Meticulous microscopic examination of the pellet is necessary to determine whether a semen sample is truly azoospermic. It is shown that sperm are identified in up to 35% of men who are thought to have NOA during an extended examination of a centrifuged specimen [34]. The definitive diagnosis of NOA relies on histological confirmation. However, a clinical diagnosis based on history, small testicular volume, and flat epididymides on physical examination, elevated serum FSH levels, and azoospermia on semen analysis can be made in many cases.

Diagnostic testicular biopsy remains the gold standard in differentiation between OA and NOA. However, the small samples obtained from diagnostic biopsy are unlikely to be representative since both testicular histology and sperm production are heterogeneous within the seminiferous tubules. Currently, many centers perform testicular biopsy for histology at the time of sperm acquisition. A separate procedure of testicular biopsy is not regarded as mandatory before sperm retrieval procedures by many male fertility specialists.

Karyotyping and Y-chromosome microdeletion (YCMD) testing typically identify the etiology of impaired spermatogenesis in 15–20% of NOA patients, and up to 17% of TESE candidates are found to have abnormal genetic evaluation [35]. YCMDs are more commonly detected in patients with lower sperm production. Ten percent of azoospermic men are noted to have YCMD, while no microdeletion is detected in men with sperm counts more than 5 × 10⁶ [36]. It is now possible for the transmission of defective genetic material to offspring with advent of ICSI and sperm retrieval. It is therefore advisable to have genetic evaluation before sperm retrieval. Results of genetic tests have been shown to alter the choice of treatment in 21% of infertile couples [35]. Donor sperm, adoption, and embryo biopsy are some options elected by patients after genetic counseling. Apart from genetic evaluation, elevated serum FSH level is one of the clinical features of men with NOA. The prognostic value of hormonal and genetic evaluation on sperm retrieval will be discussed later in this chapter. Imaging modalities are generally not indicated for the management of NOA patients unless there are abnormalities on physical examination.

Spermatogenesis should be optimized for at least 3 months prior to sperm retrieval. Any reversible causes should be corrected including avoidance to gonadal toxins. The role of varicocelectomy in our patient with NOA is not well defined. Most patients have no return of sperm to the ejaculate and require sperm retrieval despite repair of varicocele [37]. Varicocelectomy does not influence subsequent SRR in men with NOA and clinical varicoceles. The beneficial effect of varicocelectomy may take 6 months or longer to appear and, therefore, may not be a sensible choice for our patient. Hormonal disturbances, including compromised serum testosterone and increased estradiol levels, are common among men with NOA [38]. Testosterone-to-estradiol ratio (TE ratio) is commonly used clinically as an expression of the overall androgen and estrogen balance. The mean TE ratio in fertile controls is significantly higher compared to men with severe infertility [38]. Increased aromatase activity of the testes may contribute to the phenomenon [39]. By directly limiting estrogen feedback to the pituitary gland, aromatase inhibitors increase production of FSH and luteinizing hormone (LH). The correction of the endocrinopathy may enhance endogenous intratesticular testosterone levels and thus spermatogenesis. Both steroidal (testolactone) and nonsteroidal (anastrozole) aromatase inhibitors raise serum testosterone levels and correct TE ratios effectively [40]. A TE ratio of less than 10 is proposed as the cutoff to initiate treatment. Although significant improvements in the hormonal profile and semen parameters have been demonstrated in oligozoospermic men treated with testolactone, there are no studies that have demonstrated a return of sperm to the ejaculate in azoospermic men with treatment [38]. The use of aromatase inhibitors in men with NOA remains off-label. The correlation between hormone manipulation and fertility benefits remains to be defined by randomized controlled studies.

In summary, karyotyping and YCMD testing should be performed in our patient before sperm retrieval procedures. The test results carry important prognostic value. Varicocelectomy as an adjunct before sperm extraction has no evidence to improve SRR. Testing of estradiol level may be considered, and treatment initiated with TE ratio less than 10. However, the current evidence of correction of endocrinopathy with aromatase inhibitors in our patient with NOA is weak.

