This article summarizes the current literature regarding azoospermia caused by spermatogenic failure. The causes and genetic contributions to spermatogenic failure are reviewed. Medical therapies including use of hormonal manipulation, whether guided by a specific abnormality or empiric, to induce spermatogenesis are discussed. The role of surgical therapy, including a discussion of varicocelectomy in men with spermatogenic failure, as well as an in-depth review of surgical sperm retrieval with testicular sperm extraction and microdissection testicular sperm extraction, is provided. Finally, future directions of treatment for men with spermatogenic failure are discussed, namely, stem cell and gene therapy.
Key points
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Azoospermia due to spermatogenic failure can be caused by several conditions, including cryptorchidism, endocrine abnormalities, and a variety of acquired causes.
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The genetic basis of spermatogenic failure is being progressively understood, with clear definition among patients with Klinefelter syndrome, Y chromosomal microdeletions, and patients with structural chromosomal abnormalities.
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Benefit is seen in targeted medical therapy for hypogonadotrophic hypogonadism, with noncontrolled, retrospective trials suggesting improvement in spermatogenesis with other endocrine therapy.
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The optimal method of surgical sperm retrieval cannot be definitively concluded given the current literature, although there is a suggestion of superiority of microdissection testicular sperm extraction given its retrieval rates and success in salvage cases.
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Stem cell and gene therapy provide promising avenues of future research for the treatment of spermatogenic failure.
Introduction
Azoospermia is defined as the absence of spermatozoa in the ejaculate, after analysis of a centrifuged specimen. Such evaluation should be repeated on at least 2 occasions. This condition affects approximately 1% of men in the general population, and 10% to 15% of men seeking an infertility evaluation. The cause of azoospermia, whether secondary to spermatogenic failure or to obstruction of the excurrent ducts of the testis, is a key determinant of the management of these patients. The introduction of intracytoplasmic sperm injection (ICSI) in 1992 provided the first opportunity for paternity in patients with azoospermia secondary to spermatogenic failure and is currently the standard of care treatment to achieve pregnancy in couples including men with spermatogenic failure.
Causes of Spermatogenic Failure
A variety of conditions, classified as either congenital or acquired causes, are often identified as the cause of spermatogenic failure. Among the congenital causes, genetic issues, including Klinefelter syndrome, Y chromosomal microdeletions, and structural chromosomal defects, are discussed in detail below.
Cryptorchidism
Cryptorchidism, one of the most common congenital abnormalities, is reported in 1% to 4% of full-term male neonates and is a commonly described risk factor for spermatogenic failure. Among patients with untreated cryptorchidism, azoospermia rates vary from 13% in patients with a unilateral undescended testis to 89% with bilateral undescended testes. Germ cell counts have been demonstrated to be associated with the position of the testis at treatment, with intra-abdominal testes showing a greater loss in the number of germ cells compared with inguinal testes, and loss of germ cells have been noted at 6 months of age. Additional evidence suggests that the first stage of germ cell maturation, the transformation of gonocytes to Ad spermatogonia, is defective in the cryptorchid testis, potentially serving as an etiologic factor for azoospermia in these patients. Inadvertent damage to the testis during the orchiopexy procedure may further impair testicular function and contribute to many of the cases of azoospermia in cryptorchid men.
Endocrinologic abnormalities
Endocrinologic abnormalities, including hypogonadotropic hypogonadism (HH), hyperprolactinemia, and endogenous androgen excess, are additional causes of spermatogenic failure, but are rarely the primary etiologic cause of azoospermia, with a rate of diagnosis of less than 1% of men presenting for an infertility evaluation. In addition, exogenous androgen administration is a risk factor for azoospermia. The pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus controls the production of the reproductive hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), from the anterior pituitary. Congenital causes of HH include (1) Prader-Willi syndrome, a deletion or uniparental disomy on chromosome 15 associated with hypotonia, short stature, hyperphagia, mental retardation and hypogonadism; (2) Laurence-Moon syndrome, a rare autosomal-recessive disorder associated with retinitis pigmentosa, spastic paraplegia, mental retardation, and hypogonadism; (3) idiopathic HH; and most commonly, (4) Kallmann syndrome, characterized by idiopathic HH and the presence of midline defects such as anosmia. HH may also be acquired following pituitary surgery or radiation, and head trauma. Hyperprolactinemia, when pathologic, may be caused by prolactin-secreting pituitary tumors, medications including some psychotropics, antihypertensives, and opiates, hypothyroidism, and chronic renal insufficiency. This condition usually results in oligospermia rather than azoospermia and can usually be treated with medications. Exogenous androgen use, as noted in patients on testosterone replacement therapy or athletes abusing anabolic steroids, induces HH from the negative feedback of androgens on the hypothalamic-pituitary axis, leading to decreased LH and FSH concentrations and ultimately lowering the intratesticular testosterone concentrations required for the maintenance of normal spermatogenesis. Endogenous androgen excess, as seen in congenital adrenal hyperplasia, may similarly lead to suppression of gonadotropin stimulation, given excess adrenal androgen production. This condition can be managed with glucocorticoid support, but pseudotumors may develop in the testes, adversely affecting testicular function.
Varicocele
Varicoceles have a known, but variable, effect on testicular function and are identified in 35% of men presenting with primary infertility and 81% of secondary infertility patients. Among men presenting with azoospermia or severe oligospermia, varicocele is noted in 4.3% to 13.3%. The detrimental effect of varicocele on sperm function is thought to be the result of increased scrotal temperatures secondary to impaired drainage or pooling of blood around the testis, although the true cause is not understood at this time. Other proposed causes include reflux of renal and adrenal metabolites from the renal vein, decreased blood flow, and hypoxia. An in-depth discussion of varicocele can be found elsewhere in this issue.
Other acquired causes of spermatogenic failure
Acquired causes of spermatogenic failure include iatrogenic causes, such as systemic chemotherapy or radiotherapy to the testis, neoplasia of the testis, infection or inflammation of the testis as seen in postpubertal mumps orchitis, damage to the blood supply of the testis, use of drugs/gonadotoxins, as well as exposure to environmental or occupational toxins.
