14.1 Introduction
Azoospermia, or the absence of sperm in the ejaculate, affects approximately 1% of the general male population and 10–20% of men with infertility and may be caused by obstructive or nonobstructive etiologies [1]. In the past, azoospermia has been a considerable therapeutic challenge for urologists, with donor insemination or adoption being the only therapeutic options. However, the advent of intracytoplasmic sperm injection (ICSI) has enabled the potential for very small numbers of live sperm to produce offspring in conjunction with in vitro fertilization (IVF). As a result, men with azoospermia now have the ability to successfully father their own biological children.
The goal of sperm retrieval is to obtain a maximal amount of viable sperm while minimizing the amount of damage to the reproductive tract. Different techniques are available for use in men with obstructive azoospermia (OA) and for those with nonobstructive azoospermia (NOA), with each technique presenting its own advantages and disadvantages. In this chapter, we will describe the various surgical sperm retrieval techniques from the vas deferens, epididymis, and testicle of the azoospermic male and highlight important considerations regarding clinical evaluation, postoperative care, and outcomes for each.
14.2 Clinical Evaluation
Patients presenting with azoospermia or severe oligospermia need to be thoroughly evaluated in order to ascertain the cause of infertility and to identify an appropriate treatment plan. At a minimum, physicians should obtain a complete history and physical exam, two semen analyses, and hormonal studies (including testosterone and FSH). To rule out female factors for infertility, a focused evaluation or referral of the partner to a reproductive endocrinologist should also be included in the clinical evaluation of the infertile couple.
Men with congenital bilateral absence of the vas deferens on physical exam (CBAVD) should undergo genetic testing for cystic fibrosis (cystic fibrosis transmembrane regulator [CFTR]) mutations. If any mutations are detected on genetic testing, the patient and his partner should be advised to seek genetic counseling.
Men with bilateral atrophic testes on physical exam should be evaluated for primary versus secondary testicular failure. Elevated FSH levels coupled with normal or low serum testosterone levels suggest primary testicular failure whereas low FSH and serum testosterone levels are consistent with secondary testicular failure. Men with suspected primary testicular failure likely have NOA and should undergo genetic testing with karyotype and Y microdeletion testing. Secondary testicular failure, or hypogonadotrophic hypogonadism, is often caused by hypothalamic pathologies, such as Kallman syndrome or pituitary disorders (e.g., pituitary tumors). Further hormonal evaluation (with prolactin and LH) and pituitary imaging by computed tomography (CT) or magnetic resonance imaging (MRI) should be pursued as appropriate.
When the vasa and testes are normal on physical exam, the semen volume and FSH become critical in determining the cause of azoospermia. Patients with low semen volume (<1.0 mL) may have ejaculatory dysfunction or ejaculatory duct obstruction. Postejaculatory urinalysis for sperm may help diagnose retrograde ejaculation whereas the diagnostic tool of choice for ejaculatory duct obstruction is transrectal ultrasound (TRUS). The presence of midline cysts, dilated seminal vesicles (>1.5 cm), or dilated ejaculatory ducts are suggestive of ejaculatory duct obstruction. If dilated seminal vesicles are seen on TRUS, TRUS-guided seminal vesicle aspiration may be performed to confirm the diagnosis. Patients with unilateral absence of the vas deferens and low-volume azoospermia may have a variant of CBAVD and should undergo CFTR testing before TRUS.
In patients with normal semen volume, serum FSH can help delineate between OA and NOA. Markedly elevated serum FSH is diagnostic of impaired spermatogenesis and of NOA whereas low-normal serum FSH is more consistent with OA. Testicular biopsy can help to delineate OA from NOA, but because a biopsy only samples less than 5% of the seminiferous tubules within a testis, it has minimal utility in prognosticating whether or not sperm are present elsewhere in the testis of a man with NOA. Studies have shown that the testis is highly heterogeneous both histologically and in terms of spermatogenesis, which explains the poor correlation between diagnostic testicular biopsies and sperm retrieval rates in men with NOA [2]. As a result, we do not perform diagnostic testicular biopsies routinely in our clinical evaluation of azoospermia. Men with suspected OA (normal volume testes, intact vas deferens, normal serum FSH) may have diagnostic testicular biopsy performed at the time of reconstruction.
