Nonobstructive azoospermia: an etiologic review

Abstract

Azoospermia is the complete absence of spermatozoa in the ejaculate in two or more semen analyses after centrifugation. Nonobstructive azoospermia (NOA) represents the most severe form of male factor infertility accounting for 10%–15% of cases and stems from an impairment to spermatogenesis. Understanding of the hypothalamic–pituitary–testicular axis has allowed NOA to be subcategorized by anatomic and/or pathophysiologic level. The etiologies of NOA, and therefore, the differential diagnoses when considering NOA as a cause of male factor infertility, can be subcategorized and condensed into several distinct classifications. Etiologies of NOA include primary hypogonadism, secondary hypogonadism, defects in androgen synthesis and/or response, defective spermatogenesis and sperm maturation, or a mixed picture thereof. This review includes up-to-date clinical, diagnostic, cellular, and histologic features pertaining to the multitude of NOA etiologies. This in turn will provide a framework by which physicians practicing infertility can augment their clinical decision-making, patient counseling, thereby improving upon the management of men with NOA.

INTRODUCTION

Azoospermia is defined as the absence of sperm in the ejaculate. Nonobstructive azoospermia (NOA) is the absence of sperm due to testicular failure or hormonal imbalance, affecting approximately 10%–15% of the infertile male population and representing the most severe cases of male factor infertility.1,2 The etiologies of NOA can be categorized as either primary hypogonadism, secondary hypogonadism, defects in androgen synthesis and/or response, defective spermatogenesis and sperm maturation, or a mixed picture of the above categories.3 NOA is a wide-ranging diagnosis with each specific etiologic variation portending a different prognosis and management. It therefore requires the reproductive urologist and endocrinologist to have a strong understanding of where each of these disorders categorically fall, a summary of which is presented in Table 1. It is the goal of this review to detail the multitude of etiologies resulting in NOA, including the cellular and histologic features of NOA, thereby providing a scaffold of knowledge to support clinical decision-making and ultimately patient management and counseling.

Table 1.

Etiologies of nonobstructive azoospermia

Cause of NOA	Etiologies
Pretesticular causes	Pituitary causes
	Pituitary/cranial trauma
	Cranial radiation therapy
	Prolactinoma and pituitary lesions
	Drugs, medications, and hormonal therapy
	Dopaminergic and psychotropic medications
	Opioid and cannabinoid abuse
	Exogenous testosterone
	Genetic causes
	GnRH, DAX, and TAC mutations
	Congenital GnRH deficiency (Kallmann syndrome)
	Prader Willi and leptin elevation
	Endocrine-disrupting agents and idiopathic
	Chemotherapeutic agents
	Environmental toxins
	Idiopathic
Testicular causes	Infections, trauma, direct testis damage
	Infectious (mumps orchitis and HPV)
	Testicular and abdominal radiation
	Pelvic and scrotal trauma
	Undescended testis
	Varicocele
	Iatrogenic injury
	Genetic causes
	Klinefelter’s syndrome
	AZF complete deletions, gr/gr
	Partial deletions
	Translocation mutations
	Inversion mutations
	AR mutations
	Epigenetic modifications
	Endocrine-disrupting agents and idiopathic
	Chemotherapeutic agents
	Environmental toxins
	Idiopathic
Histologic findings	Hypospermatogenesis
	Maturation arrest
	Sertoli cell-only syndrome

NOA: nonobstructive azoospermia; GnRH: gonadotropin-releasing hormone; HPV: human papilloma virus; AZF: azoospermic factor; AR: androgen receptor; DAX: dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; TAC: tachykinin

PRETESTICULAR ETIOLOGIES

The hypothalamic–pituitary–testicular (HPT) axis

The root causes of NOA and male infertility overall often include dysfunction of the HPT axis. When functioning properly, it is a well-regulated hormonal communication pathway between the hypothalamus, the anterior pituitary, and the gonads. Neurons of the hypothalamus release gonadotropin-releasing hormone (GnRH) in a pulsatile manner stimulating the anterior pituitary to release follicle-stimulating hormone (FSH) as well as luteinizing hormone (LH). FSH and LH in turn regulate testicular spermatogenesis, regulatory peptides, as well as androgen production and release, which ultimately serves to modulate both positive and negative feedback inhibition (Figure 1).

