Abstract
Historically, the absence or a small number of sperm cells in the ejaculate often precluded men from fathering their genetic progeny and relegated couples to the use of donor spermatozoa insemination or adoption or childlessness. With the development of intracytoplasmic sperm injection (ICSI), men with azoospermia (absence of sperm in the ejaculate) or severe oligozoospermia (less than 5 × 106 spermatozoa in the ejaculate) are able to father a child following single sperm cell injection into the cytoplasm of a single oocyte. In the years after the development of ICSI, it was discovered that sperm retrieved directly from testicular tissue can also be used for oocyte fertilization and enable healthy embryo development.
Testicular Spermatozoa
Historically, the absence or a small number of sperm cells in the ejaculate often precluded men from fathering their genetic progeny and relegated couples to the use of donor spermatozoa insemination or adoption or childlessness. With the development of intracytoplasmic sperm injection (ICSI), men with azoospermia (absence of sperm cells in the ejaculate) or severe oligozoospermia (less than 5 × 106 spermatozoa in the ejaculate) are able to father a child following single sperm cell injection into the cytoplasm of a single oocyte. In the years after the development of ICSI, it was discovered that sperm cells retrieved directly from testicular tissue can also be used for oocyte fertilization and enable healthy embryo development.
Azoospermia is defined by a lack of spermatozoa in ejaculate identified in two separate semen specimens directly examined following centrifugation and volume condensation. While azoospermia is rare, affecting approximately 1% of the male population [1], approximately 10–15% of all infertile men are diagnosed as azoospermic [2]. Azoospermia can result due to a blockage anywhere along the sperm transit path, i.e., the efferent ducts, epididymis, vas deferens, ejaculatory duct, and urethra. When these problems in sperm cells delivery occur, azoospermia is classified as obstructive azoospermia (OA). The majority of OA cases can be determined by the patient’s medical history and physical examination alone. Other conditions that can be determined based on a review of readily available clinical evidence include vasal obstruction from prior hernia repair, congenital vassal agenesis, prior extirpative pelvic surgery, and ejaculatory duct obstruction.
Azoospermia can be classified into three categories: pre-testicular, testicular, and post-testicular. Pre-testicular causes of azoospermia include endocrine abnormalities with adverse effects on spermatogenesis (secondary testicular failure). Testicular causes of azoospermia (primary testicular failure) include disorders of spermatogenesis intrinsic to the testes. Post-testicular reasons of azoospermia relate to ejaculatory dysfunction or ductal obstruction that impairs sperm cell transit (Figure 14.1).
Figure 14.1 Etiology of azoospermia. AZF, azoospermia factor.
Cases in which the testicles do not produce spermatozoa at all or produce too few sperm cells are classified as nonobstructive azoospermia (NOA). NOA is due to defective spermatogenesis and can be classified as hypospermatogenesis, maturation arrest, or Sertoli cell-only syndrome. Defective spermatogenesis may be due to genetic abnormalities such as Y chromosome microdeletions or karyotype abnormalities but is largely idiopathic (Figure 14.2) [3]. Thus, men with NOA have fewer therapeutic options and reproduction relies on surgical spermatozoa retrieval. Common conditions that cause NOA include current or prior usage of exogenous testosterone, history of undescended testes, history of malignancy and chemotherapy, current malignancy, genetic anomalies, environmental exposures, and pituitary dysfunction.
Figure 14.2 Genetic composition of chromosome Y.
The ability to use limited numbers of testicular or epididymal sperm cells for ICSI brought a dramatic revolution in the management of couples with azoospermia. However, low embryo quality and incidence of direct uneven cleavage of embryos have been observed to be higher after ICSI with sperm cell biopsy retrieved from azoospermic males [4], compared with normozoospermic males.
The important difference of OA from NOA is that sperm cell retrieval techniques are always successful in locating sperm cells from OA patients, while locating sperm cells from NOA patients is challenging, in the best circumstances, and can be impossible in some.
