The scrotum is cleaned with antiseptic and then thoroughly washed with saline to eliminate any residual antiseptic. Under local anesthesia the head of the epididymis is palpated and stabilized between thumb and forefinger. It is then punctured directly through the scrotal skin, with a 22–26 G needle attached to a tuberculin needle containing 0.1 ml of sperm-washing medium. An air bubble is kept between the medium and the rubber stopper of the plunger to prevent direct contact between the rubber and medium. The assistant pulls the plunger all the way to the top of the syringe thus creating a suction force. The needle is gently and slowly advanced through the epididymal tubule. The needle is rotated 180° and then withdrawn partially, staying within the epididymis. It is then advanced in a different direction, all the while maintaining suction. The suction is partially released and the needle is withdrawn from the epididymis. Sometimes droplets of fluid can be seen entering the syringe, but on other occasions the epididymal aspirate may be so thin and scanty that there is no visible aspirate. Some authors have described using a larger butterfly needle on a 20-ml syringe whereby the aspirated droplets may be visualized easier in the butterfly tubing. One can expect no more than 0.3–1 ml of fluid to be obtained per aspirate [23]. The contents of the syringe are gently flushed into a dish and examined for the presence of sperm. If motile sperm are not seen, the procedure is repeated at a slightly different location moving towards the epididymal head from the tail. Since this is a blind procedure sometimes several attempts are required before good quality sperm are found. Only one paper describes the effect of PESA on subsequent reconstruction. The authors found that it did not disrupt the ability to perform reconstructive surgery [24].

The scrotum is cleaned with antiseptic and then thoroughly washed with saline to eliminate any residual antiseptic. Anxiolytics may be offered to reduce anxiety. Local anesthesia is administered in the form of spermatic cord blockade in addition to the planned testicular aspiration sites at the skin level. After adequate anesthesia is obtained, the testis is fixed with the surgeon’s hand, and a 19-G butterfly needle attached to a 20 cm³ syringe is used to aspirate testicular tissue with the aid of a Cameco piston syringe handle (see Fig. 6.2). Multiple passes are made through the same puncture site and the aspirated tissue and fluid is sent to the reproductive lab for confirmation of mature, viable sperm. Attempts are made to obtain enough tissue for at least three fresh cycles of IVF. The contralateral side may be tried if adequate tissue is not obtained. Once adequate tissue is obtained pressure is applied to the puncture sites until adequate hemostasis is achieved. Sterile dressings, Kerlix fluffs, and an athletic supporter are applied, and the patient is instructed in routine post-procedural care.

The testicular tissue may be cryopreserved. The planned cryopreservation specimens are mixed with an equal volume of test yolk buffer (TYB) and glycerol and incubated at room temperature. The specimens are transferred to cryopreservation vials in aliquots of 1 ml. The filled vials are incubated at 2–6 °C for 60–90 min. After incubation, the vials are suspended in liquid nitrogen vapor for 30 min. Then the vials are submerged in liquid nitrogen for 2 h and a post-thaw analysis was also performed.

Under adequate anesthesia (local, regional, or general), after sterile preparation of the genitalia, the testis is either delivered through a midline or transverse scrotal incision between the skin lines, if being done in conjunction with surgical exploration. Alternatively, the procedure may also be performed using the “window” technique not requiring delivery of the testis. The tunica vaginalis is opened sharply and the testis is inspected. A transverse incision along the anterior aspect of the testis between the blood vessels is made. Blood vessels may be encountered underneath the tunica are managed with bipolar electrocautery. The tubules are examined and are gently removed with a fine forceps or micro needle holder. Manual compression of the testis aids in extrusion of the testicular tissue. Once adequate tissue is delivered out of the testis it is excised sharply at its base, placed in tubal fluid media and handed off to the laboratory technician. This is repeated until adequate amounts of sperm are harvested. Hemostasis is achieved with bipolar electrocautery. The tunica albuginea is closed with 5-0 absorbable sutures. The tunica vaginalis and subsequently skin are closed with fine absorbable suture.