Failure of previous sperm retrieval does not deter further attempts in our patient. The characteristic of patchy foci of sperm production in men with NOA renders a single biopsy inadequate for identification of sperm most of the time. Multiple biopsies are essential for the successful sperm retrieval in men with NOA. Only 23% of men have sperm identified on the first biopsy, and up to 14 biopsies may be required to locate sperm in a single procedure of open testicular biopsy from 1 or both testicles [41]. Repeating testicular biopsy or testicular sperm aspiration (TESA) may be a less favorable option in view of the low SRR. Microdissection testicular sperm extraction (mTESE) after previous failed TESA or TESE procedures is more commonly practiced and studied. Patients who had 1 or 2 prior biopsies per testis have an SRR of 50% by mTESE. SRR decreases to 22% if 3 or more previous biopsies were performed per testis. This is compared to 52% of SRR with mTESE in patients who have no prior testicular surgery [42]. The minimal impact on subsequent SRR by 1 or 2 prior testicular biopsies strongly suggests that random testicular biopsies commonly miss areas of sperm production. The chance of sperm identification on repeated mTESE is 33% even when no sperm is found on the first mTESE [43]. Data show that mTESE achieves reasonable SRR after failed testicular biopsy, TESA, TESE, and mTESE in the hands of an experienced infertility surgeon. Repeated mTESE appears a viable option for our patient.

A 6-month interval between sperm retrieval procedures is recommended to our patient. This recommendation is based on the concept that spermatogenesis can be adversely affected by postoperative changes and sperm production may take 3 months to be fully restored. Although clinical data on the effect of the time interval between sperm retrieval procedures and SRR are lacking, the suggestion of a 6-month interval is supported by circumstantial evidence. It is found that 82% of abnormal sonographic findings of the testes at 3 months after TESE procedures resolve by 6 months [44]. The incidence of ultrasound findings suggestive of hematoma decreases from 5 to 7.5% and 12 to 2.5% at 1 and 6 months after conventional TESE and mTESE, respectively, suggesting that at least 6 months is needed for most of the testes to fully recover after sperm retrieval procedures [45]. However, the varying degree of testicular damage caused by different sperm retrieval procedures indicates that the optimal timing to repeat sperm retrieval procedures should be individualized. Serial ultrasound imaging of the testes may be helpful in defining the optimal time interval. While the majority of ultrasound abnormalities resolve by 6 months, endocrine function and serum testosterone level may take up to 18 months to recover [46]. The question remains unanswered, and the optimal time interval between sperm retrieval procedures is yet to be defined by further research.

The testis is the only sperm source for our patient. There are several options available for testicular sperm retrieval: (1) TESA, (2) conventional TESE, (3) mTESE, and (4) testicular mapping. TESA attempts to retrieve sperm by percutaneous technique. The procedure can be performed under cord block and local anesthesia when a fine needle is used. The low SRR renders percutaneous procedures uncommon [47, 48]. TESA is not recommended as the primary procedure of sperm retrieval for men with NOA except when used in conjunction with testicular mapping. It has been shown that percutaneous procedures are less effective than open testicular biopsy in obtaining sperm [33, 49].

TESE and mTESE are open testicular biopsy techniques and are more commonly performed in men with NOA. Multiple biopsies are usually employed to locate sperm during conventional TESE [41]. Conventional multiple biopsy TESE achieves up to 50% SRR [50]. However, it carries the risk of damage to the testicular blood supply. Complete testicular devascularization has been reported after multiple biopsies.