Figure 1.

Hypothalamic–pituitary–gonadal axis representation. Black lines indicate activation, and red lines indicate inhibition. GnRH: gonadotropin-releasing hormone; FSH: follicle-stimulating hormone; LH: luteinizing hormone.

NOA as a result of secondary hypogonadism, also referred to as hypogonadotropic hypogonadism, arises from pretesticular causes. Secondary hypogonadism can encompass a wide range of syndromes and associated conditions resulting from dysfunctional signaling along the HPT axis. In fact, it is estimated that 20% of men undergoing infertility evaluations are diagnosed with endocrinological hormonal abnormalities, with 1.7%–3% of infertile men presenting with clinically significant endocrinopathies affecting disease management.4,5

Kallmann syndrome (KAL)

An important cause of hypogonadotropic hypogonadism to understand is KAL. KAL represents a congenital GnRH deficiency, characterized by the complete absence of GnRH production. Although the incidence is uncommon overall, KAL cases constitute a large proportion of congenital secondary hypogonadism with an incidence ranging from 1 in 10 000 to 1 in 84 000 men. KAL arises from a malformation and mis-migration of neural crest cells along the midline cranial structures of the olfactory placode to the hypothalamus.6 Multiple genes have been associated with KAL. The most frequently observed genetic alterations include X-linked mutations of the Kallmann syndrome 1 (KAL-1) gene sequence at the p22.3 location.7 While KAL remains the most common congenital GnRH deficiency as discussed, there are numerous genetic mutations resulting in congenital hypogonadotropic hypogonadism. These mutations in addition to the KAL-1 mutation have extremely variable penetrances, resulting in a wide variation in presenting phenotypes. Phenotypic alterations range from normal males with subfertility, to more severe presentations of anosmia, testicular atrophy, craniofacial/skeletal dysmorphias, and NOA with hypogonadotropic infertility.7,8,9

Anterior pituitary gland trauma and tumors

Any insult or injury to the pituitary gland can cause hypogonadotropic hypogonadism with decreased levels versus a complete loss of GnRH similar to KAL. Although traumatic pituitary injuries were initially considered to be a rare cause of GnRH loss, recent studies suggest that there is upwards of 30% incidence of anterior pituitary dysfunction after trauma and traumatic brain injuries. This often results in a loss of GnRH neuronal activity and secondary hypogonadism.10,11 Through a similar mechanism of GnRH neuronal loss, pituitary infarctions, iatrogenic injuries and/or cranial radiation, as well as space-occupying pituitary lesions can cause secondary hypogonadism. This often results in loss of libido, erectile dysfunction (ED), testis atrophy, and NOA with resultant infertility.12

Many different types of pituitary tumors can be clinically encountered. However, prolactin-secreting tumors, otherwise referred to as prolactinomas, bear special mention among these lesions. Endocrinopathies including hyperprolactinemia are estimated to account for up to 2%–4% of male infertility and are the most common cause of secondary hypogonadism-induced infertility in both genders.13 Men will usually present with elevated prolactin levels, suppressed gonadotropins, occasional galactorrhea and hemianopsia, low testosterone levels, and concomitant subfertility or even NOA. While originally postulated to affect the HPT axis at predominantly the central level, supraphysiologic levels of prolactin are thought to exert effects peripherally as well. At the hypothalamic–pituitary level, elevated prolactin is thought to downregulate hypothalamic neurons expressing kisspeptin-1 (Kiss1) genes encoding the kisspeptin protein, a potent activator of GnRH-releasing neurons, thereby decreasing its concentration and ultimately resulting in infertility.14 Downstream tissues including Leydig cells, Sertoli cells, and the efferent ducts of the testicle have been shown to highly express prolactin receptors in animal models, arguing for prolactin’s importance in spermatogenesis at the end organ level.15,16