Histologic Diagnoses in Spermatogenic Failure
Histologic findings on testis biopsy use classification patterns of spermatogenesis based on its appearance, with pathologic findings varying from hypospermatogenesis, to maturation arrest (MA), to Sertoli cell–only patterns. Although published evidence is conflicting, experienced centers report varying success rates for testicular sperm retrieval based on the spermatogenic defect defined on testis biopsy. Conflict in the literature, however, may be a result of divergent histologic reporting systems as well as varying results with different surgical techniques. In addition, some studies report sperm retrieval based on the most advanced spermatogenic pattern on biopsy, whereas others report results based on the predominant pattern on diagnostic biopsy. Because an individual diagnostic biopsy samples very little of the testis, it is not surprising that variable results could be reported from histology-based evaluation of the testes of men with spermatogenic failure. In addition, there is variation in interpretation of histologic patterns, with one study reporting inconsistencies in 27% of testis biopsy results as reviewed by independent pathologists.
Hypospermatogenesis is defined by the presence of a mature spermatid in any tubule, and therefore, all stages of spermatogenesis are present to a varying degree. MA is used to describe patterns whereby there is complete interruption of spermatogenesis across all seminiferous tubules, with uniform MA characterizing spermatogenic arrest at the same stage of spermatogenesis throughout all of the tubules. It is further subcategorized into early MA, with spermatogenic arrest during the spermatogonia or spermatocyte stage, and late MA, characterized by the presence of spermatids but no spermatozoa. In contrast, Sertoli cell–only patterns are defined by the absence of any germ cells within the seminiferous tubules.
Introduction
Azoospermia is defined as the absence of spermatozoa in the ejaculate, after analysis of a centrifuged specimen. Such evaluation should be repeated on at least 2 occasions. This condition affects approximately 1% of men in the general population, and 10% to 15% of men seeking an infertility evaluation. The cause of azoospermia, whether secondary to spermatogenic failure or to obstruction of the excurrent ducts of the testis, is a key determinant of the management of these patients. The introduction of intracytoplasmic sperm injection (ICSI) in 1992 provided the first opportunity for paternity in patients with azoospermia secondary to spermatogenic failure and is currently the standard of care treatment to achieve pregnancy in couples including men with spermatogenic failure.
Causes of Spermatogenic Failure
A variety of conditions, classified as either congenital or acquired causes, are often identified as the cause of spermatogenic failure. Among the congenital causes, genetic issues, including Klinefelter syndrome, Y chromosomal microdeletions, and structural chromosomal defects, are discussed in detail below.
Cryptorchidism
Cryptorchidism, one of the most common congenital abnormalities, is reported in 1% to 4% of full-term male neonates and is a commonly described risk factor for spermatogenic failure. Among patients with untreated cryptorchidism, azoospermia rates vary from 13% in patients with a unilateral undescended testis to 89% with bilateral undescended testes. Germ cell counts have been demonstrated to be associated with the position of the testis at treatment, with intra-abdominal testes showing a greater loss in the number of germ cells compared with inguinal testes, and loss of germ cells have been noted at 6 months of age. Additional evidence suggests that the first stage of germ cell maturation, the transformation of gonocytes to Ad spermatogonia, is defective in the cryptorchid testis, potentially serving as an etiologic factor for azoospermia in these patients. Inadvertent damage to the testis during the orchiopexy procedure may further impair testicular function and contribute to many of the cases of azoospermia in cryptorchid men.
Endocrinologic abnormalities
Endocrinologic abnormalities, including hypogonadotropic hypogonadism (HH), hyperprolactinemia, and endogenous androgen excess, are additional causes of spermatogenic failure, but are rarely the primary etiologic cause of azoospermia, with a rate of diagnosis of less than 1% of men presenting for an infertility evaluation. In addition, exogenous androgen administration is a risk factor for azoospermia. The pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus controls the production of the reproductive hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), from the anterior pituitary. Congenital causes of HH include (1) Prader-Willi syndrome, a deletion or uniparental disomy on chromosome 15 associated with hypotonia, short stature, hyperphagia, mental retardation and hypogonadism; (2) Laurence-Moon syndrome, a rare autosomal-recessive disorder associated with retinitis pigmentosa, spastic paraplegia, mental retardation, and hypogonadism; (3) idiopathic HH; and most commonly, (4) Kallmann syndrome, characterized by idiopathic HH and the presence of midline defects such as anosmia. HH may also be acquired following pituitary surgery or radiation, and head trauma. Hyperprolactinemia, when pathologic, may be caused by prolactin-secreting pituitary tumors, medications including some psychotropics, antihypertensives, and opiates, hypothyroidism, and chronic renal insufficiency. This condition usually results in oligospermia rather than azoospermia and can usually be treated with medications. Exogenous androgen use, as noted in patients on testosterone replacement therapy or athletes abusing anabolic steroids, induces HH from the negative feedback of androgens on the hypothalamic-pituitary axis, leading to decreased LH and FSH concentrations and ultimately lowering the intratesticular testosterone concentrations required for the maintenance of normal spermatogenesis. Endogenous androgen excess, as seen in congenital adrenal hyperplasia, may similarly lead to suppression of gonadotropin stimulation, given excess adrenal androgen production. This condition can be managed with glucocorticoid support, but pseudotumors may develop in the testes, adversely affecting testicular function.
Varicocele
Varicoceles have a known, but variable, effect on testicular function and are identified in 35% of men presenting with primary infertility and 81% of secondary infertility patients. Among men presenting with azoospermia or severe oligospermia, varicocele is noted in 4.3% to 13.3%. The detrimental effect of varicocele on sperm function is thought to be the result of increased scrotal temperatures secondary to impaired drainage or pooling of blood around the testis, although the true cause is not understood at this time. Other proposed causes include reflux of renal and adrenal metabolites from the renal vein, decreased blood flow, and hypoxia. An in-depth discussion of varicocele can be found elsewhere in this issue.
Other acquired causes of spermatogenic failure
Acquired causes of spermatogenic failure include iatrogenic causes, such as systemic chemotherapy or radiotherapy to the testis, neoplasia of the testis, infection or inflammation of the testis as seen in postpubertal mumps orchitis, damage to the blood supply of the testis, use of drugs/gonadotoxins, as well as exposure to environmental or occupational toxins.
