Experimental materials
In this study, we utilized the absorbable protein stent extracted from nutria tails, it was first designed for incision sutures (Medical device product registration: CFDA 20173654672), has the advantages of rapid degradation, softness when exposed to water, and good histocompatibility. All material were purchased from the manufacturer (Hunan Ranyuan Medical High-tech Protein Line Co., Ltd. Changsha, Hunan, China), and according to the instructions provided by the manufacturer, the protein stent was prepared from nutria tendons by removing the skin and washing the tendons with water. The tendons were soaked in ethanol, dehydrated, and treated with NaOH solution before being washed with purified water. Then, the tendons were soaked in ethanol, dehydrated, and fixed for several hours before being stented and dried naturally. After sterilization, the stents were stored in sterile packaging bags (Figure.1 A).
Animal Preparation
Throughout the research process, we stringently followed the 3R principles (replacement, reduction, and refinement) to maintain the ethical integrity and scientific robustness of the experiment. To ensure statistically significant results for each group at three distinct time points, we incorporated a total of 54 specific-pathogen free (SPF) Sprague-Dawley rats into the study. These rats, weighing an average of 300±15g and aged 8-9 weeks, were provided by the Guangdong Experimental Animal Center. They were given sterile food and water and housed in a controlled environment at the First Affiliated Hospital of Guangzhou University of Chinese Medicine. This study was conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org) and received ethical approval from the Animal Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (Approval Number: TCMF1-2021074).
Study design
Operative techniques
The rats were randomly allocated equally to the Control, Conventional, and Stent groups. All rats underwent anesthesia with 0.3% pentobarbital sodium, injected intraperitoneally at a dose of 1mL/100g, and received sterilization procedures before a 1.5 cm incision was made in the lower midline of the abdomen to expose the right testicle. The vas deferens was ligated using 4-0 silk, and the procedure was repeated on the left testicle. To prevent bleeding, the distal and proximal arteries and veins of the vas deferens were ligated with a single-armed 10-0 nylon suture. The proximal end was located 1 cm from the epididymal tail, and the distal end was 1 cm from the proximal end.
Control group:
No further processing required.
Conventional group:
According to a previous study[9], the vas deferens was excised, releasing the white, thick contents of the lumen. Then, a vasovasostomy was performed using a double-layer suture technique under microscopy (KUY NICE SZM-7045, Beijing Tianuoxiang Scientific Instrument Co. Ltd in Beijing, China), at magnifications ranging from 4 to 25, with 10-0 nylon sutures. The mucosa layer and the muscle layer were sutured together using six and eight needles, respectively. The operation time was recorded from the first needle used for the mucosa layer suturing to the completion of the muscle layer suturing.
Stent group:
The protein stent was used to suture the full layers of the vas wall. The stent, approximately the same size as the vas deferens, was 1.5 cm in length and 0.9 mm in diameter. The first step involved carefully placing half of the stent into the vas deferens while preserving the mucosa anatomization. In the second step, the other end of the stent was inserted, and the full-layer was sutured evenly with eight needles. The operation time was recorded from the beginning of the implantation to the completion of muscle layer anastomosis. The testicles were returned to the scrotum, and the abdominal layers were closed using 4-0 silk. The entire procedure was performed by the same researcher within three days (Figure.1 B).
Patency evaluation
In weeks 2, 5, and 8, six rats from both the conventional and stent groups were randomly selected. All rats underwent anesthesia with 0.3% pentobarbital sodium, which was injected intraperitoneally at a dose of 1mL/100g. This was done before exposing the left vas deferens through blunt dissection by making an incision along the abdomen. Adhesion of the vas deferens and granuloma was recorded, and a patency test was performed using a syringe needle to determine if the vas deferens was unobstructed and if the stent remnant was present (Figure.1 C). A failed water injection test and the presence of a stent remnant in the vas deferens were considered as a failure to recanalize.
Histopathology
The vas deferens tissue was preserved in 4% paraformaldehyde for 48 hours before being embedded in paraffin tissue sections. The slices were cut into 4-µm-thick sections and stained with hematoxylin and eosin. A pathologist then examined the incisions and tube walls under a 40x and 100x optical microscope (Olympus BX53, manufactured by Olympus Corporation in Tokyo, Japan).
Effects on reproductive capacity
Sperm quantity, motility, and deformity rates were assessed to evaluate the impact of protein stents on reproductive capacity. The epididymal tail was placed in semen washing solution, and sperm quantity and motility parameters were assessed using the CASA system, while deformity rate was evaluated by identifying abnormal sperm under a microscope. The number of normal and abnormal sperm per 1,000 were calculated to estimate the sperm deformity rate.
Hormone and anti-sperm antibody (AsAb) titer
While under anesthesia, samples of serum from the abdominal aorta were collected. Following the manufacturer's guidelines, levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were measured using a commercially available rat ELISA kit (CUSABIO, Wuhan, China). Similarly, a rat ELISA test kit (MyBioSource, San Diego, CA, United States) was used to measure the serum estradiol concentration. Serum samples were added to primary mouse anti-human AsAb monoclonal IgG and secondary alkaline phosphatase-conjugated goat anti-mouse IgG (Chemicon, Temecula, CA, USA) to induce immune response and detect the serum AsAb titer (Quanta Biotech, Surrey, UK).
All rats were euthanized by intravenous injection of an overdose of pentobarbital sodium (200 mg/kg) after the tissue collection was completed.
Statistical analysis
The data processing was executed using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). Results are displayed as mean ± standard deviation. To compare multiple groups, a one-way ANOVA was performed, followed by the Tukey test to pinpoint significant differences. Statistical significance was determined by a p-value less than 0.05.