A new surgical method of vasoepididymostomy for epididymal obstructive azoospermia to improve the success rate in the rat

Introduction

Infertility is a widely recognized medical and social problem that, according to statistics, affects about 20% of couples. In the population with male infertility, the incidence of azoospermia, which is one of the most serious cases of male infertility, is 5–10% (1). Azoospermia is divided into obstructive azoospermia (OA) and nonobstructive azoospermia (NOA), with OA accounting for approximately 40% of the cases (2). OA is most commonly characterized by obstruction of the vas deferens or epididymis and accounts for 55–67% of cases (3). With advances in medical science, microscopic vasoepididymostomy has been shown to be an effective surgical method for treating epididymal OA (4-6).

The earliest intervention in history dates back to 1903 when E. Martin performed a multi-sided incision connecting the epididymis to the distal vas deferens to aid pregnancy (7). In 1978, an optically improved microsurgical anastomosis was used for the first time for anastomosis of the epididymal duct with a diameter of 0.2–0.4 mm. The diameter of the human vas deferens is similar to that of the rat vas deferens. The double longitudinal needle locking technique was first proposed in 2003 and marked a new stage of microscopic anastomosis (8). Other investigations have shown that the recanalization and conception rates of the vas deferens after epididymal vas deferens anastomosis under a microscope are 76.3% and 34.8%, respectively (9). In recent years, a new and improved single-arm sutured longitudinal intussusception microvascular epididymal anastomosis technique has been proposed, but its surgical approach has not undergone any qualitative changes (10).

Microscopic vasoepididymostomy is the most difficult surgery and requires precision. The failure rate of conventional surgical methods is as high as 30%, far from the expected surgical success rate, which is the patency rate of the anastomotic site. Therefore, we urgently need to explore a new surgical method to improve the patency rate of the anastomotic site, and reduce complications caused by anastomotic stenosis or occlusion, anastomotic tearing, and anastomotic leakage in patients, leading to failure of epididymal and vas deferens recanalization. Animal experiments have shown that the failure rate of surgery mainly depends on anastomotic blockage and a small anastomotic diameter (11). We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-91/rc).

Methods

Experimental animals and breeding male rats (type: Sprague-Dawley, SD), weighing approximately 250–300 g and at approximately 6 weeks of age, were purchased from the Animal Experimental Center of Shanxi Medical University and fed standard rat feed at an ambient temperature of 15–25 ℃ and a humidity of 40–60%. Experiments were performed under a project license granted by Ethics Committee for Experimental Animal Welfare, Shanxi Medical University, China (No. SYDL2023040), in compliance with Chinese institutional guidelines for the care and use of animals.

Experimental materials

These included isoflurane, microscope (Three eye arm stereomicroscope. MSD 205-C), microscopic instruments, surgical sutures, penicillin (400,000 units/mL), and gauze.

Surgical methods

Establishment of epididymal obstruction model

All rats were fasted for 4 h before surgery, were allowed to drink water, were anesthetized with an oxygen pump inhalation of isoflurane initiated at 3–4%, and maintained at 2–2.5% inhalation. During surgery, the vas deferens was localized and exposed along the epididymis. The researchers performed a 4-0 suture vas deferens ligation near the end of the epididymis, approximately 0.3–0.5 cm, and disconnected the vas deferens. Finally, the testes were moved back into the scrotum. Then, suture and fix the spermatic cord and scrotal muscle layer with 4-0 suture to prevent retraction of the testes into the abdominal cavity. Inhalational anesthesia was terminated 2 min before the completion of suturing. The rats were able to wake up within 2 min, and an animal model of epididymal OA was established. After 3 weeks of inducing obstruction by the modified vas deferens ligation surgery, the inflammation and edema of the surgical incision have subsided after 3 weeks.

Group I

The ligated vas deferens was freed, the ligature thread was loosened, and it was observed whether semen emerged from the distal end of the vas deferens. Methylene blue was injected into the proximal end of the vas deferens to check patency. Care was taken to control the surgical time to ensure that it was consistent with that of groups II and III. The reproductive organs were anatomically exposed for the same duration without anastomosis formation.