Histologic Diagnoses in Spermatogenic Failure
Histologic findings on testis biopsy use classification patterns of spermatogenesis based on its appearance, with pathologic findings varying from hypospermatogenesis, to maturation arrest (MA), to Sertoli cell–only patterns. Although published evidence is conflicting, experienced centers report varying success rates for testicular sperm retrieval based on the spermatogenic defect defined on testis biopsy. Conflict in the literature, however, may be a result of divergent histologic reporting systems as well as varying results with different surgical techniques. In addition, some studies report sperm retrieval based on the most advanced spermatogenic pattern on biopsy, whereas others report results based on the predominant pattern on diagnostic biopsy. Because an individual diagnostic biopsy samples very little of the testis, it is not surprising that variable results could be reported from histology-based evaluation of the testes of men with spermatogenic failure. In addition, there is variation in interpretation of histologic patterns, with one study reporting inconsistencies in 27% of testis biopsy results as reviewed by independent pathologists.
Hypospermatogenesis is defined by the presence of a mature spermatid in any tubule, and therefore, all stages of spermatogenesis are present to a varying degree. MA is used to describe patterns whereby there is complete interruption of spermatogenesis across all seminiferous tubules, with uniform MA characterizing spermatogenic arrest at the same stage of spermatogenesis throughout all of the tubules. It is further subcategorized into early MA, with spermatogenic arrest during the spermatogonia or spermatocyte stage, and late MA, characterized by the presence of spermatids but no spermatozoa. In contrast, Sertoli cell–only patterns are defined by the absence of any germ cells within the seminiferous tubules.
Genetic basis of spermatogenic failure
Spermatogenesis is dependent on the action of thousands of testis-specific genes that direct the maintenance of spermatogenic cells and satisfactory completion of meiosis in the adult man. Although a complete description of these genes and their function remains to be elucidated, it widely accepted that several genetic abnormalities are responsible for the impaired sperm production and have been associated with spermatogenic failure. Structural or numerical chromosomal abnormalities are 10 times more likely in subfertile men compared with the general population and are identified in 11% to 14% of men with nonobstructive azoospermia (NOA) or severe oligospermia. In addition to karyotype analysis, the presence of microdeletions of the long arm of the Y chromosome (Yq) can be found in these patients. A variety of single gene defects has also been identified in patients with globozoospermia, asthenospermia, and MA, which are beyond the scope of this article.
Chromosomal Abnormalities—Klinefelter Syndrome
Klinefelter syndrome (KS) is the most common chromosomal abnormality among azoospermic men, with a recent report describing a 15% prevalence in this patient group. Nonmosaic KS, 47,XXY, is found in 80% to 90% of men, with the remainder presenting with a mosaic karyotype (46,XY/47,XXY), additional X chromosomes, or structurally abnormal X chromosomes. Nearly half of KS cases are the result of a paternal nondisjunction event during meiosis. Significant phenotypic variability has been noted in KS patients, ranging from tall, eunuchoid boys with gynecomastia, delayed puberty, learning difficulties, and infertility, to well-virilized men with infertility. All patients present with very small (5 cc or less) testes and elevated gonadotropin levels regardless of testosterone output, reflecting primary testicular dysfunction. In addition, patients are noted to have a higher mortality from breast cancer, extragonadal germ cell tumors, and non-Hodgkin lymphoma, although the absolute risk of these conditions is extremely small. They are also at higher risk for diseases that have been linked to androgen deficiency, including osteoporosis and type 2 diabetes mellitus.
Sperm have been noted in the ejaculate of approximately 8% of men presenting with nonmosaic KS and are frequently found during testicular sperm extraction (TESE), described in detail below. Sex chromosomal aneuploidy rates in offspring of Klinefelter men born after TESE-ICSI are reported to be less than 1% in one report, even though sperm aneuploidy rates in these patients are potentially increased in comparison to fertile controls. It has been hypothesized that spermatogenesis in Klinefelter men is completed according to 2 potential pathways: (1) 47,XXY spermatogonia have the potential to complete meiosis, and (2) cryptic 46,XY spermatogonial stem cells (SSC) in the testes, representing a low-level gonadal mosaicism, with increased sperm aneuploidy rates caused by meiotic errors due to a compromised testicular environment. Evidence for both hypotheses is strong but conflicting, and as such, definitive conclusions as to the source of spermatogenesis in men with KS cannot be made.
Other Chromosomal Structural Abnormalities
Cytogenetically apparent structural chromosomal abnormalities are also described among severely oligospermic or azoospermic men. They are generally characterized by the exchange of genetic material between 2 or more chromosomes, usually after chromosomal breakage. Balanced translocations are noted in 5% of severely oligospermic men presenting for infertility.
Reciprocal translocations are defined as a break or rearrangement between 2 or more chromosomes, not involving the centromere. Although the total chromosome content may remain the same, 2 pairs of homologous chromosomes differ in composition. Depending on the location of the break point, particular gene sequences may be disrupted, potentially leading to infertility. They are reported to occur in 0.7% of severely oligospermic or azoospermic men.
Robertsonian translocations, on the other hand, occur when acrocentric chromosomes (number 13, 14, 15, 21, and 22) fuse at their centromere. Minimal essential genetic material is present on the short arm of the acrocentric chromosomes, and those heterozygotes generally remain phenotypically normal. However, effects are frequently noted during spermatogenesis. Robertsonian translocations are noted in 0.8% of severely oligospermic and azoospermic men requiring ICSI.
Other abnormalities can be found on the Yq, including ring Yq, which may lead to deletions in the azoospermia factor (AZF) regions of the Yq, as discussed further below. 46,XX males will also present with spermatogenic failure, as they have usually had translocation of the sex-determining region of the Yq but do not have any of the AZF region required for spermatogenesis.
Y Chromosomal Microdeletions
The role of the Yq in spermatogenesis has been increasingly elucidated since the mid 1970s, when gross deletions of long arm of the Yq were identified in a group of men with spermatogenic failure, followed by the report of submicroscopic Yq microdeletions in 1992. These microdeletions have been spatially and topographically classified within the AZF regions and are undetectable by karyotyping, requiring polymerase chain reaction testing for their evaluation. The presence of Yq microdeletion has been noted to have significant geographic variability and is seen in approximately 10% of U.S. men with severe oligospermia or azoospermia, and 7.4% of oligospermic/azoospermic men reported throughout the literature in a meta-analysis.