Testosterone replacement therapy (TRT) and medications

Although not a direct toxin, TRT can also precipitate NOA. Exogenous androgen use, whether patients on TRT or those using androgen-augmenting medications, induces a negative feedback arc on the HPT axis which then decreases FSH and LH, inhibiting intratesticular testosterone and spermatogenesis.17 This multilevel disruption of the HPT axis results in Leydig cell alterations, diminished Leydig cell counts, sperm ultrastructural changes including aneuploidy on fluorescence in situ hybridization, and even sperm apoptosis at high enough levels.18,19,20 These hormonal and structural abnormalities will often manifest as NOA or cryptozoospermia. Although up to 65% of patients with azoospermia at presentation after exogenous androgen administration will recover some degree of spermatogenesis, this is often inconsistent and dependent on the patient’s age and the duration of androgen therapy.21 Inquiring about exogenous androgen use or TRT is, therefore, a requisite to a proper azoospermia workup and differential etiologies.

Among the various hormonal modulating therapies, there remains another common men’s health medication that has potential fertility implications. Finasteride is a type-2 5α-reductase inhibitor that blocks the conversion of testosterone to its more active form dihydrotestosterone. Finasteride is a common medication prescribed for both benign prostatic hyperplasia and male pattern baldness, with the latter indication attracting a much younger age demographic to starting the medication. The findings of a recent study showed that a nondose-dependent reduction of fertility parameters and worsening hormone profile was associated with finasteride use.22 Although azoospermia was overall a rare occurrence, these effects were found to be reversible within the limits of their study. Consideration should therefore be given to holding finasteride in a male with poor semen parameters or fertility concerns who is concurrently taking finasteride.

Medication-induced hyperprolactinemia is additionally an important consideration in men presenting with infertility/NOA and elevated prolactin levels. There are a multitude of different medications that elevate serum prolactin levels; the most common are the antipsychotics/antidepressants, opiates, gastric dopaminergic blockers, and certain antihypertensive medication classes.23 Opiates in a similar fashion cause infertility and hypogonadism by stimulating prolactin secretion and subsequent inhibition of the HPT axis (Figure 1). Long-term opioid use has been shown to centrally suppress hypothalamic secretion of GnRH, ultimately leading to diminished intratesticular androgens and poor spermatogenesis.24 A study in humans found reduced sperm count, reduced motility, abnormal morphology, and azoospermia.25 In animal models, chronic tramadol administration resulted in increased apoptosis of sperm, Leydig, and Sertoli cells with corresponding losses of FSH and LH versus untreated controls, further demonstrating the deleterious effects of chronic opioid abuse on fertility.26

Leptin pathway and molecular genetics

Leptin is a ubiquitous fat-derived mammalian hormone with numerous functions including the regulation of food intake and bodyweight, as well as the regulation of multiple hormonal axes including the HPT axis.27 Leptin is a potent hormone that affects spermatogenesis and steroidogenesis at both central and peripheral levels. It exerts an almost behavioral modulation on mammals, conveying information about energy abundance to the hypothalamus in order to modulate reproductive function and drive.28 Similar to neurokinin, leptin exerts a stimulatory effect on kisspeptin neurons in the premamillary nucleus of the hypothalamus representing a permissive factor in the onset of puberty, thereby coordinating GnRH and LH secretion, especially in the setting of energy abundance.27,29 While the loss of leptin and therefore gonadotropic agents is intuitive as a cause of central-endocrine-based infertility, it has far reaching effects peripherally as well. There are at least three known leptin receptor isoforms found within gonadal tissues in both fertile and infertile men, exerting an effect on both testosterone synthesis and spermatogenesis.30 Leptin plays a role in normal mammalian spermatogenesis as seen in mouse leptin knock-out models which demonstrated reduced seminiferous tubule area, increased apoptotic factors, and fewer tubules exhibiting spermatids/mature spermatozoa.31 Hence, it is abundantly clear that loss of leptin activity is detrimental to sexual health, yet it also seems to function along a spectrum of concentrations. High levels of leptin, found in obesity and metabolic syndromes, have a direct cytotoxic effect on Leydig cells and LH production in vitro. Elevated levels, furthermore, result in diminished sperm concentration and sperm viability, with an inversely elevated sperm DNA fragmentation in obese males versus controls.32,33 Alterations in the leptin pathway can profoundly affect normal spermatogenesis leading to subfertility and NOA.