Group II

The vas deferens was incised obliquely at a 45° angle (Figure 1A), an 8-0 suture was pulled, and the serosal layer of the vas deferens and the epididymal capsule were fixed. Two identical 10-0 nylon sutures were sutured from the outside to the inside of the vas deferens at the puncture site. After insertion of the needle, it was threaded parallel through the epididymal duct, and the needle was temporarily not removed to prevent collapse of the epididymal duct. Another 10–0 stitches were sutured in the middle of the two stitches (Figure 1B), the knot was tied, and the epididymal duct was lifted (Figure 1C). The epididymal duct was cut using microscopic scissors and a circular incision was made (Figure 1D). The leaked epididymal fluid was collected with a capillary glass tube, placed on a microscope slide, and examined for sperm under a microscope. If no sperm was found, the dilated epididymal duct was searched for sperm from the epididymal tail, epididymal body, to the epididymal head. If sperm were found, the suture was continued into the mucosal layer of the vas deferens with 10-0 needles at both ends, as shown in Figure 2. The suture was tightened, the epididymal duct was pulled into the vas deferens cavity, and a knot was tied to fix the epididymal duct and vas deferens. The serosal layer of the vas deferens and epididymal capsule was reinforced with approximately six stitches and sutured with 8-0 intermittent circular reinforcement. The incision was then closed.

Figure 1 Schematic diagram of the new surgical procedure CIVE. (A) The vas deferens was cut obliquely at 45°, enlarged by 4×. (B) Another 10-0 stitch was sutured in the middle of the two stitches, enlarged by 15×. (C) The knot was tied and the epididymal duct was lifted, enlarged by 15×. (D) Microscopic scissors were used to cut open the epididymal duct. The circular incision was made, enlarged by 15×. CIVE, 2-suture circular intussusception vasoepididymostomy.

Figure 2 Schematic diagram of suture sequence for new and traditional surgeries. Vas deferens a1 and b1 are the entry points, and a2 and b2 are the exit points. Two 10-0 single-needle nylon sutures were passed from the outside to the inside through the lumen of the vas deferens at the marked points (a1 and b1) and from the inside to the outside through the muscle layer at the marked points (a2 and b2); c: elliptical incision; d: 45° oblique resection of the vas deferens; e: the surgical incision for the epididymal duct was innovatively changed from the traditional linear surgical incision to the circular surgical incision in the new surgical method.

Group III

The vas deferens was transected and anastomosed vertically at an angle of 90° (Figure 3A). The serosal layer of the vas deferens and epididymal capsule were fixed with 8-0 sutures. Two identical 10-0 nylon sutures were injected in the direction indicated (Figure 3B). The two 10-0 suture needles were first inserted parallel to each other and sutured into the epididymal tube. Care was taken to ensure that the needles were not pulled out temporarily to prevent the collapse of the epididymal tube. Using a sharp blade, the wall of the epididymal tube was cut longitudinally between the two needles (Figure 3C) to ensure that the cut was linear (Figure 3D). A capillary glass tube was used to collect the overflowing epididymal fluid and was placed on a microscope slide to search for sperm under a microscope. The same steps as group II were performed. If sperm were found, the two ends of the needle were sutured further into the mucosal layer of the vas deferens, as shown in Figure 2. The suture was tightened, and the epididymal duct was pulled into the vas deferens cavity. The epididymal duct and the vas deferens were fixed using a knot. The serosal layer of the vas deferens and epididymal capsule was reinforced and sutured with approximately six stitches with 8-0 intermittent circular reinforcement. Finally, the incision was closed.

Figure 3 Schematic diagram of traditional LIVE surgical procedures. (A) The vas deferens was cut vertically at 90°, enlarged by 4×. (B) Two needles were inserted and guided in parallel through the epididymal duct, enlarged by 15×. (C) A sharp blade was used to longitudinally cut through the wall of the epididymal duct between the two needles, enlarged by 15×. (D) The linear incision was made, enlarged by 15×. LIVE, 2-suture longitudinal intussusception vasoepididymostomy.

Postoperative treatment and monitoring

Each group of eleven models can reach a balance point between effect size, sample size, significance level, and actual limitations. Care was taken to observe the mental state and changes in the hair color of the rats. If the rats were mentally lethargic, an appropriate intraperitoneal injection of 0.9% physiological saline (3–5 mL) was administered as a fluid replacement, and the wound was checked.

Statistical analysis

Statistical analysis was performed using SPSS 25.0, and the research data followed a normal distribution, using (mean ± SD) to describe the central tendency and dispersion of surgical time. For the comparison of recanalization rates, we used Fisher’s exact test to compare the difference in recanalization rates between two groups, with a single-tailed test as the analysis direction, and statistical significance was set at P<0.05.