Since the introduction of mTESE in 1999 [33], the procedure has gained popularity due to several advantages over conventional TESE. The use of a microscope allows identification of subtunical blood vessels and decreases the risk of damage to the testicular blood supply [51]. A higher SRR of 45–65% is associated with mTESE compared to 30–45% with conventional TESE [33, 47, 51]. Moreover, mTESE is more effective in recovering sperm from men with testicular volume of less than 10 mL [52]. Larger quantity of sperm is obtained during mTESE with less testicular tissue removed. An average of 160,000 spermatozoa are obtained in samples that weigh 9.4 mg during mTESE, compared to 64,000 spermatozoa yielded by 720 mg of testicular tissue from conventional TESE [33]. However, it was concluded in a systematic review that mTESE performs better than conventional TESE only in cases showing Sertoli-cell-only pattern on histology where tubules containing foci of active spermatogenesis can be identified by the microscopic appearance of larger and more opaque tubules [53].

mTESE also has the lowest complication rates compared to other sperm retrieval techniques [53]. mTESE results in less intratesticular reaction than conventional TESE despite the wide equatorial incision along the tunica albuginea and extensive dissection. The achievement of complete hemostasis during mTESE results in less acute and chronic sonographic changes on scrotal ultrasound. Less postoperative pain after mTESE has been reported due to less retraction of tunica albuginea and compression of testicular parenchyma [54].

Despite the advancement in sperm retrieval techniques, lasting effects on testicular function after testicular sperm extraction should not be overlooked. Serum testosterone levels drop by 20% of preoperative levels at 3–6 months after sperm retrieval procedures and are not completely recovered at 18 months postoperatively [46]. It also has been reported that mTESE leads to reduction in serum testosterone levels and increase in FSH and LH levels [55]. Histologic studies of the testes after sperm extraction procedures reveal a 7 and 5% decrease in seminiferous tubule volume and germ cell density, respectively [56].

Another option for obtaining sperm from our patient with NOA is testicular mapping. It consists of systematic fine needle aspiration (FNA) following a 22-site template of bilateral testes. Further management is stratified by the test results. Patients who have no sperm identified are offered the options of adoption and donor insemination, and attempt to use a sperm retrieval procedure is generally not recommended in expert centers. On the other hand, a directed sperm retrieval procedure will be offered in the presence of sperm. The location and quantity of sperm identified on mapping guide the subsequent sperm retrieval procedures. Testicular mapping is an outpatient procedure performed under local anesthesia. The procedure is well tolerated, and patients usually resume normal activity within a day [57]. An early study has demonstrated the potential use of FNA to identify sperm in men with NOA with 2–3 samples from each testis [58]. The role of FNA is further supported by a report of 60% SRR in men with NOA with up to 15 samples from each testes, but the quantity is insufficient to inject all a partner’s ova in most cases [59]. Therefore, testicular mapping/FNA as the sole sperm retrieval procedure is not recommended. The optimal number of sites of diagnostic aspiration remains unclear. Despite the advantage in avoiding or minimizing the invasiveness of sperm retrieval procedures, the wide application of testicular mapping is hindered by the significant cytologic experience required in identifying sperm in a smear of aspirated seminiferous tubules.

Subsequent sperm retrieval is executed from the least to most technically demanding procedures in the sequence of TESA, conventional TESE, and mTESE based on the map. It has been demonstrated that sufficient sperm for injection of all available oocytes can be retrieved in 95% of cases [60]. Bilateral procedure was only required in 22% of patients. Complex sperm retrieval with mTESE was performed in 23% of men, while the majority had sperm acquired by TESA and TESE [60]. It is of note that the high SRR was reported from patients with positive FNA results to begin with. Currently, there is no head-to-head studies comparing the different strategies of mTESE and testicular mapping ± sperm retrieval. The advantages and disadvantages of various sperm retrieval techniques in men with NOA are presented in Table 11.2.

The importance of intraoperative specimen handling in increasing sperm yield has been addressed. The mechanical disruption of individual tubules by aggressive mincing in the medium and repeated passage of testicular suspension via angiocatheter increases sperm yield by up to 300-fold [61]. The procedure of sperm retrieval can be terminated once sufficient sperm are identified in the operating theater by surgeon or embryologist under microscope. The increased efficacy in sperm identification prevents unnecessary damage to the already compromised testis of our patient.