Examination of several preoperative variables, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, and testicular size and volume, was initially employed to diagnose the category of azoospermia and predict the outcome of sperm cell retrieval. However, later, these factors were demonstrated to have low sensitivity and specificity in predicting the success of sperm cell retrieval [5]. Testosterone, while not very useful as an isolated test, is helpful in relation to FSH, LH, prolactin, and estradiol levels in determining the overall function of the male reproductive gonadal–pituitary system. Analyses of FSH (most experts consider FSH >7.6 mIU/ml as abnormal for spermatogenesis) and LH levels have been shown to differentiate OA from NOA with some degree of accuracy. While a semen analysis is, in most cases, a crude indicator of a man’s fertility potential, the consistent finding of a total absence of sperm cells on multiple analyses indicates that the man is infertile. Semen parameters such as pH, semen volume, and the presence/absence of fructose are additional and useful clinical data [6].
Men with low ejaculate volume should repeat the semen analysis, paying close attention to complete and proper collection after a 2–3-day abstinence [7]. In men with low ejaculate volume (less than 1.5 ml) and normal FSH and testis volume, evaluation of the collection process, and post-ejaculate urine analysis to evaluate possible retrograde ejaculation are essential first steps. Usually, millions of sperm cells found in the post-ejaculate urine manifest retrograde ejaculation. When men with low ejaculate volume, oligozoospermia or azoospermia, and palpable vasa do not suffer from retrograde ejaculation and semen pH is <7.2, a transrectal ultrasound (TRUS) to evaluate dilation of seminal vesicles or ejaculatory ducts is a useful diagnostic test to identify ejaculatory duct obstruction [8]. TRUS is a vital tool in the evaluation of men with azoospermia. Ejaculatory duct obstruction is an unusual and difficult-to-diagnose disorder. On transrectal ultrasonography, one can often, but by no means always, see dilated seminal vesicles and a dilated ejaculatory duct. While there are no absolute diagnostic features for this condition, when a patient has sufficient findings (e.g., midline prostatic cyst, dilation of the seminal vesicle, and seminal vesicle agenesis), obstruction is likely to have occurred. Additional seminal indicators of ejaculatory duct obstruction are seminal acidic pH (<7.2) and fructose absence, whereas at normal status seminal vesicle secretions are alkaline and contain fructose.
Because normal vas (ductus) can be easily palpated within the scrotum, the diagnosis of unilateral or bilateral vasal agenesis is made by physical examination. Approximately 25% of men with unilateral vasal agenesis and about 10–15% with congenital bilateral absence of the vas deferens (CBAVD) usually have unilateral renal agenesis, which can be identified by ultrasonography [9]. In azoospermic men with unilateral vasal agenesis, TRUS may assist in demonstrating a related contralateral segmental atresia of the seminal vesicle or vas deferens [10]. There is a robust association between CBAVD and mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene [11]; almost all men with clinical cystic fibrosis have CBAVD. Conversely, at least three-quarters of men with CBAVD have mutations of the CFTR gene [12]. However, failure to detect a CFTR abnormality in a man with CBAVD does not completely rule out a mutation entirely, because 10–40% are undetectable using conventional clinically available methods. Rates of CFTR mutations are not higher in patients with renal anomalies and unilateral or bilateral vassal agenesis compared with the overall population [13–14]; these patients likely have a non-CFTR etiology for these anomalies. Most men with CBAVD have regular spermatogenesis, but other potential coexisting causes of impaired spermatogenesis must be investigated before collecting sperm cells for assisted reproduction [15].
In azoospermic men with average semen volume, serum FSH and testicular volume are the most important determinants of whether a diagnostic testicular biopsy may help assess spermatogenesis. A marked increase in serum FSH and low testicular volume strongly suggest NOA. Although serum FSH levels reflect the predominant pattern of spermatogenesis, they may not reflect isolated areas of spermatogenesis within the testis and are not related to the more advanced stages of spermatogenesis. The relationship between FSH and the presence of any spermatogenesis is not straightforward in men with NOA. Some men with normal-ejaculate-volume azoospermia have a regular testicular test, normal FSH, and a testicular biopsy that exhibits a spermatogenesis defect (most often maturation arrest). These men have NOA and should not be offered scrotal exploration and reconstruction. Inhibin B has also been found to be slightly more sensitive than FSH as an index of spermatogenic status. However, it was concluded that the inhibin B level alone, or in combination with the serum FSH level, fails to predict successful biopsy for spermatozoa in patients with NOA. By the evaluation of the azoospermic male, the key question is whether spermatogenesis is occurring in the testes and to what degree.
