A Technique for Percutaneous Epididymal Spermatozoa Aspiration and Processing

Begin with a male patient with azoospermia, a condition in which semen lacks male gametes or spermatozoa.

The genital area is disinfected and covered with a drape to expose only the scrotum, the organ that houses the testicles, which produce spermatozoa. 

Illuminate the region and identify the epididymis, a structure responsible for the maturation and storage of spermatozoa.

Insert a syringe containing a specialized culture solution for gametes into the epididymal tissue.

Apply negative pressure while puncturing the tissue to extract epididymal fluid containing spermatozoa.

Transfer the collected fluid to a dish containing the culture solution to preserve spermatozoa viability.

Using a microscope, count the mature, healthy spermatozoa that exhibit motility.

Transfer the solution to a tube, then centrifuge to separate the spermatozoa and remove the supernatant.

Resuspend the cells in a fresh culture solution.

The isolated spermatozoa are now ready for use in infertility treatment procedures. 

To begin, gather a surgical instruments package, two syringes, disinfectant solution, sterile rubber gloves, and adhesive dressing. Examine the surgical instruments package that underwent high pressure steam sterilization for damage. Place the package on the instrument table and open the outer layer. Next, ask the andrologists to put on sterile rubber gloves.

Open the inner layer of the surgical instruments package and check the sterilization indicator tape for proper sterilization. Then, prepare two 5 milliliters syringes equipped with 0.7 millilitres needles for anesthesia and surgery. Open the ampulla containing lidocaine injection. Use a 5 milliliter sterile syringe to aspirate the lidocaine solution and eliminate the air bubbles from the syringe.

Now, position the prepped patient on the operation table in a lithotomy position. After removing the pants, secure the upper garment above the navel to expose the perineum and lower abdomen below the navel fully. Then, adjust the angle of the shadowless lamp to illuminate the patient's scrotum.

Use oval forceps to grip a sterile gauze ball. Immerse the gauze ball fully in a disinfectant solution. And disinfect the skin, starting from the lower abdomen and moving towards the front and inner thighs, perineal area, penis, and scrotum. Then, place a sterile square towel under the patient's buttocks. Spread a sterile hold towel around the surgical area, fully exposing the scrotum in the whole area.

Using a sterile Pasteur pipette, transfer approximately 2 milliliters of G-MPOS-PLUS culture solution into the culture dish. After closing the lid, deliver the dish to the nurse in the operating room through the transfer window. Now, place the culture dish on the sterile towel on the instrument table and open the lid.

Using another 5 milliliters syringe, aspirate approximately 0.2 milliliters of G-MPOS-PLUS culture solution and remove the air bubbles. Then, hold the patient's scrotum with the left hand, securing the epididymis that requires puncture. Palpate the epididymis with the right hand to confirm the surgical puncture location.

Next, with a 5 millilitres syringe containing G-MPOS-PLUS culture solution in the right hand, puncture perpendicular to the skin into the epididymal tissue. While maintaining negative pressure, repetitively puncture the epididymal tissue to allow fluid to enter through the needle, then withdraw the puncture needle.

Inject the extracted epididymal fluid into the culture dish. After that, aspirate nearby G-MPOS-PLUS culture solution to rinse the syringe to maximize the amount of collected epididymal fluid. Now, transfer the culture dish containing G-MPOS-PLUS culture fluid and epididymal fluid to the embryo laboratory technician through the transfer window.

Place the culture dish on the stage of an inverted optical microscope and secure it. Turn on the power switch of the microscope and adjust the light intensity appropriately. Then, rotate the converter of the inverted optical microscope. Select the 10 times objective lens, and rotate the coarse and fine focus spirals to adjust the focal length.

Once the epididymal spermatozoa are located, observe them at 40 times magnification to evaluate the number and quality. Using a sterile Pasteur pipette, transfer the liquid from the culture dish to a 15 milliliter centrifuge tube after observing sufficient active spermatozoa. Place the centrifuge tube on the balance tray and use a centrifuge tube of the same specification containing sterile water to level.

Then open the centrifuge door. Place the tubes in the card slot symmetrically and close the door. Press the Start button to initiate the centrifugation process. Once the process is completed, remove the tube containing the epididymal fluid from the centrifuge. Then, gently aspirate the supernatant with a sterile Pasteur pipette and discard the drawn supernatant.

Using a sterile Pasteur pipette, transfer the sediment from the bottom of the centrifuge tube to a new centrifuge tube containing approximately 2 milliliters of G-IVF Plus culture solution. Gently pipette the solution to thoroughly resuspend the sediment. Transfer approximately 0.5 milliliters of G-IVF Plus culture solution to the centrifuge tube. Gently blow the epididymal spermatozoa at the bottom to make it thoroughly suspended, and use them for the ICSI procedure.