On the day of oocyte retrieval, scrotal exploration is performed through a median raphe incision under local or general anesthesia, and sperm are retrieved using an open testicular biopsy technique. In order to confirm accurate identification of the testis and to avoid any injury to the epididymis, delivery of the testis is routinely performed. Testicular blood vessels in the tunica albuginea are identified with 8-15x optical magnification. An avascular region near the midportion of the medial, lateral or anterior surface of the testis is chosen, and a generous incision in the tunica albuginea is created with a 15o ultrasharp knife, avoiding any capsular testicular vessels.
With this approach, larger (450-500 mg. samples) of testicular parenchyma can be harvested, instead of retrieving the usual diagnostic biopsy volume of 50-100 mg. However, a recent microdissection technique that we have applied allows the removal of tiny (2-3 mm; 3-5 mg volumes) of testicular tissue with improved sperm yield. The tubules containing sperm can often be visually identified under an operating microscope after opening the testis, when 15-25x magnification is used to assist the biopsies. This approach 1) improves the yield of spermatozoa per biopsy, 2) results in less tissue removal (and loss of testicle), and 3) allows identification of blood vessels within the testicle, minimizing the risk of vascular injury and loss of other areas of the testis. The excised testicular biopsy specimen is placed in human tubal fluid culture medium supplemented with 6% Plasmanate. Isolation of individual tubules from the mass of coiled testicular tissue is achieved by initial dispersal of the testis biopsy specimen with two sterile glass slides, stretching the testicular parenchyma to isolate individual somniferous tubules.
Subsequently, mechanical disruption of the tubules is accomplished by mincing the extended tubules with a sterile scissors in HTF/Plasmanate medium. Additional dispersion of tubules is achieved by passing the suspension of testicular tissue through a 24 gauge angiocatheter. For minimal tissue specimens, little dissection is performed in the operating room, since the tissue sample is so small and opening of the individual tubules must be done in the embryology laboratory, immediately prior to ICSI. Intraoperatively, a "wet preparation" of the suspension is examined under phase contrast microscopy at 100x and 400x power. If no spermatozoa were seen, then (1) additional biopsies of tissue are obtained through the same tunical incision, (2) biopsies are performed using additional incisions, and (3) contralateral biopsies are obtained, if needed. After dispersal, immediate intraoperative evaluation of the specimens was performed by a member of the IVF laboratory in the operating room. Subsequent processing of the testicular tissue suspension, including microdissection of the specimens is performed in the IVF laboratory. Aliquots of tissue are also processed for cryopreservation.