Clinical Conditions

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.

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.

Considerations Before Treatment

Sperm retrieval by TESE can be performed prior to or coincident with an IVF cycle for the female partner. We have performed all initial TESE procedures on the day of oocyte (egg) retrieval during a programmed IVF cycle to maximize the potential to retrieve viable spermatozoa for use with ICSI. Therefore, the female partner must be evaluated by the IVF center before considering a TESE procedure. Since men with NOA have marginal sperm production, TESE procedures should be delayed for at least 6 months after any intervention such as a prior biopsy or TESE procedure, or other inguinal/scrotal surgery. Since the testicular blood supply penetrates the tunica albuginea and then disperses in a series of end-arteries that spread over the testicular parenchyma, multiple biopsies should be avoided to minimize the risk of devascularization of the testis. The use of optical magnification may also minimize the risk of testicular injury. We have seen two men who have had significant devascularization of the testis with atrophy after TESE performed at other centers using multiple biopsy technique.

TESE (Testicular Sperm Extraction)

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.

IVF and ICSI

The general ICSI procedure has been described previously. Aggressive immobilization of spermatozoa, a technique that increases fertilization rates for immature sperm, was carried out for all testicular spermatozoa.

Results

During the last 81 attempted TESE-ICSI cycles, sperm were retrieved for injection in 47 cycles (47/81; 58% retrieval rate). Mean male age of patients entering treatment was 38.9 years (range 29-53), with a mean female age of 34.2 years (range 25-44). Men had initial mean serum FSH levels of 17.7 IU/L(normal 1-9), and average testicular volumes of 11.3 cc. Six patients had only one testis, due to a history of radical orchiectomy for testis tumor. Five patients were previously treated with chemotherapy for non-Hodgkin's lymphoma(3) or nonseminomatous germ cell tumor(2), respectively. Testis biopsies were abnormal in all patients, and most patients had predominantly Sertoli cell-only pattern on biopsy. Since the chance of finding spermatozoa is dependent on at least one area of spermatogenic activity, the testis biopsies were graded based on the most advanced pattern of spermatogenesis identified, not the predominant pattern. Sperm were initially identified as non-motile in almost all procedures. After additional processing, all samples with spermatozoa exhibited some form of sperm motility.

Table 1: Sperm Retrieval: Non-obstructive Azoospermia

Histologic Category Sperm Retrieval Rate with TESE ( % )
Hypospermatogenesis 79% (31/39)
Maturation Arrest (n=19) 47% ( 9/19)
Sertoli Cell-only ( n=21) 24% (5/21)

The normal fertilization rate per metaphase II oocyte injected was 268/439(61%). Fertilization and subsequent embryo transfer occurred for all forty-seven couples with sperm retrieved. Clinical pregnancies (fetal heart seen on ultrasound) were established for 26/47 (55%) couples with retrieved sperm [26/81 (32%) of entire TESE cohort], with a single fetal heart beat seen in 23/26 cycles and twin gestations in three couples. Spontaneous abortion after achieving a clinical pregnancy has occurred for six couples 6/26 (23%), and an ectopic pregnancy was terminated for one couple. Ongoing or delivered pregnancies occurred for 19/47 (40%) attempts of TESE-ICSI. Thirteen healthy children have been born to date from Cornell after TESE-ICSI for NOA.

Table 2: TESE-ICSI Non-obstructive Azoospermia

Retrieval Attempts N=81
Sperm Retrieval 58% (47/81)
Fertilization Rate 61% (268/439)
Clinical Pregnancy 55% (26/47)
Ongoing/Delivery 40% (19/47)
Overall Pregnancy 32% (26/81)

The most advanced spermatogenic pattern (but not the predominant pattern) appears to affect the results of sperm retrieval. For men who had at least one area of hypospermatogenesis present on diagnostic testis biopsy, retrieval of spermatozoa was achieved in 79% (31/39) of attempts, whereas for men with maturation arrest as the most advanced pattern, only 47% (9/19) of men had sperm retrieved by TESE. If the entire diagnostic biopsy had a Sertoli cell-only pattern, then sperm were retrieved in 24% (5/21) TESE attempts. Although no finding absolutely determined sperm retrieval or negated the possibility of successful TESE, the findings of diagnostic biopsy were helpful in evaluating the chance of success with TESE. In addition to the role of diagnostic biopsy in identifying the rare cases of intratubular germ cell neoplasia (carcinoma-in-situ) and confirming the diagnosis of non-obstructive azoospermia, diagnostic biopsy helps to provide prognostic information regarding the chance of a successful TESE procedure to obtain sperm.

Seven TESE-ICSI cycles involved men with classic Klinefelter's syndrome(47,XXY karyotype, or mosaic patterns that do not include 46,XY). Three of seven cycles resulted in ongoing pregnancies or deliveries, despite the appearance of Sertoli cell-only in one man on diagnostic biopsy prior to attempted sperm retrieval. The delivery of a healthy, normal boy for one couple, twins (boy and girl) and an ongoing pregnancy with two normal fetal karyotypes for a third couple have been achieved for these men with Klinefelter's syndrome. These findings illustrate the importance of genetic evaluation prior to treatment and the potential of TESE-ICSI to provide fertility for couples despite underlying genetic abnormalities. Spermatid retrieval and injection provides some hope for treatment of couples where no sperm can be retrieved with TESE.

Summary

Sperm retrieval for use with the advanced form of assisted reproduction, ICSI, is now possible for many men with non-obstructive azoospermia. Men with NOA may have unique genetic defects that should be evaluated prior to an attempt at conception. At New York Hospital-Cornell, 58% of couples have sperm retrieved from the testis with TESE, and 55% of couples achieve a clinical pregnancy using sperm retrieved with TESE, using the advanced reproductive technique of ICSI. Since some couples will not have sperm retrieved with TESE, the potential use of back-up frozen donor spermatozoa should be discussed with couples prior to simultaneous TESE-ICSI attempts.

References

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Kahraman S, Ozgur S, Alatas C, Aksoy S, Tasdemir M, Nuhoglu A, et al. Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men. Hum Reprod 11:756-760, 1996.

3. Novero V, Goossens A, Tournaye H, Silber S, Van Steirteghem AC, Devroey P: Seminoma discovered in two males undergoing successful testicular sperm extraction for intracytoplasmic sperm injection. Fertil Steril 65:1051-4, 1996.

4. Palermo GD, Schlegel PN, Colombero LT, Zaninovic N, Moy F, Rosenwaks Z. Aggressive sperm immobilization in ICSI using epididymal and testicular spermatozoa improves fertilization and pregnancy rates. Hum Reprod 11:1023-1029, 1996.

5. Palermo GD, Schlegel PN, Sills ES, Menendez S, Veeck LL, Rosenwaks Z. Births following testicular sperm used with intracytoplasmic sperm injection in non-mosaic Klinefelter's syndrome. N Engl J Med 338:588-590, 1998.

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