As our understanding of molecular genetics has evolved, several types of hypogonadotropic hypogonadism that were once thought to be “idiopathic” have now been characterized. These mutations often have an earlier onset of symptoms with delayed puberty, infertility/NOA, and typical low serum gonadotropins. Mutations in the dosage-sensitive sex-reversal, adrenal hypoplasia congenita critical region on the X-chromosome, gene 1 (DAX-1) steroid receptor family occurs at a critical region of the X chromosome which is responsible for proper development of the adrenal cortex and pituitary gonadotropins.34 DAX-1 mutations are associated with congenital adrenal hyperplasia, which on its own can lead to infertility via hypogonadotropic hypogonadism, delayed puberty, and cryptorchidism. However, it also appears to have direct effects on the HPT axis’ pulsatile secretion of GnRH, leading to secondary hypogonadism and NOA.35

Mutations that affect either the GnRH receptor-ligand complex, FSH/LH subunit mutations, or mutations to the leptin signaling pathway can all cause secondary hypogonadism and subsequent infertility. All of these mutations can result in variable phenotypes ranging from features of KAL to isolated hypogonadotropic hypogonadism depending on the mutation and penetrance. There are numerous defects that can occur at the GnRH level including problems with release, synthesis, and resistance. Deactivating mutations can occur in genes coding for G protein-54, kisspeptin ligand, and the tachykinin-3 (TAC3) gene-encoding neurokinin-B protein. The G protein-54 coupled receptor is responsive to the kisspeptin ligand (which as aforementioned is a potent stimulator of GnRH secretion) that when inactive causes a loss of an LH surge resulting in undetectable GnRH, micropenis, cryptorchidism, and infertility at birth.36,37 TAC3 gene mutations coding neurokinin-B cause a loss of pulsatile GnRH secretion with a similar phenotypic presentation of micropenis and infertility, however with a more variable temporal presentation often affecting pubertal development as well.38,39,40

There are additionally extremely rare mutations to the β-subunit of the FSH and LH genes which resulted in azoospermia and low testosterone in the ten male patients as reported.41,42 Of the genetic causes of FSH and LH deficiency, most have been found to be point mutations in exons causing frameshift mutations and disruptions of mRNA expression of the gonadotropins β-subunit. This, in turn, disrupts the androgen axis and normal sperm production, which can ultimately result in secondary hypogonadism and NOA.34

TESTICULAR ETIOLOGIES

While there are abundant pretesticular causes of NOA, intrinsic testicular causes account for a significant proportion of cumulative NOA cases, estimated to affect 1% of all men and 10% of infertile men.43 Causes of primary hypergonadotropic hypogonadism range from direct damage to testicular tissues (such as infections, trauma, gonadotoxins, or even varicoceles) to more surreptitious causes in the form of genetic or idiopathic defects. Although a surplus of testicular etiologies can result in NOA, in a general sense, most variations end with intrinsic spermatogenic failure. It therefore behooves the treating urologist or endocrinologist to have an understanding of the potential causes of primary testicular failure.

