Pathological Influences of Twelve Months Vasectomy on the Reproductive Tissues in Rabbits

doi:10.4236/asm.2012.21002

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Advances in Sexual Medicine, 2012, 2, 3-9

http://dx.doi.org/10.4236/asm.2012.21002 Published Online January 2012 (http://www.SciRP.org/journal/asm) 3

Pathological Influences of Twelve Months Va sectomy on

the Reproductive Tissues in Rabbits

Xinggang Wang, Yuanfeng Zhang, Zonglin Chen, Xunbin Huang*

1Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology,

Wuhan, China

Email: *[email protected]

Received November 25, 2011; revised December 27, 2011; accepted January 10, 2011

ABSTRACT

Objective—The effects of vasectomy on testes and related structures are disputable. The aim of this study was to de-

termine whether the reproductive organs of rabbits were influenced after 12 months vasectomy. Study design—Bilat-

eral vasectomy was performed on 10 male adult New Zealand Big Eared White Rabbits (another 10 rabbits set as sham

operated group) and tissue samples were examined by light microscopy after 12 months treatment. The histologic al-

terations were observed and analyzed by stereology method. Cell apoptosis by Terminal Deoxynucleotidyl Transferase

Mediated dUTP Nick End Labeling (TUNEL) assay were employed. Result—Bilateral vasectomy for 12 months af-

fected the morphology of testis, epididymis and vas deferens tissues. Significant changes were noted in the histological

stereology analysis. The apoptosis of the cells on the testis, epididymis and vas deferens increased compared with the

sham operated group. Conclusion—Our findings suggest that the reproductive tissues of rabbits were influenced in a

relatively longer term vasectomy.

Keywords: Vasectomy; Testis; Epididymis; Pathology; Rabbit

1. Introduction

Vasectomy was first used as a procedure for male contra-

ception in 1899 [1]. Currently, about more than 40 mil-

lion couples rely on it to prevent pregnancy over the

world [2]. Traditionally, this operation is thought to be a

convenience and high safety method. However, side ef-

fects such as the stasis of epididymis, painful nodules and

sperm granuloma limit the acceptability of using this te-

chnique [3].

The effect of vasectomy on testis and related organs

between species are disputable. Flickinger et al [4]. Re-

ported that after vasectomized the rats, significant altera-

tions of testis were observed. In the contrast of this find-

ing, another report reported by McDonald found no chan-

ges in testis of rats [5]. There are also contradictory re-

ports regarding the short-term and long-term effect.

At the same time, the requirements of vasecto my rever-

sal increase every year [6], therefore it is mandatory to

evaluate the function of the reproductive organs follow-

ing vasectomy for long term using.

So, monitoring the progressive changes in the testis and

related structures after long-term operation brook no de-

lay. In order to evaluate these damages in the testis, epi-

didymis and vas deferens at post-operation, the bilateral

vasectomy and sham operation were carried out on 10

male rabbits respectively and the samples were obtained

after 12 months. Attempts were also made to confirm the

effects of vasectomy on cell apoptosis of these organs.

2. Materials and Methods

2.1. Animal and Experimental Design

Twenty normal male New Zealand Big Eared White Ra-

bbits, aged 5 - 6 months, weighting 2.1 - 3.7 kg, were ob-

tained from the Experimental Animal Centre, Huazhong

University of Science and Technology (HUST). The

animals were allowed to acclimate to their new surroun-

dings for at least 1 week. Then the rabbits were marked

according to their weight and divided into two groups

(vasectomy and sham operated groups) randomly. The

animal experiment was approved by the Institution al Re-

view Board at Huazhong University of Science and Te-

chnology.

2.2. Vasectomy and Sham Operation

Anesthesia was induced by intra-peritoneal injection of

sodium pentobarbital at a dose of 30 mg/kg body weight.

Under sterile conditions, a scrotal incision was made to

expose the vas deferens and about 2 - 3 cm length of it

was excised with the two ends ligated in the vasectomy

X. G. WANG ET AL.

group. In the sham operated group, however, the vas de-

ferens was only exposed without cutting off. After opera-

tions, each animal was treated with 80,000U penicillin

with intramuscular injection for three days and kept the

wounds dry to avoid infection. The rabbits were raised as

routine in the animal center for 12 months.