Men with NOA or severe oligospermia (<5 million sperm/mL) should undergo genetic testing. Genetic testing, namely karyotype and Y microdeletion testing, can help identify the cause of impaired spermatogenesis in 15–20% of men with NOA and provide important prognostic information. Men with Klinefelter syndrome (47XXY) or AZFc deletions have a 65–75% chance of sperm retrieval with microtesticular sperm extraction (microTESE) while those with complete deletions of AZFa, AZFb, or AZFb+c have a very poor prognosis (near 0% chance of sperm retrieval) [3,4].
14.3 Indications
The critical classification that will determine the type of surgical sperm retrieval method used is whether the patient has OA or NOA. Vasal and epididymal sperm retrievals can only be considered for those presenting with OA while testicular sperm retrievals (especially microdissection testicular sperm extraction) can be considered for those with OA or NOA.
14.4 Preoperative Preparation
Because men with NOA have severely impaired spermatogenesis, their limited production should be optimized prior to surgery. Men with prior scrotal surgery should wait at least 6 months before surgical sperm retrieval to allow for resolution of any hematoma or inflammation that may impair sperm production, which usually takes 3 months [5]. Those with varicocele may also benefit from varicocelectomy to optimize sperm production and potentially allow for the return of sperm to the ejaculate. However, the benefits from varicocelectomy may take 6 months or longer to be realized and while up to 39% of men may have return of motile sperm to the ejaculate, less than 10% of men have sufficient sperm in the ejaculate to avoid TESE [6,7]. While early studies suggest that performance of varicocelectomy has no effect on sperm retrieval rates at TESE, more recent studies have noted significantly higher rates of sperm retrieval at TESE following varicocelectomy [7–9]. Finally, men with NOA often have compromised serum testosterone levels and relatively increased estradiol levels that could be caused by increased testicular aromatase activity. Treatment of these men with low serum testosterone (<300 mg/dL) and decreased testosterone:estradiol ratios (<10) with an aromatase inhibitor (such as anastrozole, 1 mg P.O. daily) reliably increases circulating testosterone and decreases estradiol, thereby enhancing intratesticular testosterone levels and improving spermatogenesis [10].
Additionally, all patients scheduled for surgical sperm retrieval procedures should refrain from taking aspirin and nonsteroidal anti-inflammatory medications for 1 week preoperatively and discontinue anticoagulants perioperatively. Patients may receive a single dose of IV antibiotics 30 minutes prior to incision.
14.5 Techniques
14.5.1 Vasal Aspiration
Following administration of adequate anesthesia (either regional or general), the patient is placed in the supine position and the genitalia prepped as per the usual aseptic technique. The testicle is delivered via either a midline raphe or a high transverse incision along the scrotal skin lines. The straight vas deferens is palpated along the spermatic cord using the thumb and forefinger and stabilized using a Babcock clamp. Blunt dissection of the vasal adventitia is carried out using a curved mosquito clamp, taking care to not damage the perivasal vasculature. Bipolar cautery can be used to maintain adequate hemostasis. Once the vas is completely isolated, a Penrose drain can be used to separate the bare vas from its adventitia. A straight mosquito clamp is then placed below the bare vas to serve as a platform on which the vas can be hemitransected using a 15º ultrasharp microknife in a single motion. A 24G angiocatheter attached to a tuberculin syringe containing 0.1 mL of sperm wash medium can be used to aspirate sperm from the testicular vas lumen. A small sample should be placed on a slide for examination under a bench microscope for the presence of motile sperm while the rest is gently flushed into sperm wash medium.
Once adequate amount of motile sperm are aspirated, the vasotomy can be closed in two layers. First, three 10-0 nylon sutures are used to reapproximate the vasal mucosa before three 9-0 nylon sutures are used to close the vasal muscularis and adventitia, taking great care to not obliterate the vasal lumen. A 0.25% Marcaine solution is infused within the spermatic cord and the testicle is replaced within the scrotum. The dartos fascia and skin are both closed separately with fine absorbable suture. The incision can be infiltrated with 0.25% Marcaine solution with epinephrine to ensure adequate postprocedural anesthesia. Finally, Bacitracin ointment, fluff gauze, and a scrotal supporter are applied.