Infectious processes

Infections affecting the testis can occur throughout one’s life, with varying effects on fertility depending on the type of infection and the timing. Mumps orchitis has one of the strongest classical associations with hypogonadism and infertility, and although it was largely uncommon in the postmeasles–mumps–rubella vaccine era, it has recently begun to have a resurgence beginning in the early 2000s.44 Mumps is known to cause parotitis, aseptic meningitis, pancreatitis, encephalitis, and commonly orchitis in males around 1 week after symptomatic onset.45 Of all the extra-salivary manifestations, orchitis is the most common. Mumps orchitis in prepubertal boys is generally self-limiting with minimal long-term sequelae. However, infections after puberty are more devastating with 36% developing some levels of testicular atrophy and infertility/subfertility ranging from 13% to 33% in some studies. Infertility in this regard is thought to be mediated by inflammatory cytokine damage to the testis and inhibition of testosterone synthesis.46,47 Barták48 examined the ejaculates of 298 patients 1–3 months after a mumps orchitis infection. They found that 50% of these men had severe disruptions in spermatogenesis, including oligospermia, asthenospermia, teratospermia, and NOA. Additionally, men with unilateral orchitis carried a better fertility prognosis when compared to those with bilateral orchitis or orchitis of a solitary testicle.48

Mumps orchitis is far from the only virus with known gonadotoxic effects. Both human papillomavirus (HPV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have strong associations with infertility and NOA. HPV is a DNA virus that is thought to be the most common sexually transmitted infection globally.49 HPV is frequently found in the seminal fluid (predominantly within the sperm head itself) of not only HPV-infected infertile patients but also fertile controls with infected partners.50,51 Local upregulation of pro-inflammatory mediators, such as interleukins and tumor necrosis factors, leads to the generation of radical oxidative species (ROS). ROS, when present within the testicle and semen itself, lead to increased DNA fragmentation and sperm apoptosis, both having strong associations with NOA.52 Given the incredible prevalence of HPV in society, a true incidence rate of NOA in relation to HPV infection is difficult to ascertain. However, there have been associated infections in upward of 40% of NOA men in retrospective studies. Testicular biopsies of 185 men with NOA found DNA sequences of HPV in the Leydig cells, Sertoli cells, and even the germline cells, substantiating its strong correlation with azoospermia.53,54

Although the SARS-CoV-2 has been prevalent for a far shorter time than the abovementioned pathogens, it has garnered significant attention within the men’s health and infertility disciplines. Several studies have shown that the virus reaches high levels in the male genitourinary tract despite no discreet viral particle identification within the seminal fluids.55,56,57 Postmortem examination of testicular tissues in 12 men with SARS-CoV-2 infection demonstrated significant injury to Sertoli and Leydig cells, lymphocytic infiltration, and seminiferous tubule destruction, potentially resulting from viral immunologic damage.58 Recent evidence looking at how this damage may affect sperm parameters found that out of 45 men recovered from SARS-CoV-2, 8 (18.6%) of them remained azoospermic.59 As alarming as this appears to be, conflicting evidence implies that impairment of spermatogenesis may in fact be transient. Reproductive damage from SARS-CoV-2 may be more akin to the decreased spermatogenesis found during other acute viral or febrile illnesses, thus necessitating more high-level studies.60

Chemoradiation therapy

Other important testicular etiologies of NOA involve varying forms of toxic and traumatic exposures to the testicular parenchyma. Chemoradiation is a highly prevalent form of oncologic treatment known to be deleterious to fertility in often a dose- and therapeutic regimen-dependent manner. Chemotherapy acting at the level of the testicle causes preferential damage to Sertoli cells, leading to elevated FSH and thereby oligo- or azoospermia.61 Whether or not chemotherapy causes prolonged azoospermia becomes a question of the type of agent used, if used in combination or not, the dose of said regimen, and the timing/duration utilized. Despite a multitude of chemotherapeutic agents available, in actuality, most merely cause reversible or transient azoospermia. Prolonged and irreversible azoospermia is most often seen and associated with alkylating agents, platinum-based agents, and cyclophosphamide. Among these therapeutic medications, cyclophosphamide is used as a reference standard producing a “cyclophosphamide equivalent dose (CED)” by which other agents are measured against.62,63 Using the CED also allows one to understand the effect of dosage on fertility, where it has been shown that there is an increasing risk of azoospermia with each additional 1000 mg m⁻² of CED. In a general sense, there seems to be a stronger association between infertility and the risk of azoospermia in younger patients when compared to older patients undergoing chemotherapy; however, recent evidence seems to imply that this connection may not be as strong as once thought.64 That said, these potent therapeutic regimes have a definite effect on male infertility, and as aforementioned most azoospermia tends to be transient. In fact, an animal study has shown that sperm DNA aneuploidy tends to resolve to baseline levels around 18–24 months from the termination of treatment, lending credence to this as an appropriate timeframe to use contraception and avoid pregnancy.65 Radiation similarly has a dose-, fractionation-, and location-dependent effect on spermatogenesis. While low doses of radiation ≤1 Gray (Gy) often have a reversible effect on fertility, upwards of 6 Gy of direct testicular radiation generally causes irreversible NOA.66 The deleterious effects on fertility usually stem from direct damage to Leydig and Sertoli cells and direct sperm damage, with type B spermatogonia being the most sensitive and showing changes at essentially all doses of radiation.66