In general, testicular tissue may be sampled for diagnostic purposes via multi-site fine-needle aspiration (or “testicular mapping”) and open testicular biopsy. The success of sperm cell retrieval is dependent on the status of spermatogenesis in the testes, rendering it essential to characterize the status [16]. Testicular sperm aspiration (TESA) or testicular fine-needle aspiration (TFNA) are aspirations performed using a butterfly needle of 21 gauge or thinner attached to a 20 ml syringe. The testis is punctured and negative pressure is applied on the syringe primed with nutrient medium. The needle is moved back and forth four to five times in different directions without removing it from the puncture site. The negative pressure is then reduced over 30–60 seconds and the needle is withdrawn. Any protruding tubules from the puncture site are transferred to a small plate or tube containing a sperm nutrient medium. The procedure enables minimally invasive sampling of the tubules from various portions of the testis. An ideal sperm cell retrieval method should allow for retrieval of sufficient spermatozoa with minimal trauma and with a single intervention. Usually, 10–20 needle passes are required to obtain adequate specimens by TESA/TFNA. Sperm cell retrieval rates vary depending upon the type of azoospermia, with success rates of ~100% in OA patients and only about 30% success in NOA cases [17].
Testicular sperm extraction (TESE) differs from TESA in that it requires an incision to reach the testicular tissue. During the TESE method the hemiscrotum is opened and the testis is extruded to the surface of the incision point, the protruding tissue is excised sharply and placed in sperm nutrient medium. The process is repeated several times in other areas of the testis. Obtained testicular tissue is then processed in the laboratory. Two 23–25 gauge insulin needles on 1 ml insulin syringes are bent to 70–80 degrees. These bent needles are used to press and spread the testicular tissue and tubules. Within a few minutes, a delicate suspension with very little particulate matter is obtained. The suspension is drawn up leaving the tissue fragments behind. The suspension is allowed to settle in the tube for 0.5–1 hour at room temperature, and the supernatant is aspirated, without debris, and then centrifuged at 900 × g for 5 minutes, and the formatted pellet is resuspended in a small volume (~50 µl) of sperm medium. Droplets of 5 µl are placed in an ICSI dish. The testicular suspension is kept at <37°C. Storing human testicular tissue at 37°C causes a significant increase in the number of apoptotic cells [18].
For all azoospermia patients, the remaining testicular tissue fragments should be tested by a pathologist for conclusive determination of azoospermia type and for possible early cancer diagnosis. The European Germ Cell Cancer Consensus Group recommends the use of Stieve’s or Bouin’s solution for the fixation of testicular tissue [19]. Testicular germ cell tumor (TGCT) is the most common cancer in men aged 15–40 years. The incidence of TGCT has more than doubled over the past 50 years; however, the underlying etiology is unknown. Tumors are found in 4% of men who present to the fertility center for infertility evaluation and 50% of men with testis cancer are infertile/subfertile at the time of diagnosis [20–21]. Carcinoma in situ (CIS) is challenging to identify in testicular biopsies. Several markers for CIS of the testis have been developed, including placental alkaline phosphatase, AP2-gamma, and stem cell factor receptor c-KIT. OCT3/4 is a highly specific marker for CIS and currently the best marker for noninvasive diagnosis [22].
Today, TESE is the most frequently used technique for sperm cell extraction in azoospermic men with a mean sperm cell recovery rate of ~50% [23]. Repeat TESE has been found to be progressively more difficult and a minimum 6-month period is recommended between repeat procedures [24]. For men with ongoing spermatogenesis, sperm cell banking must be offered as a first-line management, therefore in azoospermic men cryopreservation of a testicular biopsy provides the option of using testicular spermatozoa for fertilization after thawing.
Obstructive Azoospermia (OA)
In the case of an obstruction somewhere along the sperm journey path, spermatozoa can be obtained from the epididymis or the testicular tissue. The epididymis is readily imaged on ultrasound and epididymal pathology can easily be determined. As the typical thickness of a human epididymal head is in the 7–8 mm range, enlargement can be an indication of an obstructive etiology [25]. Missing epididymis segments or total absence of the epididymis on ultrasound suggest an obstructive etiology as well. Solid tumors of the epididymis, though rare, would also suggest an obstructive etiology. As genital tract infection can also cause epididymal obstruction, the ultrasound findings may differentiate between OA and NOA, rather than a singular find.