Results

All recruited rats completed the respective surgical procedures and survived. One rat model experienced transient weight loss due to prolonged anesthesia, but this did not affect the reliability of the data. All models were included in the analysis, and no animals or data units were excluded. The surgical time and the mechanical and functional patency rates for each anastomosis were recorded. Two investigators reviewed the measurement of all the results.

The average surgical time was 86.4±12.9 min. According to the elliptical calculation, the area of the CIVE anastomosis is 3.14 times larger than that of LIVE (Figure 4). At 3 months postoperatively, all animals were tested in blinded fashion. Expose reproductive organs to evaluate whether the anastomosis is unobstructed. The recanalization rate of group II was 90.9%, compared with 63.6% in group III. The P value of the single-tailed test is equal to 0.042. There was a statistically significant difference (P<0.05) in finding sperms at the distal end of the vas deferens (mechanical patency rate) and in retrograde methylene blue imaging (functional patency rate) (Figure 5A,5B).

Figure 4 Comparison diagram of CIVE and LIVE anastomosis. A₁', A₂', B₁', and B₂' are the injection points for CIVE, and the red circle represents the CIVE anastomotic site; A₁, A₂, B₁, and B₂ are the injection points for LIVE, and CDC1E is the anastomotic site after the LIVE epididymal duct is pulled into the vas deferens; CC₁ is the linear incision line of the LIVE anastomosis. CIVE, 2-suture circular intussusception vasoepididymostomy; LIVE, 2-suture longitudinal intussusception vasoepididymostomy.

Figure 5 Schematic diagram comparing the anastomosis area between CIVE and LIVE. (A) Retrograde injection of methylene blue into the distal vas deferens, showing the epididymal duct. This represents successful surgery after epididymal vas deferens anastomosis, enlarged by 15×. (B) Retrograde injection of methylene blue into the distal vas deferens, with the epididymal duct not shown, enlarged by 15×. This represents surgical failure. CIVE, 2-suture circular intussusception vasoepididymostomy; LIVE, 2-suture longitudinal intussusception vasoepididymostomy.

Complications

The incidence of sperm granulomas in postoperative groups II and III was 0% and 18.2%, respectively, with a single-tailed test P value of 0.24. There is currently no statistical difference, but the observed difference (0% vs. 18.2%) is clinically significant, and point estimates indicate a lower incidence of complications in the new surgery group. Due to insufficient sample size, the sample size can be expanded for further verification in the later stage.

In summary, there was no statistically significant difference in the average surgical time between the two groups. Nevertheless, the anastomotic patency rate of the new surgical method in group II was significantly higher than that of the traditional surgical method in group III. All surgical incisions in rats healed well.

Discussion

Epididymal anastomosis of the vas deferens under a microscope is an effective surgical method for treating OA of the epididymis. It requires 15–20× magnification under the microscope to accurately target the vas deferens lumen. It is currently considered the most demanding surgical method in male urology. In addition, granulomas formed by surgical incision take up a certain volume. Second, in conventional surgery (LIVE), needles are inserted on both sides about a half of the diameter of the epididymal tubules. Suppose the needles of LIVE are inserted from both sides of the diameter of the epididymal tubules or A₁', A₂', B₁', and B₂' in Figure 4 when the epididymal canal is inserted into the vas deferens. In that case, the incision of LIVE’s epididymal duct closes due to insufficient tension, resulting in closure of the anastomosis or even failure of the surgical anastomosis. With our new surgical method, four-fifths or even the entire diameter of epididymal tubules can be inserted into the vas deferens. Since the anastomosis site is circular, worrying about the closure of the epididymal incision is unnecessary due to insufficient tension. This new surgical method can also increase the anastomotic area. Therefore, we believe that the key technology for the success of surgery is to expand the anastomotic area as much as possible. In our previous research on the patency of pipeline anastomosis, we validated the oblique 45° tangential anastomosis model, which significantly reduced the problem of anastomotic stenosis (11,12).

The newly developed surgical method we designed involves three major innovations. First, the traditional epididymal duct was cut open in a straight line using an extremely thin surgical blade. The epididymal duct is extremely thin, with a diameter of only about 0.2–0.4 mm. It cannot be cut even with microscopic scissors. Therefore, for the first time, Tian Quanfa proposed the use of the Tian method, which is the single needle puncture traction cutting method to change the incision of the epididymal canal from linear to circular. Removing a part of the epididymal duct wall greatly reduces the likelihood of wound closure, and the anastomotic area and patency rate are increased. Second, in conventional LIVE surgery, needles are inserted on both sides about a half of the diameter of the epididymal tubules. With new surgical method of CIVE, four-fifths or even the entire diameter of epididymal tubules can be inserted into the vas deferens. Since the anastomosis site is circular, worrying about the closure of the epididymal incision is unnecessary due to insufficient tension. This new surgical method can also increase the anastomotic area. Third, the oblique 45° incision of the anastomotic site can be closed based on the elliptical area formula; it can increase the area of the epididymal vas deferens anastomosis site by a factor of approximately 1.41 times while avoiding the accumulation of new granulation tissue in the same plane and further narrowing of the vas deferens anastomosis site (11).