Complete deletions of the AZFa, AZFb, or AZFb+c regions are associated with the complete absence of spermatozoa at TESE. Among patients with Yq microdeletions, AZFa, AZFb, AZFa+b, AZFb+c, and AZFa+b+c microdeletions account for approximately 30% of the microdeletions evaluated in a meta-analysis of 13,000 patients with spermatogenic failure. On the other hand, microdeletions encompassing the AZFc region have been associated with more favorable outcomes as these microdeletions quantitatively impair spermatogenesis, with isolated reports of natural paternity. A recent population-based study reviewed DNA samples from greater than 20,000 men from 5 population centers throughout the world and analyzed for the presence of interstitial deletions involving AZFc. DNA donors included healthy controls and patients being evaluated for a multitude of diseases, without discussion of fertility status. Any deletion in the AZFc region was noted in 3.7% of patients. Given the rates and the identification of specific AZFc deletions within this population, the authors reported a 0.04% rate of b2/b4 deletions, which they report is universally associated with severe spermatogenic failure. Of note, they further described more favorable regional deletions within AZFc and estimated that about 1.4% and 1.8% of men with gr/gr and b1/b3 deletions, respectively, present with severe spermatogenic failure. These data serve to understand the prevalence of subdeletions of the AZFc region better, but deletions of these subregions, especially gr/gr, do not seem to cause spermatogenic failure.
Medical therapy for spermatogenic failure
Given the obligate role of hormonal stimulation for complete spermatogenesis, medical therapy has been used in the treatment of spermatogenic failure, with a specific target, as in HH, for treatment of low serum testosterone levels and empirically (although empiric medical therapy is currently largely replaced by assisted reproductive techniques).
Specific Endocrine Therapy
Men with HH are a subset of patients presenting with spermatogenic failure who have specific medical therapy available, with little controversy about the role of endocrine therapy and the restoration of spermatogenesis in most men.
Gonadotropin replacement therapy
Gonadotropin replacement is usually performed with one of a limited number of gonadotropin drugs, with no clear advantage of any one formulation established in the literature. Human chorionic gonadotropin (hCG), an LH analog, is typically administered intramuscularly or subcutaneously at doses from 1000 to 3000 IU 2 to 3 times weekly, to first correct the LH deficiency. Dosing is adjusted to achieve serum total testosterone levels in the mid-normal range. Once testosterone levels have normalized or spermatogenesis is initiated, FSH is added in the form of (1) human menopausal gonadotropin (hMG), containing both LH and FSH activity, in doses of 75 IU subcutaneously 2 to 3 times weekly; or (2) recombinant human FSH (rFSH) in doses of 75 to 150 IU subcutaneously 2 to 5 times weekly. Therapy with both hCG and FSH is required for optimal spermatogenesis in patients with the onset of HH before puberty, as indicated by testis volumes less than 4 mL. Alternatively, GnRH–injecting pumps can be used to inject 25 ng/kg of GnRH subcutaneously every 2 hours, although this is used less commonly.
Combined gonadotropin replacement therapy has been demonstrated to induce spermatogenesis in up to 84% of patients in a combined data analysis of a total of 81 patients, one of the largest available studies. Among these patients, 69% were noted to have sperm concentrations greater than 1.5 million/mL. Predictors of response to gonadotropin therapy have been evaluated by a few studies. Overall, larger testis volume and the spontaneous transit through puberty are generally noted to be favorable factors for the success of gonadotropin replacement therapy. One small study suggested that prior gonadotropin use was also a favorable prognostic factor for sperm production. By way of plausibility, testis volume, largely determined by the amount of seminiferous tubules within the testis, is influenced by prior exposure to gonadotropins and logically can be used to predict sperm output. It is possible that the presence of gonadotropins at puberty “primes” the testis for spermatogenesis with future endocrine manipulation, whether endogenously by way of the pituitary gland, or with hormone replacement therapy.
Dopamine receptor agonists
Among patients presenting with hyperprolactinemia, medical therapy should initially be provided with dopamine receptor agonists. Hyperprolactinemia suppresses the release of GnRH from the hypothalamus. Cabergoline (0.5–1.0 mg orally twice weekly) or bromocriptine (2.5–10 mg orally daily) isused. Cabergoline therapy is generally preferred due to increased efficacy and lower risk of side effects. In addition, it has been specifically demonstrated to improve fertility by normalizing sperm quality in men with hyperprolactinemia.
Empiric Endocrine Therapy
No established hormonal therapy has been proven to increase spermatogenesis for men with NOA/severe oligospermia. Patients with spermatogenic failure usually present with elevated plasma gonadotropin levels, and it had been generally thought that medical therapy in this setting would prove ineffective. However, men with low serum testosterone levels may also have low intratesticular testosterone levels, providing a rationale for medical therapy for these men to enhance intratesticular testosterone levels and spermatogenesis.
Gonadotropin replacement therapy
Interest in the use of rFSH for patients with spermatogenic failure stems from initial case reports describing the recovery of sperm in the ejaculate of men with previously diagnosed NOA, although it is unclear if extended analysis including evaluation of a centrifuged semen analysis was performed. Foresta and colleagues have studied the effects of rFSH in a population of 97 men with severe oligozoospermia. Patients were prospectively randomized to receive either pretreatment with GnRH agonists followed by rFSH versus rFSH alone and were evaluated for hormone response, including plasma inhibin B levels, used as a surrogate for Sertoli cell function. Patients with high pretreatment FSH levels and low inhibin B demonstrated significant increases inhibin B levels over baseline, insinuating a potential for “gonadotropin reset” in these patients. It must be noted, however, that semen parameters were not evaluated following treatment.