2.3. Tissue Processing

Twelve months after operation, organs removal was per-

formed under anesthesia. Testes were immersed in Bouin

fluid, and epididymis and the vas deferens were fixed

with 10% formalin solution for 24 hours. Then the sam-

ples were dehydrated in ethanol and immersed in paraf-

fin.

Testis, epididymis and vas deferens were cut into par-

allel slices about 10 µm thickness. Some of the slices were

stained with hematoxylin and eosin (H & E) and then ob-

served the changes under the light microscopy.

For the apoptosis study, the slices were processed under

the manufacture’s introduction (Roche Company, Swit-

zerland). Slices were de-waxed and dehydrated. Then the

specimens were digested with proteinase-K (20 µg in

Tris/HCl) for 20 min and then dried in the air condition.

The slices were fixed in 4% paraformaldehyde solution 5

min. After the slices were fixed, slices were washed with

Phosphate Buffered Saline (PBS) and incubated in 0.3%

formaldehyde solution for 30 min at the room tempera-

ture, then washed with PBS again. Then incubated the

slices with 50 µl of TUNEL reaction mixture solution at

37˚C in a wet box for 60 min, and then washed with PBS

three times. After the slides were dried, 50 µl of biotin

labeled peroxidase (POD) was added and incubated for

30 min at 37˚C in the wet box, washed three times with

PBS and the incubated with 200 µl DAB (diaminoben-

zidine) for 10 min at room temperature. Until the back-

ground of slices became brown, the reactions were

stopped and washed DAB with deionized water for sev-

eral times.

2.4. Stereological Analysis

Slices stained with HE were taken photos with the image

acquisition system (Olympus IX71, Japan). The whole

tubules area, lumen area and cellular thickness were mea-

sured. The mean values of every measured morphologi-

cal parameter, the rate of lumen area to whole area and

the rate of cellular thickness to whole area were calcula-

ted. Ultimately, the parameters were statistically analyz-

ed between the vasectomy group and the sham operated

group.

2.5. Statistical Analysis

All data were analyzed with SPSS (v 13.0; Chicago, IL,

USA) using Independent-Samples Student’s test. p <

0.05 was considered statistically sig nificance. Data in the

table are shown as mean ± SEM (standard of error of the

mean).

3. Results

3.1. Effects of Vasectomy on Morphology

Changes

The histological changes under the light microscopy in the

testis, epididymis and vas deferens after vasectomy were

compared and shown in Figure 1.

The germinal epithelium did not show any sign of da-

mages, and the arrangements, counts of spermatogenesis

was not altered substantially in any animal of the sham

operated group (Figure 1(a)). While in the vasectomy group,

seminiferous epithelium became thinned and loose, cells

were in disorder. The seminife rous tubules were expand-

ed. Spermatids and spermatozoa could not be observed

clearly in the seminiferous tubules (Figure 1(b)). In the

sham operated gr oup , th e ciliu m in the wa ll of the epidid y-

mis duct was arranged orderly, pseudostratified columnar

epithelial cells arranged in neat rows without slightest

deformation (Figure 1(c)). However, the epididymis was

significantly distended and enlarged in the vasectomy

group. The pseudostratified columnar epithelial cells were

compressed and deformed. There were plenty of frag-

mentations deposited in the epididymis lumen (Figure

1(d)).

In the sham operated group, the epithelium cells of the

vas and the muscular layers were both intact (Figure

1(e)). While in the vasectomy group, the epithelium was

compressed and deformed, and at the basement, fibropla-

sias could be obser ved (Figure 1(f)).

3.2. Effect of Vasectomy on the Cell Apoptosis

The germinal epithelial cells of testis of the sham operat-

ed group did not go apoptosis. The spermatogenic cells at

all levels existed and arranged in neat rows. In the testis

of the sham-operation group, only 4% of spermatogenic

cells underwent apoptosis among 500 cells (Figure 2(a)).

In contrast, only a few normal cells could be observed in

the vasectomy group. About 89% of the spermatogenic

cells in the vasectomy group underwent apoptosis, inclu-

ding most spermatogonia, spermatocytes, spermatids and

sperm (Figure 2(b)).

Only small amount cells about 16 % of epididymis epi-

thelial cells in the sham operated group went apoptosis,

which might be the normal programmed cell death (Figure

2(c)). While the cells were severed significantly apopto-

sis after the vas deferens obligated for 12 months. Almo-

st all the cells were stained brown which indicated the

DNA of these cells broken (Figure 2(d)). The apoptosis

index in the vasectomy group was 96%.