Endocrine-disrupting chemicals

Along the same line, there is also a strong interplay of environmental factors and toxins that has drawn renewed consideration given the population level decline of semen parameters in Western men.67 There are truly a litany of endocrine-disrupting chemicals with links to male infertility including heavy metals, pollutants, tobacco products, marijuana, and plastic products. Environmental exposures can occur and cause harm throughout human development spanning prenatally to adulthood. The most devastating exposures have been found to occur early in life and typically cause HPT axis dysfunction. Environmental exposures in adulthood cause direct testicular damage, interference with Sertoli and Leydig cells, as well as dysregulation of the HPT axis resulting oligoazoospermia.68 While true NOA is less frequent in adult exposures than that in childhood exposures, profound abnormalities in semen parameters are commonplace and should be included in the differential of physicians managing infertile couples.68

Testicular trauma and direct testicular damage

A well-known spectrum of conditions causing NOA stems from trauma. This can include accidental trauma, but importantly comprise iatrogenic trauma as well. Accidental trauma is an overall rare cause of azoospermia. In a military study of 139 men with azoospermia, of which 99 presented with NOA, four (4%) of the men with NOA had testicular trauma as their causative etiology.69 Testicular torsion is relatively common in men under 25 years old (estimated as 5.9 per 100 000 males between 1 year and 17 years of age, and 1.3 per 100 000 males over 18 years old) and represents a true urologic emergency.70 Occasionally, with delayed presentations, a prolonged torsion may necessitate orchiectomy which can understandably impair fertility.71 Much of the damage, which is incurred when a testicle is salvaged, is thought to be mediated by ischemia-reperfusion injury and generation of ROS, with evidence by Arap et al.72 implicating the potential role of anti-sperm antibodies as a potential mechanism of infertility as well. Outright infertility and NOA are uncommon in unilateral torsion with a contralateral functioning testis, subfertility, and sperm abnormalities occur in 36%–39% of patients post-torsion with some form of abnormalities on semen parameters being common in up to 50% lifelong.71 The risk of azoospermia is a direct result of the duration of torsion and functionality of the contralateral testicle. A study by Almekaty et al.73 found that of 62 postpubertal boys with a median of 32.5-h torsion duration, 33.8% had azoospermia with the presence of an abnormal contralateral testicle being a separate independent predictor of NOA development. Moreover, 57%–88% of contralateral biopsies taken during unilateral torsion events showed pathology including maturation arrest, an indication that other preexisting abnormalities in the other testicle may be strongly contributing to NOA as well in this patient population.74