Various heterogeneous reports exist in the literature looking at the use of gonadotropins. A Cochrane meta-analysis was performed in 2007 looking at 4 randomized controlled trials with a total of 278 patients with varying degrees of oligospermia and reported a significantly higher pregnancy rate (13.4%) for patients on gonadotropins compared with those on placebo or no treatment (4.4%) within 3 months of completing therapy (OR 3.03, 95% CI 1.3–7.09). Small studies evaluating the use of gonadotropin therapy as a rescue treatment for patients initially failing conventional TESE and microdissection TESE (micro-TESE) have reported surgical sperm retrieval at a second setting in 20% to 25% of patients. However, this retrieval rate is similar to an approximately 30% to 45% rate of sperm retrieval with repeat conventional TESE or micro-TESE after failure of initial conventional TESE. In addition, Aydos and colleagues retrospectively evaluated the use of pure FSH before micro-TESE in 63 patients with spermatogenic failure and FSH levels less than 8 mIU/mL and compared with 45 patients who were not treated with pure FSH. Patients opting for treatment had higher sperm retrieval rates compared with controls (64% vs 33%, P <.01). Ultimately, the retrospective, selective nature of the above-reported trials suggests that further study with randomized controlled trials is necessary before recommending the use of expensive empiric gonadotropin therapy in patients with spermatogenic failure.
Empiric selective estrogen receptor modulators (SERM)
Clomiphene citrate and tamoxifen are nonsteroidal estrogen receptor modulators that block the negative feedback exerted by estrogen at the pituitary and hypothalamus and therefore increasing GnRH pulses and the magnitude of LH/FSH release per GnRH pulse. These agents are frequently used in idiopathic infertility, with few articles focusing specifically on the use of SERMs in NOA patients. Hussein and colleagues reviewed the use of clomiphene citrate in 42 NOA patients from 3 international centers, with the diagnosis of NOA confirmed by initial testis biopsy. Patients with Sertoli cell–only patterns or testicular malignancies were excluded, with 43% of treated patients demonstrating MA and 57% with hypospermatogenesis on prior diagnostic biopsy. After a mean treatment of 5.2 months, whereby dosages were titrated to obtain serum testosterone levels of 600 to 800 ng/dL, sperm were recovered in the ejaculate of 64% of patients, and the remainder demonstrated favorable changes in testicular histology at TESE. Although promising, this study does not have a control group as a comparator, making it difficult to evaluate the magnitude of benefit, if any, from such treatment. Published literature on tamoxifen for men with NOA is limited to a single study that provided tamoxifen to 32 men with NOA (no control arm) and reported a nearly 20% rate of recovery of sperm in the ejaculate.
Hussein and colleagues also recently published a study evaluating the benefit of clomiphene with or without the addition of hCG and hMG on the treatment of 612 NOA patients at a single center. These patients had at least 3 extended semen analyses confirming azoospermia and remained azoospermic for at least 12 months without intervention. Patients with FSH levels elevated to greater than 1.5 times laboratory reference levels were excluded. Patients either opted for no medical treatment before micro-TESE (116 patients) or treatment (496 patients) that consisted of varying regimens of combinations of clomiphene, hCG, and hMG based on changes in hormone parameters. Sperm were noted in the ejaculate of 11% of patients following treatment, and retrieval rates at micro-TESE in the various treatment groups varied from 52% to 58%, compared with 34% in the nontreatment group. The nonrandomized nature of the study precludes any conclusions regarding these treatment regimens. The role of empiric SERM therapy among men with spermatogenic failure is not well understood at this time.
Aromatase inhibitor therapy
Aromatase inhibitors block the conversion of testosterone to estradiol and androstenedione to estrone, therefore reducing the negative feedback of estradiol on the hypothalamus and increasing GnRH, LH, and FSH levels. Available formulations include testolactone (50–100 mg/day), anastrozole (1 mg/day), and letrozole (2.5 mg/day). They have been frequently used among patients with defective spermatogenesis associated with low serum testosterone levels and testosterone/estradiol (T/E) ratios less than 10, based on research by Pavlovich and colleagues. Ramasamy and colleagues have reported an effect of T/E ratio on prognosis and likelihood of successful micro-TESE in KS among men with low baseline testosterone levels (<300 ng/dL) who were subsequently started on aromatase inhibitors. Patients with successful sperm retrieval at micro-TESE had higher pretreatment (before aromatase inhibitors) T/E ratios compared with those with failed micro-TESE (7.1 vs 4.9, P = .03). In addition, preoperative T/E ratios were significantly higher in those with successful micro-TESE (13.6 vs 10.0, P = .04).
A recent study has evaluated the use of empiric therapy with letrozole for men with spermatogenic failure, following a case report documenting the induction of spermatogenesis on TESE after a 4-month treatment with letrozole in a man with initial percutaneous testis biopsy demonstrating hypospermatogenesis. Cavallini and colleagues reported on 4 NOA patients with FSH levels less than 10 IU/L empirically treated with letrozole and reported recovery of sperm in the ejaculate of all 4 men, with concentrations varying from 40,000 to 90,000 sperm/mL after 3 months of treatment. Significant increases were noted in FSH, LH, and testosterone values during treatment. Again, no control group was identified. Reifsnyder and colleagues evaluated the role of optimizing testosterone before micro-TESE, using aromatase inhibitors for men with serum testosterone less than 300 ng/dL and T/E ratio less than 10, and clomiphene citrate in those with T/E ratios greater than 10. Comparing those patients opting for any medical therapy and versus those who did not, the authors found no difference in sperm retrieval rates, pregnancies, and live births following micro-TESE. Further research on larger patient populations is required before adoption of aromatase inhibitor therapy as empiric treatment of spermatogenic failure.
Surgical therapy for spermatogenic failure
Since the advent of ICSI in the treatment of men presenting with NOA, even the azoospermic men once deemed sterile now have the opportunity to father biologic children with surgical sperm retrieval. Despite the absence of ejaculated sperm, the testes of men with spermatogenic failure will often demonstrate isolated foci of spermatogenesis. In men with spermatogenic failure and a clinical varicocele, the role of varicocelectomy in the improvement of sperm production remains debatable. The true clinical challenge, however, centers around identifying and retrieving the isolated pockets of spermatogenesis, and as a consequence, several sperm retrieval techniques have been described.