14.5.2 Percutaneous Epididymal Sperm Aspiration
Percutaneous epididymal sperm aspiration (PESA) can be performed in an office setting with local anesthesia. To perform PESA, one requires a 22–26G needle attached to a tuberculin syringe containing 0.1 mL of sperm-washing medium. There should be an air bubble maintained within the syringe to prevent contact between the rubber and the sperm-washing medium.
After the patient is placed in the supine position, the scrotum is cleaned with an antiseptic solution and rinsed with saline to remove any residual antiseptic. Local anesthesia is administered via cord block and skin infiltration using 2% lidocaine. The head of the epididymis is then palpated and isolated between the thumb and index finger. The 22–26G needle is then used to puncture the epididymis through the scrotal skin. Once within the epididymis, the plunger of the tuberculin syringe should be pulled all the way back to create a suction force. While maintaining the suction force, the needle is carefully advanced and withdrawn partially throughout the epididymis, taking care to remain within the epididymis at all times. The needle can be rotated 180º and the angle of entry can be varied slightly with each pass (while maintaining suction) to ensure that multiple areas of the epididymis are sampled. Once this position is satisfied, the suction can be partially released and the needle removed from the epididymis. One should not expect a significant amount of epididymal fluid following PESA (can be anywhere from 0.3 to 1 mL) and the epididymal aspirate may, in fact, be invisible.
The epididymal fluid should be flushed into an Eppendorf tube containing sperm-washing medium and a small sampled placed on a slide for examination under the bench microscope. If no motile sperm are detected, the procedure can be repeated at a more proximal portion of the epididymis (toward the epididymal head). Multiple passes may be required before motile sperm are found. If no motile sperm are found, PESA can be performed on the contralateral side or one can proceed to testicular sperm aspiration (TESA). If no motile sperm are found on PESA or TESA, the patient can be booked for future microsurgical epididymal sperm aspiration (MESA) or microdissection testicular sperm extraction (microTESE).
14.5.3 Microsurgical Epididymal Sperm Aspiration
Unlike PESA, MESA requires microscopic magnification and is ideally performed with general anesthesia. Following induction of anesthesia, the patient is placed in the supine position and the testis is delivered through either a scrotal midline raphe incision or a high-transverse incision along the scrotal skin lines. The tunica vaginalis is incised using electrocautery and the epididymis is inspected under the operating microscope in order to identify dilated epididymal tubules containing golden, semitranslucent fluid for aspiration. Opaque, yellow tubules typically contain sperm heads and debris and should be avoided.
The structure of the epididymis consists of numerous ducts that coalesce at the junction of the epididymal head and body into a single epididymal duct that travels through the epididymal tail into the vas deferens. In order to minimize the risk of inducing an epididymal obstruction, the safest area to perform a MESA is in the epididymal head, where damage to a single duct will not cause obstruction to the entire outflow tract. In OA, the likelihood of finding motile sperm in the epididymal head is higher than normal [2].
Jeweler forceps are used to pick up the tunica of the epididymis, which is then incised with a small microscopic curved scissors to reveal the dilated tubule. Blunt dissection with the microscopic needle holder can be used to further expose the dilated tubule. Bipolar cautery can be used judiciously to ensure meticulous hemostasis, taking care to not damage the exposed tubule. At this point, a glass micropipette with a sharpened tip can be used to both puncture the tubule and aspirate the epididymal fluid. Alternatively, a 15º ultrasharp microknife can be used to incise the exposed tubule and a regular glass 0.5 μL micropipette (0.5 mm luminal diameter and 0.9 mm outer diameter) used to collect the epididymal fluid using capillary action (Figure 14.1).
Figure 14.1 After incision of the dilated epididymal tubule, a glass micropipette is used to aspirate the epididymal fluid using simple capillary action.
A small sample of the epididymal fluid should be placed on a slide and examined immediately under a bench microscope to confirm the presence of motile sperm. Once motile sperm are visualized, the epididymal fluid can be flushed from the micropipettes into sperm wash media and given to the IVF lab personnel for processing and storage. If no motile sperm are visualized, a more proximal epididymal tubule can be isolated and punctured until motile sperm are found. Manual compression of the testis and/or epididymis during aspiration can maximize the amount of fluid retrieved. Aspiration is continued until no more fluid can be obtained. About 10–20 μL of epididymal fluid can usually be aspirated.