X. G. WANG ET AL.

(a) (b)

(f)

(e)

Sections were prepared and stained with H & E. In the sham group, the spermatogenic cells of testicular seminiferous tubules arranged in neat rows and no

significant changes were seen (a); Spermatogenic cells of the seminiferous tubules became loosen and irregularly arranged in the vasectomy group (b). Epithe-

lial cells in epididymis of t he sham operated gr oup arranged in neat rows, cilia can b e seen clearly (c) ; The epididymis of the vasectomy group was expanded

with epithelium compressed. A lot of fragments deposited and cilia were disappeared (d); The epithelial cells of vas deferens in the sham operated group were

arranged orderly, no cell deformed (e); Mucosal folds of the vas deferens lumen in the vasectomy group became shallower or even disappear, muscular fibrosis

can be see n (f). a, b, c, d × 400, e, f × 200.

Figure 1. Histological changes of the testis, epididymis and vas deferens of two groups.

X. G. WANG ET AL.

(a) (b)

(d)

(c)

(e) (f)

In the sham operated group, spermatogenic cells at all levels existed and were arranged densely in neat rows, cells went apoptosis can be seen occasionally (a);

Spermatogonia, spermatocytes, spermatids in the vasectomy group went apoptosis (nucleus brown stained) (b); Only a small amount of cell went apoptosis in

the epididymis of the sham operated group (c); Most of the nucleus of the epitheliums in the epididymis was brown stained in the vasectomy group (d); No

differenc es of cell apoptosis were observed between the two gr o ups (sham operated and v asectomy g ro up, e, f). a, b, c, d, e, f × 400.

Figure 2. Cell apoptosis of tissues of two groups.

X. G. WANG ET AL.

By careful observation of the ep ithelium and muscular

cells in both groups, brown staining of nucleus was not

clear in both groups. Most of the brown staining obser-

ved in the plasma of the cells. So we think there were not

significant differences of cell apoptosis between the two

groups. (Figure 2(f)).

3.3. Stereological Analysis Results

The area of spermatozoa/epididymis tubules, the area of

lumen, the thickness of cellular, the rate of lumen area to

whole area and the rate of cellular thickness to whole

area were shown in Table 1. Most of the items measured

shown significant differences (P < 0.05). Compared with

the sham group, the whole area of epididymis and lumen

areas of tubules of the vasectomy group were great in-

creased. The cellu lar th ickness of ep ididymi s b ecame thin-

ner. Although the celluar layer thickness of testis didn’t

show the significance between the sham group, the rate

of cellular thickness to whole area shown significant dif-

ference (P = 0.0054). These changes may be caused by

the continuous increasing pressure for long term.

4. Discussion

The observed morphologic and stereological changes in-

duced by vasectomy in rabbits in our study demonstrated

an obvious damage to the reproductive tissues for 12 mon-

ths. Increasing pressure in the vas deferens may destroy

the normal structure of the seminiferous epithelium and

influence the function of spermatogenesis. The effects of

vasectomy on epididymis may also influence the capaci-

tation or maturation of sperms.

Urry et al [7]. found that vasectomy could influence

spermatogenesis in adult dogs. Jarow et al [8]. reported

that severe histological changes could be observed at post-

vasectomy testicular tissues and autoimmune response en-

hancement was also verified. These results are consistent

with our experiments.

A large number of studies performed on different spe-

cies in order to find the reasons of these damages. Until

now, the etiologies of reproductive damages after vasec-

tomy were still not very clear. They may be mechanical,

hormonal, immunologic, or any combination of these or

other factors.

After vasectomy, the continuous production of sperm

which could not be drained, increase the pressure of the

seminiferous tubules. The inner pressure of testis, epidi-

dymis and vas deferens significantly increased and cause

the expansion of epididymis and vas deferens [9]. Vare

suggested that the changes of the seminiferous tubules

probably result from blood stasis, which, is caused by in-

creased internal pressurec [10].