Varicocelectomy
With the advent of assisted reproductive techniques, the role of varicocelectomy in patients with spermatogenic failure has changed. For men with NOA, it has been presumed by many authors that modest improvements in semen quality or spermatogenesis can improve surgical sperm retrieval rates, lead to the recovery of sperm from the ejaculate, or even result in natural conception.
Success, defined as the presence of sperm in the ejaculate, in the treatment of spermatogenic failure with varicocelectomy, has been variable across studies. Outcomes in the recent literature report any sperm in 21% to 56% of postoperative semen analyses, with a maximum sperm concentration of 3.8 million sperm/mL. These retrospective reviews evaluated a total of 262 patients with fairly limited follow-up and did not include a control arm of patients not undergoing varicocelectomy. It must also be noted that among NOA patients with multiple azoospermic semen analyses, 5% to 35% of patients will occasionally have sperm found in the semen, without any further treatment. Follow-up in these studies is generally limited to a maximum of 6 months (among those who report follow-up), with relapse of azoospermia after apparent induction of spermatogenesis in about 25% of patients.
Ultimately, although induction of spermatogenesis is an ideal outcome, the success of varicocelectomy in achieving spontaneous pregnancy, avoiding ICSI, and avoiding surgical sperm retrieval among patients with spermatogenic failure needs to be assessed more rigorously. A review from a meta-analysis, including several of the articles cited herein, reported a spontaneous pregnancy rate of 6% following varicocelectomy in NOA patients. Instead of merely reporting on the presence of any sperm on any semen analysis as the outcome measure of varicocele repair for NOA, a more clinically relevant outcome is the presence of adequate sperm to avoid TESE after varicocele repair. The need for TESE following varicocelectomy was evaluated in an article by Schlegel and Kaufman, who reported on their experience on 31 men diagnosed with NOA and a clinical varicocele who subsequently underwent varicocele repair. Sperm in the ejaculate were noted in 22% of these men postoperatively at a mean follow-up of 14.7 months. However, less than 10% of the overall population had viable sperm in the ejaculate either previously cryopreserved or at the time of ICSI and were able to actually avoid TESE. In addition, these authors reported identical testicular sperm retrieval rates of 60% using micro-TESE among men with spermatogenic failure and varicoceles who either did or did not undergo varicocele repair. These retrieval rates differ from reports by other authors of improved retrieval rates of 53% to 61% following varicocelectomy, versus 30% to 38% in those without varicocelectomy. Although some benefit occurs for some men with NOA who undergo varicocele repair, most men have subsequent ICSI treatment delayed for at least 6 months to realize the potential benefits of varicocele repair and the vast majority continue to require TESE.
Sperm Retrieval Techniques
The most common methods for sperm retrieval include fine-needle aspiration (FNA)/testicular sperm aspiration (TESA), conventional (multi-biopsy) TESE, and micro-TESE. A recent Cochrane review of surgical sperm retrieval modalities reported insufficient data from randomized trials for recommendation of any one retrieval technique for men with NOA.
FNA/TESA
Lewin and colleagues reported the initial description of TESA leading to live delivery following ICSI in 1996. TESA is a relatively straightforward technique, whereby an 18- to 21-gauge needle connected to steady negative pressure is used to perform several testicular punctures to retrieve sperm. The procedure can be performed under local anesthesia and is reported to have less postoperative pain than TESE. Varying sperm retrieval rates have been reported using TESA, from 7% up to 60% in different patient populations, with no studies reporting on more than 87 patients. Most studies did not include histopathological diagnoses in these patients. Khadra and colleagues reported their experience in 84 men, whereby patients failing TESA had a conventional TESE, and described a 68% retrieval rate with TESA in men with hypospermatogenesis, 29% in MA and 2% for Sertoli cell–only pathologic abnormality. Unfortunately, TESE retrieval rates were not reported for comparison. Several authors in prospective controlled and retrospective studies have reported significantly lower retrieval rates with TESA as compared with TESE, while other descriptive studies report retrieval rates using multiple aspirations to perform TESA that seem similar to TESE in an NOA population (50%–60%). Taken together, these data suggest that TESA/TEFNA (testicular fine needle aspiration) has sperm retrieval rates that are inferior to that obtained with TESE, likely because the aspiration technique does not reliably sample all areas of the testis.
Given the wide range of results with TESA, several investigators have used the concept of FNA “mapping” to identify and map sites of spermatogenesis, following an initial report by Gottschalk-Sabag and colleagues in 1993 demonstrating the correlation between FNA and open testis biopsy. Testicular FNA mapping is performed under local anesthesia, with multiple percutaneous aspiration sites marked on the scrotum, followed by FNA with a 23-gauge needle to aspirate tissue fragments. Tissue fragments are expelled and smeared onto a slide and assessed for the presence of mature spermatozoa. Results are then used to determine where and how sperm retrieval is executed, with TESA used in patients with globally distributed sperm patterns, conventional TESE directed to active sites of spermatogenesis in patients with few sites of spermatogenesis, and micro-TESE in those with rare foci of spermatogenesis. Sperm detection rates vary according the number of FNA sites observed. Turek and colleagues have reported an approximately 50% detection rate in men receiving a mean of 8 to 14 FNA sites per testis, and a 60% rate using 18 sites per testis. Sperm retrieval rates following mapping are expected to be good, because sperm have already been seen, but patients with less than 2 positive sites were able to have sperm found only 81% of the time, and those with greater than 2 sites had sperm found in only 90% of cases.
Testicular sperm extraction
Conventional TESE, performed with either a single open biopsy or multiple biopsies, can be performed with local, regional, or general anesthetic. The tunica albuginea is incised, with either a single or multiple incisions, and a relatively large volume of tissue is obtained for retrieval, or through multiple smaller incisions in different locations in the tunica albuginea, where the testis is gently squeezed and the protruding tissues are excised. It was initially reported in 1993 in cases of obstructive azoospermia and later used for treatment of men with spermatogenic failure. Donoso and colleagues recently performed a systematic review of the literature assessing sperm retrieval techniques among men with NOA and reported a mean sperm retrieval rate weighted by sample size of 49.5% across several different authors and institutions. Many of these studies did not repeat semen analysis on the day of planned sperm retrieval, whereby many of the men who had sperm found with TESE could have had sperm found in the ejaculate and perhaps never needed surgery.