Once aspiration is complete, the incised epididymal tubule can be closed using either 10-0 nylon sutures or the bipolar cautery. The tunica vaginalis is closed with absorbable suture in a running fashion and the intratunical space is infused with 0.25% Marcaine before the testicle is returned to the scrotum. The dartos fascia is also closed with fine absorbable suture in a running fashion and the subdermal layer is infused with 0.25% Marcaine with epinephrine prior to skin closure with a 5-0 Monocryl suture. Finally, Bacitracin ointment, fluff gauze, and a scrotal supporter are applied.
14.5.4 Testicular Sperm Aspiration
One can perform TESA in an office setting using two different methods requiring different pieces of equipment – either a biopsy gun or a 19G butterfly needle attached to a 20 cm3 syringe and Cameco piston syringe handle. Both techniques will be described in the following section.
Once the patient is placed in the supine position, the scrotum is cleaned with an antiseptic solution and rinsed with saline to remove any residual antiseptic. Local anesthesia is administered via cord block and skin infiltration using 2% lidocaine at the planned aspiration sites. In order to minimize potential damage to the epididymis, we recommend using the inferior pole of the testicle as the entry site for aspiration. The testis is then grasped up near the scrotal skin and stabilized with the nondominant hand. If using a biopsy gun, the biopsy gun is fired with the entry point of the needle at the inferior pole of the testis (Figure 14.2). Once removed, pressure is held at the site of the biopsy while the testicular tissue is placed in sperm-wash medium. If the 19G butterfly needle attached to the 20 cm3 syringe is used, then the needle is used to puncture the testicle at the inferior pole through the scrotal skin. The Cameco piston syringe is used to produce and maintain a suction force as multiple passes are made through the same puncture site. The aspirated tissue and fluid are then gently flushed into sperm-wash medium.
Figure 14.2 With the testicle stabilized using the nondominant hand, a biopsy gun is fired into the inferior pole.
The testicular tissue aspirated should then be passed through a 24G angiocatheter to promote suspension of sperm from the testicular tissue into the sperm-wash medium. A sample can then be placed on a slide and examined immediately under a bench microscope for the presence of mature, viable sperm. Multiple aspirations may be taken to ensure enough tissue and sperm are obtained for at least three fresh IVF cycles. If no mature, viable sperm are detected, TESA may be performed on the contralateral testicle.
Once satisfied, continuous pressure should be applied to the biopsy sites to promote adequate hemostasis. A sterile dressing and fluff gauze are applied and held in place by a scrotal supporter.
14.5.5 Testicular Sperm Extraction
TESE, also known as an open testicular biopsy, can be done under local, regional, or general anesthesia and with or without the assistance of a microscope. A microdissection TESE (or microTESE) will be discussed later on.
Once appropriate anesthesia is administered, the patient is placed in a supine position and the genitalia are prepped with an antiseptic solution. Using either a midline raphe incision or a high-transverse incision along the scrotal skin lines, the testis is delivered. The tunica vaginalis is incised using electrocautery and the testis is inspected. Use of an operating microscope during this stage greatly enhances visibility of the testicular blood supply and allows for the subsequent transverse incision of the tunica albuginea to occur between blood vessels. A 1 cm transverse incision is then made along the anterior surface of the testicle between the blood vessels using a 15º ultrasharp microknife. Bipolar cautery is used to ensure adequate hemostasis, especially of subtunical blood vessels. Testicular tubules are extruded from the testicle using manual compression and are excised sharply from the base using curved iris scissors (Figure 14.3). The specimen is then placed in an Eppendorf tube containing tubal fluid media. The testicular tissue can be minced sharply and then passed sequentially via a 24G angiocatheter to release the sperm from the seminiferous tubules into the tubal fluid media. Processing of the testicular tissue in this manner increases sperm yield by up to 470% [11]. A small sample is placed on a slide for microscopic examination of mature, viable sperm.