Percutaneous epididymal sperm cell aspiration (PESA) for ICSI was described in 1994 by Tsirigotis and colleagues [26]. It offers a relatively fast, minimally invasive, and relatively inexpensive method for sperm cell retrieval and is the first choice due to its minimally invasive nature. Epididymal sperm cells offer the advantage of great maturity and motility relative to testicular sperm cells. The PESA technique is similar to that of TESA. The needle is moved back and forth inside the epididymis. Once the fluid is seen just above the needle hub, it is expelled into a tube with a sperm nutrient medium. If PESA does not retrieve sperm cells, microsurgical epididymal sperm cell aspiration (MESA) is suggested in the setting of multiple prior surgeries and extensive scarring. Relative to PESA, MESA offers the advantage of controlled exposure of the epididymal tubule with the ability to extract far more significant quantities of motile sperm cells. Usually, large numbers of sperm cells can be collected from the epididymis. If a sufficient number of epididymal sperm cells are collected, density gradient centrifugation can be used to prepare the spermatozoa for assisted reproductive technology.
In the case of no spermatozoa being obtained from the epididymis, sperm cells can be retrieved from the testes by open biopsy or by percutaneous needle biopsy. TFNA, using a small butterfly needle attached to a syringe, may be used to harvest spermatozoa for ICSI, especially in men with OA. The advantage of TFNA is that it does not require surgical equipment and experience, and can be performed in an outpatient setting under local anesthesia. Testicular samples contain large numbers of non-germ cells, such as erythrocytes, leukocytes, and Sertoli cells, and therefore spermatozoa must be separated from these non-germ cells. Sperm cells collected from the testes are used for ICSI and not for classic in vitro fertilization (IVF) because the samples generally contain only small numbers of spermatozoa, with poor motility. Pentoxifylline and theophylline, phosphodiesterase inhibitors, both inhibit the breakdown of cAMP and are occasionally applied to increase the motility of epididymal and testicular spermatozoa before ICSI [27–28].
Re-evaluation of an ejaculated semen specimen on the day of planned sperm retrieval is critical for the management of men with azoospermia. Approximately 5–10% of men with presumed azoospermia bear rare sperm cells, which can be found on careful evaluation of a semen sample, using an extended sperm preparation technique. Extended sperm examination of additional microdroplets (under oil to prevent evaporation) allows observation of the entire specimen, and not merely a limited part of the sample. Identification of such rare sperm cells can avoid an unnecessary sperm cell retrieval operation.
Nonobstructive Azoospermia (NOA)
Most patients with primary testicular dysfunction resulting in azoospermia show a Sertoli cell-only pattern in their testicular histology. Others have maturation arrest or have sclerosed and/or atrophied seminiferous tubules. A few seminiferous tubules may still show occasional foci of active spermatogenesis. Incomplete Sertoli cell-only syndrome describes cases in which the current histopathology is germ cell aplasia (Sertoli cell-only syndrome), but some tubules do show active spermatogenesis. The same applies to maturation arrest: when full spermatogenesis is present in several tubules, the condition is referred to as incomplete maturation arrest. A biopsy can identify five main histological patterns of spermatogenesis: the absence of seminiferous tubules (tubular sclerosis), no germ cells within the seminiferous tubules (Sertoli cell-only syndrome), incomplete spermatogenesis (spermatogenic arrest), all germ cell stages present with decline in spermatozoa count (hypospermatogenesis), and normal spermatogenesis [22]. The diagnosis of “nonobstructive azoospermia” should be made according to the histopathological findings, rather than based on clinical indicators only, such as FSH levels or testicular size.
Histological diagnosis of NOA revealed Sertoli cell-only syndrome in 60.6% of patients, while 23.8% had hypospermatogenesis and 15.4% of patients had maturation arrest [29]. The sperm cell retrieval rate is higher in patients with hypospermatogenesis compared with patients with Sertoli cell-only syndrome or maturation arrest (88.2% vs. 30.5% and 30.9%, respectively) [29]. The observation that sperm cells could be identified in testis biopsies of men with NOA drove the development of TESE. Indeed, even the most intense forms of NOA, such as Klinefelter syndrome (XXY), were effectively treated with TESE and ICSI, with the successful sperm cell retrieval rate ranging from 27% to 69% [30]. Sperm cell retrieval rates in all cases of NOA range from 16.7% to 44.3% [29]. The use of TFNA was shown to provide limited numbers of sperm cells in men with NOA.