The question of single versus multiple testis biopsies has been the subject of one randomized trial and several observational studies. Intuitively, given the patchy distribution of spermatogenesis in men with spermatogenic failure, one would assume that taking multiple samples from different sites within the testis would improve sperm retrieval rates. Fahmy and colleagues reported data from a randomized trial, with comparable retrieval rates using a single extended incision versus multiple incisions in a group of 89 men without sperm on the initial incision, 29.5% versus 26.7%, respectively. It should be noted, however, that this retrieval rate (whether single or multiple biopsies) is on the lower end of published results and may be secondary to population or surgical technique differences. This result differs from data noted in observational studies in several articles, where multiple biopsies (at least 3) are associated with progressively increasing sperm retrieval rates. Amer and colleagues reported on 316 NOA men, where 216 underwent single bilateral testicular biopsy and 100 underwent multiple biopsies, with similar histologic findings in both groups. Patients undergoing multiple biopsies had significantly higher retrieval rates overall (49% vs 38%), as well as in MA and mixed pathology patients. Patients with unfavorable pathologies, Sertoli cell–only and diffuse tubular sclerosis, did not have a difference between the 2 techniques.
Microdissection TESE
Micro-TESE, initially described by Schlegel in 1999, applies microsurgical techniques and the assistance of the operating microscope to identify individual seminiferous tubules most likely to contain active spermatogenesis. Following local, regional, or general anesthetic, the testis is delivered through the scrotum and opened widely in an equatorial plane, taking care to avoid testicular blood vessels. Using 15× to 25× optical magnification, seminiferous tubules that are larger and more opaque, therefore more likely to contain sperm, are identified and removed, thereby improving retrieval rates and removing a 70-fold smaller volume of testicular tissue as compared with conventional TESE. If no spermatozoa are seen, subsequent samples are taken until the entire testis has been safely surveyed, and if needed, the contralateral testis is evaluated as well. In the initial trial, micro-TESE had a sperm retrieval rate of 62% versus 46% using conventional TESE in the contralateral testis of the same patient.
Experience with micro-TESE at Weill Cornell has continued to be encouraging, with a 56% overall sperm retrieval rate in 1495 micro-TESEs. Patients had a mean age of 35.6 years, with a mean FSH of 27.7 IU/L. Retrospective comparative studies using micro-TESE as described herein suggest a possible improved likelihood of retrieval with micro-TESE as compared with conventional TESE. In a histopathologically unmatched retrospective study, Okada and colleagues compared 24 patients undergoing conventional TESE to 74 with micro-TESE and reported significantly higher retrieval rates overall with micro-TESE (45% vs 17%) as well as among patients with Sertoli cell–only syndrome (34% vs 6%), although the improvement among men with MA did not reach statistical significance (75% vs 38%). Tsujimura and colleagues reported retrospective results from 56 patients with micro-TESE and 37 with multiple TESE and found no statistically significant difference in sperm retrieval rates between the 2 surgical approaches (43% vs 35%, respectively) among patients with matched preoperative characteristics. Ramasamy and colleagues compared outcomes from 543 TESE attempts, whereby the initial 83 were done conventionally and the following 460 done by microdissection, and reported a significantly higher retrieval rate with micro-TESE (57% vs 32%). Amer and colleagues performed a prospective study comparing 100 patients with concordant bilateral testicular histopathologies, who underwent micro-TESE on one testis and conventional TESE in the contralateral testis. They reported significantly higher retrieval rates with micro-TESE compared with conventional TESE (47% vs 30%).
Several investigators have additionally evaluated the role of micro-TESE as a salvage procedure when conventional TESE has failed, whereby TESE was performed at the same institution or elsewhere. Retrieval rates for micro-TESE in these patients have been reported to be about 45% to 57%. Ramasamy and Schlegel looked specifically at the effect of prior failed testicular biopsies (performed at other institutions) and reported that failure to find sperm with 1 to 2 negative biopsies did not significantly affect success of micro-TESE (51% retrieval rate), and significantly lower but a still considerable retrieval rate in men undergoing 3 to 4 negative biopsies (23%). Although these retrospective studies represent heterogeneous patient populations with possible subtle differences in surgical technique, the published data further suggest a superior retrieval rate with micro-TESE compared to conventional TESE.
Complications
Complications secondary to surgical sperm retrieval include development of hematoma, intratesticular fibrosis, and testicular atrophy/hypogonadism. As expected, there are no randomized controlled studies to compare short-term and long-term consequences of the various treatment options. A report by Schlegel and Su described the presence of ultrasonographic abnormalities in the testes in 82% of men at 3 months following open testicular biopsy. The frequency of development of a hematoma and acute ultrasonographic testicular changes following micro-TESE were lower than that observed with conventional TESE in the short-term period in several reports, varying from 7% to 44% versus 30% to 80%. This lower complication rate is likely secondary to better hemostatic control during microdissection as opposed to open surgical extraction, as well as to a decrease in the overall amount of tissue being removed with micro-TESE. Decreases in serum testosterone levels have been noted following both micro-TESE and conventional TESE, with returns to pretreatment testosterone levels in 85% of men after 12 months and 95% after 18 months for both approaches. Decreased testosterone levels are of concern because many men who present with severe defects in sperm production already have defective androgen production, and many would be candidates for immediate testosterone replacement therapy if they were not interested in fertility. After sperm retrieval for NOA, approximately 5% of men require testosterone replacement therapy.
Fresh Versus Frozen Sperm
The question of whether to use fresh testicular sperm or frozen thawed sperm for ICSI has been the object of several studies. Of note, however, is that most of these studies include both obstructed (who make up most of the patients) and NOA patients lumped together in the analysis, making definitive conclusions difficult in the management of the man with spermatogenic failure. Among NOA patients, several studies have reported statistically similar pregnancy and live delivery rates between fresh and frozen sperm. However, these studies, as with all studies looking at men with spermatogenic failure, are plagued by small population sizes, making definitive conclusions difficult.