Furthermore, when vasectomy was done, the sperm ex-

posed and the exposure sperm were phagocytized by pha-

gocytes and the epididymis epithelium, then stimulate

their own immune system and promote the formation of

anti-sperm antibodies [11]. At the same time, the sperm

cannot be eliminated from the body after vasectomy, the

sperm become death and they contribute to the produce

of anti-sperm antibodies. The sperm antibodies can also

promote the occurrence of sperm specific immune respon-

se [12]. Some researchers have reported that the percent-

tage of anti-sperm antibodies after vasectomy formed was

in 60% - 80% [13]. Many studies have shown that anti-

sperm antibodies have a relationship with the changes of

testicular morphology. Chehval et al [14] found that anti-

sperm antibody levels were significantly increased after

unilateral vasec tomy in adult rats. Hence, lea ding to chan-

ges of testicular structure or infer that anti-sperm anti-

bodies may damage the testicular structure.

Table 1. Stereological analysis results of two groups male rabbits after twelve months.

Items Sham group

(

XSD

)

Vasectomy group

(

XSD

)

P value

Whole area (µm2)

Testis

Epididymis

16847.80 ± 2400. 85

55750.71 ± 8412. 33

46122.69 ± 6205. 24

129420.05 ± 1771 6.90

0.0901

0.0451

Lacunar area (µm2)

Testis

Epididymis

462.54 ± 143.23

15479.01 ± 469.3 1

24257.89 ± 5804. 83

111747.79 ± 1159 0.90

0.0021

0.0039

Lacunar area/Whoel area

Testis

Epididymis

0.0274 ± 0.0068

0.2829 ± 0.0438

0.5255 ± 0.1034

0.8685 ± 0.0699

0.0069

0.5609

Cellular layer thickness (µm)

Testis

Epididymis

66.4600 ± 11.895 8

59.0938 ± 6.9416

23.0580 ± 2.9048

11.1390 ± 0.9479

0.1356

0.0006

Cellular layer thickness/Whole area (1/µm)

Testis

Epididymis

0.0039 ± 0.0002

0.0011 ± 0.0002

0.0005 ± 0.0001

0.0001 ± 0.00001

0.0054

0.0084

X. G. WANG ET AL.

The production of reactive oxygen species (ROS) after

vasectomy might be another reason for these damages.

After vas deferens ligated, the sper m deposition cou ld cause

local inflammatory response and generate ROS. ROS gen-

eration may have a certain relationship with the testicular

tissue damage [15]. Excessive ROS products may also

through the peroxida tion of sperm membrane unsaturated

fatty acids to cause sperm dysfunction [16]. Some reports

suggested that the changes of reproductive hormones

level might be associated with these damages [17-19].

Other reports have also confirmed that the level of repro-

ductive hormones in rats changed after vasectomy [19],

the pressure of the vas deferens increased [9], the anti-

sperm antibodies produced [11]. All these can promote

the expression of gene p53 and Bax [20]. P53 along with

Bax and these damages may be the main reasons of the

degree of apoptosis increasing.

Vasectomy has been generally considered as a safe and

effective male contraception producer. However, long-

term blockage of the vas deferens may cause damages to

reproductive organs along with the time increasing. As

shown in our results, the most direct consequences of this

damage were led to the decline in male fertility and also

can cause the produce of anti-sperm antibodies. The anti-

sperm antibodies can exist in 20 - 30 years in some pa-

tients and this can cause the autoimmune infertility after

vasectomy reversal [21].

In the research of Mcvicar et al., patients who had un-

dergone a vasectomy more than 5 years were examined,

and the clinical pregnancy rate was significant reduced

[1]. Furthermore, the epididymis plays a crucial role in

the storage and maturation of spermatozoa produced by

the testis [22]. After the vasectomy, the normal histology

of the epididymis was damaged. Thus the function of the

epididymis might be affected. The spermatozoa could not

maturate and then affect the recovery rate of pregnant

after reversal.

Meanwhile, the requirements of vasectomy reversal

increase every year, more than 6% of men who undergo

vasectomy will request reversal [6]. Although the contin-

uous progress with the microsurgery, the success rate of

vas deferens can rise up to 96%, but the recovery rate of

pregnant is only 57% [23].

As far as concerned, the safety of this procedure needs

more attentions. Our findings infer that longer term va-

sectomy might influence fertility recovery in an irrever-

sible way.

5. Conclusion

In summary, the dangers of long term vasectomy should

not be ignored, the vas deferens targeting contraception

technology need to be focused on none-complete-block-

ing methods, such as one-end-opened vasectomy or fil-

tering intra-vas devices research and development.

6. Acknowledgements

This work was supported by the funds from the 11th Five-

Year National technology R & D Program of China

(grant no.2006BAI03B03).

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