure, the denser the fibrous tissue would be and the more likelihood the possibility of vas occlusion would be (Table 2). The duration and the histological alteration were significantly related (Fisher exact test: P<0.01).

Figure 3. (a) Longitudinal section of rabbit vas deferens removed 75 days after intravasal laser irradiation: Vas completely occluded by fibrous tissue. At one end (right), vasal lumen filled with densely packed spermatozoa (i) but without apparent vasal epithelium; at left end, lumen filled with epithelial cells rather than spermatozoa. (b) and (c): higher magnification of part of (a). k: densely packed spermatozoa outside the vas; l: muscularis; #: nylon thread. Lengths (left to right) of (a), (b) and (c): 3.86 mm, 0.38 mm and 0.76 mm, respectively.

Table 2. In vivo rabbit vas segments after intravasal laser irradiation. Duration and histological alternations are significantly related (Fisher exact test: P<0.01).

Duration after irradiation	Vas segments with normal histology or mucosal damage	Vas segments with lumen occluded
15 days ~ 30 days	7	1
60 days ~ 180 days	4	13

The patent vasa had either a normal histology or certain damages to the mucosal layer (Figure 4). Three of the 7 patent vasa at day 15 ~ day 30 and 2 of the 4 patent vasa at day 60 ~ day 180 had a normal histology.

Figure 4. (a1) Longitudinal section of rabbit vas deferens removed 30 days after intravasal laser irradiation; (a2): higher magnification of part of (a1). Most of mucosa destroyed but vasal lumen not occluded (*). #: nylon thread. Lengths (left to right) of (a1) and (a2): 1.90 mm and 0.76mm, respectively.

Upon careful and thorough histological observation, groups of spermatozoa, i.e. sperm granuloma, were observed outside the vas (Figure 3). Such granuloma was observed in 10 of the 14 occluded vas segments and 1 (at day 120) of the 11 patent segments.

3.4 In vivo human group

The vas from one side was almost completely occluded by the connective tissue, however, the other vas had all the mucosa and inner longitudinal muscle layers and part of the middle circular muscle layer destroyed and the vasal lumen was filled with granulomatous tissue, consisting of loosely packed spermatozoa and other tissue cells (Figure 5, 6). Outside both vasa there was sperm granuloma consisting primarily of densely packed spermatozoa and some other tissue cells (Figure 6).

Figure 5. Transverse sections of two human vasa deferentia removed 15 days after intravasal laser irradiation. (a2), (b2), (c2) and (d2), each with length (left to right) 0.38 mm, are at higher magnification of part of (a1), (b1), (c1) and (d1), respectively, each with length 1.90 mm. (a1) and (b1): from two different sections of one vas; (c1) and (d1): from the other vas. * indicating the same place in vas between each pair of micrographs, e.g. (a1) and (a2). Vasal lumen is (i) almost completely occluded by connective tissue (a1), (ii) filled with relatively dense (b1) or loose (c1) sperm granulomatous tissue, (iii) basically normal (d1).

Figure 6. Transverse sections of human vas deferens removed 15 days after intravasal laser irradiation. Vasal lumen (a) filled with relatively loose sperm granulomatous tissue [cf. micrograph (c2) in Figure 5]; (b) is from sperm granuloma outside vas: sperm densely packed with many non-sperm cells. H: sperm head with typical morphology; N: non-sperm nucleus. Length (left to right) of either micrograph is 76 m.

4 Discussion

The study demonstrated that intravasal laser irradiation could completely occlude the vasal lumen with fibrous tissue. The presence of sperm granuloma outside the occluded vas further suggested vas blockage [16]. Although 1 rabbit vas and 1 human vas were occluded 15 days after photocoagulation, complete vas occlusion occurred more likely at 60 or more days. However, the contralateral human vas was not occluded 15 days after treatment with a tissue destruction of the mid-muscular layer and the lumen was filled with loose granulomatous tissue (Figure 5, 6). We speculated that this vas would be finally occluded by fibrous tissue as a result of partial destruction of the muscularis. The speculation was supported by the finding that there was either vas occlusion, mucosal destruction or nothing abnormal in the laser treated rabbit vas, suggesting that those vasa with muscular destruction had been occluded. Note that one fourth of the in vitro rabbit vas segments (Table 1) and most of the in vitro human vas segments (see section 3.2 of the text) subjected to laser irradiation had an acute destruction of the muscularis and three fourths of the in vivo rabbit vasa were occluded 60 days ~180 days after irradiation (Table 2).

It was previously indicated that the threshold lesion for the development of effective vas occlusion might be a destruction up to the middle circular muscle layer [14] and the present results suggested that an ideal laser amount for rapid vas occlusion would be a dose that could completely burn off the mucosal layer without much damage to the muscular layer. It would take a longer time for the vasal cavity to be occluded if a considerable amount of the muscularis was destroyed. It has been indicated that an incomplete mucosal destruction may also lead to complete vasal occlusion, which is, however, inconclusive as the remaining epithelium may regenerate and repair the mucosal damage.

The fact that only 14 of the 25 rabbit vas segments were occluded 15 days ~180 days after laser irradiation indicated a noticeable variation in laser-induced vas occlusion. This may be attributable to factors such as the laser dosage and/or the variable insertion of laser-delivering optical fiber into the vasal lumen. For example, when the optical fiber is guided through into the muscular layer of the vas, luminal occlusion by connective tissue would be less likely. The conclusion is therefore that transcutaneous intravasal photocoagulation can be a good alternative to vasectomy, but appropriate dosage of laser and precise insertion of the optical fiber will be critical factors determining its safety and effectiveness. More research will be needed on the standardization of the irradiation procedure before its use as a safe and reliable male contraceptive.

Technically, the importance of the use of methacrylate embedded longitudinal serial sections should be emphasized. Compared with paraffin embedded sections, the resin sections can be cut thicker and have better microscopic effects. For example, artificial tissue separation was less likely to occur in methacrylate thick sections, because the resin is not resolved in staining. Artificial tissue separation would be a disadvantage to determine whether the vas is occluded or not. Although it is difficult to trace the histological changes of the vas using longitudinal sections, observing the entire vas segment on transverse sections may need much more efforts (perhaps 7 times). Therefore, only the vasal segments in the human in vivo group were transversely sectioned and observed.

Acknowledgements

The authors are grateful to photographer Li Lin from Nanchong Medical School for preparation of prints of Figures 1 to 6. This study was supported by grants from the Sichuan Committee of Family Planning (#99-4-2), the Sichuan Youth Foundation of Science and Technology (Chuan Ke Ji [2001] 2) and the Tenth Five Year National Key Grant of Science and Technology (2002BA-709B04b).

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Correspondence to: Prof. Zheng-Wei Yang, Director, Morphometric Research Laboratory, North Sichuan Medical College, 234 Fujiang Road, Nanchong, Sichuan 637007, China.
Tel: +86-817-224 2781, Fax: +86-817-224 2600
Received 2003-03-19 Accepted 2003-09-16