A Technique for Percutaneous Epididymal Spermatozoa Aspiration and Processing

Abstract

Source:
Li Xiaoming¹, Liu Xiao¹, Li Haiyang¹, Cai Xiaohua¹, Ou Jianping<sup>1

1</sup>Center for Reproductive Medicine, The Third Affiliated Hospital of Sun Yat-sen University.

This video demonstrates the percutaneous aspiration of epididymal spermatozoa from a patient with azoospermia, a condition characterized by the absence of spermatozoa in the semen. The procedure involves disinfecting the scrotal area, aspirating epididymal fluid using a syringe with a specialized culture solution and processing the collected sample to isolate viable spermatozoa. The prepared spermatozoa are then ready for use in advanced infertility treatment procedures.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Preoperative preparation

1. Preparation of surgical supplies
   1. Prepare the necessary supplies for the surgical procedure, including one surgical instruments package, two 5 mL syringes equipped with a 0.7 mm needle (0.7 x 32 Thin Wall Long Bevel (TWLB)), 60 mL disinfectant solution, two pairs of sterile rubber gloves, and one piece of sterile adhesive dressing.
   2. Ask the nurses to examine the surgical instruments package subjected to high-pressure steam sterilization for any damage or expiration. Once confirmed to be free of any issues, place it on the instrument table and open the outer layer.
   3. For the andrologists: put on sterile rubber gloves, then open the inner layer of the surgical instruments package to check whether or not the sterilization indicator tape is qualified, as well as ensure that all instruments inside are complete.
   4. Prepare two 5 mL syringes equipped with 0.7 mm needles (0.7 x 32 TWLB) for anesthesia and surgery, and then open the ampoule containing lidocaine injection.
   5. Utilize a 5 mL sterile syringe to aspirate the lidocaine injection, subsequently eliminating the air bubbles from the syringe.
2. Preparation of patient undergoing surgery
   1. Arrange skin preparation for the patient. Shave the perineum pubes using a disposable razor and wash perineum and surrounding skin with soapy water to ensure that there is no excess pubes or stains.
   2. Position the patient on the operation table in a lithotomy position, remove the pants, and secure the upper garment above the navel to fully expose the perineum and lower abdomen below the navel.
   3. Adjust the angle of the shadowless lamp in order to illuminate the patient's scrotum.
   4. Utilize oval forceps to grip a sterile gauze ball, which is then fully immersed into disinfectant solution to initiate the disinfection process. Carry out skin disinfection from the periphery to the scrotum in the following order: lower abdomen, front and inner of the thighs, perineal area, penis, and scrotum.
   5. After the initial disinfection, replace the sterile gauze ball and repeat the disinfection process. After three rounds of disinfection, the preoperative disinfection process is completed.
   6. Start the process of laying sterile towels after the disinfection is completed. First, place a sterile square towel under the patient's buttocks, then spread a sterile holed towel around the surgical area and its surroundings, fully exposing the scrotum in the hole area.
3. Preparation of G-MOPS-PLUS culture solution
   1. Ask technicians of the embryonic culture room to transfer approximately 2 mL of G-MOPS-PLUS culture solution into the culture dish using a sterile Pasteur pipette. Properly close the lid, and subsequently deliver it to the nurse in the operating room through the transfer window.
   2. Place the culture dish on the sterile towel on the instrument table and open the lid.
   3. Using another 5 mL syringe, aspirate approximately 0.2 mL of G-MOPS-PLUS culture solution, and subsequently remove the air bubbles for future utilization.

2. Anesthesia and surgery

1. For the andrologists: hold the syringe filled with lidocaine injection in the right hand while fixing the patient's spermatic cord with the left hand. Puncture subcutaneously and inject a small amount of lidocaine to form a wheal. Then, puncture subcutaneously and deepen into the tissue of the spermatic cord, starting to inject approximately 0.5 mL of lidocaine after confirming no blood return. To achieve a better effect of spermatic cord block anesthesia, change the direction of the injection needle 2 to 3 times at the same puncture point and repeat the operation of injecting lidocaine into the spermatic cord.
2. Hold the patient's scrotum with the left hand, securing the epididymis that requires puncture. By palpating, confirm the location of the epididymis where the surgical puncture is needed using the right hand. Then, hold the syringe containing lidocaine mentioned above and inject it under the skin at the puncture site to achieve local infiltration anesthesia.
3. Hold the patient's scrotum with the left hand, securing the epididymis that requires puncture. Then, hold the 5 mL syringe containing G-MOPS-PLUS culture solution (0.2 mL) in the right hand and puncture perpendicular to the skin, penetrating into the epididymal tissue. Maintain negative pressure in the syringe and repetitively puncture the epididymal tissue so that the epididymal fluid enters through the needle under the effect of negative pressure.
4. Continue to maintain negative pressure and slowly withdraw the puncture needle. Inject the extracted epididymal fluid into the culture dish. After completing the injection, aspirate nearby G-MOPS-PLUS culture solution to rinse the syringe in order to maximize the amount of collected epididymal fluid injected into the culture dish.

3. Treatment of surgical specimens

1. Search and observation of epididymal spermatozoa
   1. Transfer the culture dish containing G-MOPS-PLUS culture fluid and epididymal fluid to the embryo laboratory technician through the transfer window.
   2. The technicians of the embryonic culture room take over the culture dish and place it on the stage of an inverted optical microscope, securing it. Turn ON the power switch of the microscope and rotate the light adjustment button to an appropriate light intensity.
   3. Rotate the converter of the inverted optical microscope, select the 10x objective lens, rotate the coarse and fine focus spirals in turn to adjust the focal length. Observe the focusing situation and search for epididymal spermatozoa under the microscope.
   4. Continue to rotate the converter of the inverted microscope to the 40x objective lens, rotate the fine focus spiral to adjust the focal length, and observe the epididymal spermatozoa field by field. Evaluate the number and quality of epididymal spermatozoa. It can be seen that the normal epididymal spermatozoa have matured and obtained the ability to move.
2. Treatment of epididymal spermatozoa
   1. On observing enough active epididymal spermatozoa, transfer the liquid in the culture dish to a 15 mL centrifuge tube through a sterile Pasteur pipette.
   2. Place the above centrifuge tube on the balance tray and use a centrifuge tube of the same specification containing sterile water to level.
   3. Open the centrifuge door, place the centrifuge tubes in the card slot symmetrically, and then close the centrifuge door. Set the centrifugal force to 500 x g and the centrifugation time to 5 min through the control panel. Press the START button on the control panel to initiate the centrifugation process.
   4. After the centrifugation process is completed, open the centrifuge door, and remove the centrifuge tube containing the epididymal fluid. Unscrew the lid, gently aspirate the supernatant with a sterile Pasteur pipette, and discard the drawn supernatant.
   5. Utilizing a sterile Pasteur pipette, transfer approximately 2 mL of G-IVF-PLUS culture solution to the centrifuge tube. Then gently pipette the solution in the centrifuge tube to resuspend thoroughly.
   6. Repeat the processes of balancing, centrifugation, and removal of the upper layer of liquid.
   7. According to the needs of intra-cytoplasmic spermatozoa injection (ICSI), transfer approximately 0.5 mL of G-IVF-PLUS culture solution to the centrifuge tube utilizing a sterile Pasteur pipette, and gently blow the epididymal spermatozoa at the bottom of the centrifuge tube to make it suspended thoroughly.
   8. Use the thoroughly suspended epididymal spermatozoa directly for the ICSI process.