TESE Introduction

Testicular failure affects approximately 1% of the male population and 10% of men who seek fertility evaluation. Azoospermic men with testicular failure (non-obstructive azoospermia) have either Sertoli cell-only pattern, maturation arrest, or hypospermatogenesis on testis biopsy. Until recently, it was assumed that men with non-obstructive azoospermia were untreatable. The only options offered to these couples to have children were the use of donor spermatozoa or adoption. Several clinically relevant findings have changed our approach to this condition. First, we have observed that direct evaluation of testis biopsy specimens often demonstrates sperm in men with non-obstructive azoospermia, despite severe defects in spermatogenesis.

In addition, it was previously thought that sperm must traverse the male reproductive tract before acquiring the ability to normally fertilize an egg. Our experience with men who have unreconstructable obstructive azoospermia, including congenital absence of the vas deferens suggests that complete transit through the epididymis is not a prerequisite for fertilization. Retrieval of sperm from the testis or epididymis was associated with good pregnancy rates using in vitro fertilization. Our subsequent experience indicated that micromanipulation of gametes during assisted reproduction could improve these pregnancy rates. Subsequently, we have observed that optimized in vitro micromanipulation techniques can further enhance fertilization and pregnancy rates using epididymal or testicular sperm. This leads us to the second important clinical observation, that sperm can be retrieved from the testes of men with obstructive azoospermia and used with the assisted reproductive procedure of intracytoplasmic sperm injection (ICSI) during in vitro fertilization (IVF) with a high chance of achieving pregnancies and deliveries of normal children. These findings have led investigators from Belgium and elsewhere to perform testicular sperm extraction (TESE) with ICSI for men with non-obstructive azoospermia. Low pregnancy rates of 20 to 21% per attempt have been reported.

TESE Patient Evaluation

Azoospermia may be due to either abnormal sperm production (non-obstructive azoospermia) or normal sperm production in the presence of obstruction (obstructive azoospermia). To determine that a semen sample is truly azoospermic, centrifugation of the semen sample with meticulous microscopic examination of the pellet is necessary. For all patients with azoospermia, a complete history and physical examination is necessary to identify potentially correctable causes of male factor infertility. Typically, the man with non-obstructive azoospermia will have small testes (< 15 cc) with a flat epididymis. Some men may have a history of cryptorchidism. Hormonal evaluation of a man with non-obstructive azoospermia (NOA) will typically demonstrate an elevated serum FSH, with normal or nearly normal testosterone and estradiol levels. Prior to further intervention, we will usually treat any correctable abnormalities that are found on evaluation of a man with NOA, including surgical repair of large varicoceles, correction of hormonal abnormalities, and avoidance of gonadal toxins for at least three months prior to attempted TESE. The diagnosis of NOA can only be definitively made on testicular biopsy, which may be helpful to rule out the unlikely possibility of testicular intratubular germ cell neoplasia (carcinoma-in-situ).

Men with NOA are also at increased risk for harboring genetic defects that can be transmitted to their children with treatment. These abnormalities include both chromosomal abnormalities, detectable with routine karyotype testing, and Y chromosome microdeletions, so called "AZF(azoospermic factor) defects." Other rare genetic causes of male infertility are nicely reviewed by Mak and Jarvi in J Urology 156:1245-57, 1996. Karyotype testing is available at most major medical centers, as well as at Dianon Laboratories in Stamford, Connecticut. Y chromosome microdeletion analysis, a PCR-based assay of peripheral leukocytes, is available at a few academic centers, including locally at Cornell and NYU as well as at the Genetics & IVF Institute of Fairfax, Virginia. For men who are found to have a genetic abnormality contributing to their infertility, formal genetic counseling is mandatory prior to treatment with assisted reproduction.

TESE

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.