Regarding the occurrence of sperm granulomas, the author considers that compared to the LIVE, the CIVE circular anastomosis is closer to the inner wall of the vas deferens and is less likely to leak out from the gap between the walls. Secondly, due to the higher patency rate and larger anastomotic opening area. The probability of CIVE developing sperm granulomas will be lower.

Owing to the specificity of the surgical method, it is difficult for the surgeon to accurately determine the specific cause of anastomosis failure after surgery. If recanalization fails, it may be due to the following reasons. First, the anastomotic site is the most important factor: (I) Narrowing or occlusion of the anastomotic site: an opening of epididymal duct that is too large can easily lead to anastomotic leakage, whereas an opening or scar formation at the opening of the epididymal duct can easily lead to re-obstruction. Therefore, the diameter of the epididymal duct opening during surgery should be approximately equal to the oblique inner diameter of the vas deferens cavity. mainly the new surgical method changes the opening of the epididymal duct into a circular shape, significantly reducing the likelihood of anastomotic occlusion and increasing the anastomotic area compared with the traditional linear anastomotic approach. (II) Anastomotic tear: if the vas deferens are too free and too short, it can increase anastomotic tension. Excessive force when suturing the outer membrane of the vas deferens and epididymis can result in pulling at the anastomotic site. Therefore, all surgeons must undergo rigorous training before performing microsurgery. (III) Anastomotic leakage: anastomotic leakage can easily lead to sperm granuloma formation. This may be caused by shaking when inserting and withdrawing the needle or by repeated insertion and withdrawal, resulting in leakage at the suture site. Besides, it may be due to the opening of the epididymal canal being too large and some openings of the epididymal canal not being fully drawn into the vas deferens, as well as the overlapping of the vas deferens and epididymis with two needles tied too loosely. There are too few fixed suture needles for the outer membrane of the vas deferens and epididymis. Second, the factors of the vas deferens are also very important: separation of the vas deferens from the vas deferens arteries should avoid excessive “nakedness,” which can lead to ischemic necrosis. Repeated punctures of the vas deferens can lead to damage to the distal vas deferens and scarring.

Surgical points to consider: (I) In microscopic vasoepididymostomy, two needles should be inserted into the epididymal tubules without pulling them out. This method can prevent the fluid collapse (8). (II) The depth of the puncture site for the vas deferens is approximately 0.3–0.4 mm. If it is too deep, the epididymal duct is easily pulled out and torn. If it is too shallow, the liquid is prone to leakage. (III) The vas deferens artery is separated by 1–2 mm, and excessive separation may cause ischemia and necrosis of the vas deferens.

This is a limitation of the animal studies. Because this experiment involved a rat model treated with vasectomy, the vas deferens artery and vein were not preserved. However, in humans, the vas deferens artery and vein can be preserved during surgery. Nevertheless, the authors believe that the success or failure of vasoepididymostomy under the microscope is directly affected by the level of microsurgical intervention by the surgeon and is closely related to the size of the anastomosis.

Conclusions

The aim of this study is to propose a new surgical approach called ‘CIVE’. Compared with LIVE, CIVE at a 45° oblique incision of the vas deferens provides a larger anastomotic area and has a higher recanalization rate, which is worthy of further investigation and promotion. The ultimate goal is to provide patients with a safer and more effective treatment plan.

Acknowledgments

The authors are grateful to the anonymous reviewers who contributed to this study.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-91/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-91/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-91/prf

Funding: This work was supported by the Key R&D projects in Lvliang City (No. 2022SHFZ12) and Research Project of Shanxi Provincial Health Commission (No. 2024222). The funder only provides research funding and does not intervene in any aspect of the research.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-91/coif). Q.T. receives consulting fees from the Key R&D projects in Lvliang City (No. 2022SHFZ12) and Research Project of Shanxi Provincial Health Commission (No. 2024222). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license granted by Ethics Committee for Experimental Animal Welfare, Shanxi Medical University, China (No. SYDL2023040), in compliance with Chinese institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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