Multiple-biopsy sperm cell retrieval was also shown to be effective, but in some cases, up to 20 biopsies were required to identify sperm cells needed for ICSI [31]. In TESA, the aspirations are usually carried out using a needle attached to a syringe. The needle is introduced through the scrotal skin into the testis (Figure 14.3). The testicular parenchyma is aspirated by creating negative pressure. In general, for multiple-biopsy TESE, the tunica albuginea is incised transversely at several locations of the center and upper and lower poles of each testis. The testis is then gently squeezed, and the protruding tissues are excised. The multiple-biopsy TESE results in the interruption of testicular blood supply and even to devascularization of the testis since the vessels supplying the testicular parenchyma migrate under the surface of the tunica albuginea. This operation can lead to a risk of damage to the testis.
Figure 14.3 Types of testicular biopsy.
Testicular microdissection, micro-TESE (mTESE), as developed by Schlegel and Li in 1998 [32], was a revolutionary innovative technique that has enabled men with the most severe defects in sperm cell production to become biological fathers. mTESE involves a wide transverse opening of the tunica albuginea of the testis, which exposes all areas of seminiferous tubules for inspection under high power operative microsurgery. The technique relies on the ability of a surgical microscope to identify the individual seminiferous tubules that are engorged with sperm cells. This technique seems to offer the most comprehensive search for sperm cells in the testis of men with NOA [33]. After exposing the seminiferous tubules, a systematic search is performed with a microscope (~×20–40 magnification) (Figure 14.4). Larger tubules thought to contain sperm cells are isolated, placed into the nutrient medium, and examined. Successful mTESE, as a salvage procedure in cases of failed TESE, has been reported [34], while others have reported a limited chance of sperm cell retrieval using mTESE in such men [35]. A surgical procedure such as TESE has possible complications: hematoma, devascularization, inflammation, testicular microlithiasis, and a transient but significant decrease in total testosterone levels, which recover to baseline levels between 18 and 26 months after TESE preparation [36]. The use of an operating microscope to identify the sites of sperm cell production is a more effective way of finding sperm cells compared with multi-biopsy TESE, and provides for sperm cells with less tissue removed [33]. Using microsurgical inspection, the more distended tubules can be selected for excision. mTESE is considered the standard for sperm cell retrieval among NOA patients, with the highest sperm cell retrieval rates while minimizing tissue loss [37]. Subsequent studies have documented that mTESE is 1.5-fold more effective than random multiple-biopsy TESE, which was twofold more effective than TESA in controlled trials [38]. The vast majority of seminiferous tubules in men with NOA lack significant numbers of germ cells and demonstrate a collapsed and thin morphology on microscopic appearance. However, rare seminiferous tubules with a full complement of spermatogenesis are larger and more opaque on microscopic appearance and are therefore selected from the surrounding tubules. mTESE succeeded in increasing the sperm cell retrieval rate to the range of 42.9–63% [39]. The sperm cell retrieval was most significant from dilated seminiferous tubules (90%), followed by testes where tubules were slightly dilated (47%), and finally, from testes with no dilated tubules (7%) [40]. Another study with NOA men supported these findings and showed with mTESE a sperm cell retrieval rate of 31% among tubules less than 200 µm in diameter, compared with 44% and 84% among 200–300 µm-diameter and greater than 300 µm-diameter tubules, respectively [41]. Anatomical studies have demonstrated that the highly coiled seminiferous tubules originate and terminate in the center of the testis (in the mediastinum or intratesticular rete region), traveling out to the periphery of the testicle. A typically very fine filamentous septum separates each tubule with blood vessels running parallel to the tubules. This organization allows dissection deep within the testicular parenchyma, which, in turn, enables direct observation of nearly every region of the hundreds of seminiferous tubules within the testis. If the sperm cells are not found on one side of the testis, the other side of the testis is opened.