The experience at Weill Cornell shows that sperm retrieved from NOA patients are frequently limited in number and typically do not survive thawing. An abstract presented by Hopps and colleagues evaluated 95 attempted ICSI cycles in men with documented motile sperm before cryopreservation, with only 33% presenting with viable sperm from the frozen-thawed sample. Pregnancy rates among the 830 micro-TESEs resulting in sperm retrieval at Cornell was 48%, compared with a pregnancy rate of 38% at this same center, in the 33% of men with clearly viable sperm available after thaw. Other centers using just frozen-thawed samples in smaller reports have shown a pregnancy rate of only 26%, suggesting better pregnancy outcomes using freshly retrieved sperm. Because most samples with no documented viability were not used, it can only be presumed that the pregnancy rate after ICSI with these sperm would have only been lower. The authors continue to use fresh sperm for ICSI, typically retrieved on the day before oocyte retrieval for men with NOA, unless frozen-thawed sperm have documented viability.
Surgical Sperm Retrieval Rates According to Etiology of Spermatogenic Failure
Given the success noted with surgical sperm retrieval, attention has turned to delineating the likelihood of retrieval according to the presumed cause of spermatogenic failure.
KS
The advent of ICSI and surgical sperm retrieval, with successful delivery of healthy children to men with nonmosaic KS initially described in 1996, has significantly changed the prognosis for paternity in men with KS and has led the authors of recent publications to question whether these men should be labeled infertile at all. Mosaic 46,XX/47,XXY KS has been reported in up to 3% of men, with reports of the presence of sperm in the ejaculate and subsequent paternity in the past. A systematic review of articles containing only patients with nonmosaic KS (338 patients in all, range 10–74 patients) has reported a 44% overall successful retrieval rate in men undergoing TESE or micro-TESE. Patients undergoing micro-TESE had significantly higher retrieval rates than those undergoing TESE (55% vs 42%). It must be noted, however, that these results reflect a wide variety of different institutions with varying surgical techniques and laboratory experience.
Recently updated results from Weill Cornell represent the largest experience of micro-TESE in KS men, looking at 127 men with classic and mosaic KS (mosaic patterns do not include 46,XY) undergoing simultaneous retrieval during 155 ICSI cycles. Overall retrieval rates in the 155 cycles were 65%, with a 61% retrieval rate per patient. The pregnancy rate was 40%, with 40 children born to date (multiple gestation rate, 31%), all of whom have been healthy 46,XX girls and 46,XY boys. Given these promising results, men with KS seeking paternity should be offered surgical sperm retrieval to address their fertility concerns.
Cryptorchidism
Successful use of surgical sperm retrieval has been demonstrated in several studies of azoospermic men with a history of cryptorchidism. A variety of studies (range 15–79 patients/study) have reported retrieval rates varying from 52% to 72% using conventional or micro-TESE. The authors’ experience in men with a history of cryptorchidism includes 151 men, in whom 181 micro-TESE procedures were performed, and had a 64% overall retrieval rate, with a 62% retrieval rate per patient. The retrieval rate in men with a history of bilateral cryptorchidism was similarly 62%. In analyzing the literature, however, one must note that not all “cryptorchid” men have the same condition, and that in fact several patients throughout these studies may have undergone orchidopexy for a retractile, but otherwise healthy testis. Although a couple of the studies looked specifically at men with NOA, men undergoing orchiopexy are at risk for obstructive azoospermia, which is not clearly controlled for in other studies in the literature.
Postchemotherapy azoospermia
Studies evaluating sperm retrieval from azoospermic men following chemotherapy are generally limited to case series with small overall patient numbers. In addition, given that cancer is less frequently seen in younger men more likely to be interested in fertility, and the wide variety of underlying cancers as well as a myriad of chemotherapeutic treatment options, it has thus far been impossible to determine the specific effect of any one agent or oncologic diagnosis on the likelihood of surgical sperm retrieval in those men rendered azoospermic. Results from a study in 2002 including a total of 23 men from 2 centers, one with conventional TESE and another with FNA mapping and TESE, reported a 65% retrieval rate, although time from chemotherapy to surgical retrieval was understandably not controlled. In the cohort of men at Weill Cornell, the authors identified 93 men at least 6 years after chemotherapy who underwent 114 retrieval attempts and report a 48% retrieval rate overall, with a 30% retrieval rate for men treated with chemotherapy including alkylating agents, but a much higher retrieval rate for men treated for prior germ cell tumors.
Microdeletions in the AZF region of the Yq
As described above, men with complete deletions of the AZFa and AZFb regions of the Yq are invariably azoospermic with no sperm anywhere in the testis, and as such, micro-TESE is not performed on such patients. One study looking at the use of TESE and micro-TESE in 42 oligospermic and azoospermic men with AZFc deletions reported a 66% retrieval rate among the 21 azoospermic men. A report of 21 patients with AZFc microdeletions from a single Korean center described a 43% retrieval rate with TESE. The authors have had successful sperm retrieval in 72% of 54 micro-TESE procedures in men with complete AZFc deletions, with a 46% pregnancy rate.
Uniform MA
Men with uniform MA reflect a subset of men with spermatogenic failure with a higher likelihood of genetic abnormalities, and with generally lower sperm retrieval rates than other men presenting with NOA. Retrospective studies varying from 15 to 151 patients have reported sperm retrieval rates from 23% to 51% with varying surgical techniques. In addition, Weedin and colleagues have reported decreased retrieval rates among men with early MA as compared with those with late MA. In the authors’ cohort, successful sperm retrieval has been noted in 50% of 119 micro-TESE procedures in men with uniform MA, with a per-patient retrieval rate of 45% and clinical pregnancy rate of 29% across treatment attempts.
Sertoli cell only
Patients presenting with Sertoli cell only on testicular histopathology similarly represent a distinct cohort of men, with lower sperm retrieval rates, varying from 29% to 43%, noted in the overall literature as compared with other men presenting with spermatogenic failure. In the cohort at Weill Cornell 670 micro-TESE procedures in men with pure Sertoli cell–only pathologic abnormality have been identified and a 44% overall sperm retrieval rate has been achieved, with a clinical pregnancy rate of 46% among patients with sperm retrieved. Of note, a subset of men within this cohort, patients with normal volume testes (greater than 15 cc) and FSH levels between 10 and 15 IU/L, have a much poorer prognosis, with a retrieval rate of 5.9%.
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