Among the many disorders that can cause direct testicular damage and NOA, perhaps none have been better studied than cryptorchidism and varicoceles of the cord. Cryptorchidism is a highly prevalent malformation in young boys. Unilateral cryptorchidism has similar paternity rates when compared to the general population; however, bilateral cryptorchidism has been found to have azoospermia rates at approximately 40% and paternity rates between 35% and 53%.75 It is postulated that increased heat during the first 12 months of life retards the development of neonatal gonocytes into adult dark spermatogonia, a process which is heat dependent as evidenced by higher rates of germ cell loss in abdominal testicles versus scrotal or inguinal testicles.76,77 Comparably, varicoceles are very common in the male population and are estimated to be a contributor to 35%–44% of men with primary infertility.78 There are many proposed mechanisms, by which varicoceles cause infertility and most of which have the end result of ROS generation. Disruption of the venous countercurrent exchanger causes scrotal hyperthermia/hypoxia, with generation of ROS causing oxidative stress to Sertoli and Leydig cells. ROS additionally inhibits spermatogenesis through direct harm to sperm and HPT axis dysregulation.78 Accumulation of oxidative damage can cause infertility through azoospermia or severe oligospermia in 4%–13% of men with varicoceles.79

GENETIC CAUSES OF TESTICULAR FAILURE

It has been well established that male infertility carries with it a risk for other medical diseases; in fact, it has been estimated that of men presenting with infertility and an abnormal semen analysis, approximately 1%–6% will have an undiagnosed medical condition.80 Genetic abnormalities are not an exception to this rule and are encountered much more frequently in infertile men where an estimated 11%–14% of azoospermic men have some sort of genomic alteration present on testing.81,82

Among the numerous possible chromosomal abnormalities found in infertile men with NOA, the most common are 47,XXY variations causing Klinefelter’s syndrome (KS). Due to around a 10% rate of mosaicism, there is a wide range of phenotypic presentations ranging from tall eunuchoid males with gynecomastia, delayed puberty, and NOA, to phenotypically normal men with subfertility.83 Despite KS being a relatively frequent genetic diagnosis in the setting of NOA, the exact mechanistic causes of infertility are still largely unknown. It is hypothesized that the aneuploidy causes the sperm themselves to degenerate, while additionally, there appears to be a communication breakdown between Sertoli cells and immature germ cells during development. Furthermore, in a pathogenesis similar to Fragile X syndrome, CAG DNA nucleotide repeat polymorphisms have been discovered whose length is inversely correlated with androgen inactivation. In fact, the presence of gynecomastia and a patient’s total potential height is reciprocally related to CAG repeat length, lending a possible explanation for the high phenotypic variability present with KS.84

Even though there have been scarce reports of spontaneous pregnancies in mosaic KS men, the overwhelming majority require microscopic testicular sperm extraction (micro-TESE) and assisted reproductive techniques. The advent of micro-TESE has allowed previously “sterile” men a chance at pregnancy; however, with this technology arose the need to predict which men would actually benefit from surgical sperm retrieval. Since the initial discovery of microscopic gene deletions along the long-arm of the Y chromosome (Yq) in areas controlling spermatogenesis, dubbed azoospermic factors (AZF), significant strides have been made in regard to its role in fertility. Complete AZF deletions are most commonly due to a deletion or duplication during the homologous recombination process. These mutations are rare overall from a population standpoint but are estimated to be responsible for infertility in up to 10% of patients with idiopathic NOA.85 Which AZF variant becomes deleted has profound implications on spermatogenesis and the probability of finding sperm on micro-TESE. Complete deletions of AZFa, AZFb, AZFb+c, and AZFa+b+c are associated with true NOA and a 0% chance of finding sperm on micro-TESE for example.86 While this has obvious implications and immediacy to the affected infertile couple, these cluster of mutations are directly heritable to their male progeny. Although there is little long-term follow-up on parturition from fathers with AZF mutations, there is loose evidence of progressive loss of spermatogenesis and other potential genetic abnormalities most notably Turner’s syndrome among progeny.87 A strong understanding of the pathogenesis of these mutations can therefore be fundamental to counseling the subfertile male in addition to any future progeny of the affected male.

A more recently understood deleterious mutation of the AZF regions of the Y chromosome involve homologous recombination related 1.6 mega-base partial deletions of g1/g2, r1/r3, and r2/r4 (gr/gr deletions) regions within the AZFc region. Given the inherent variance of the homologous recombination repair system, there is a large diversity of deletions that can occur including reductions in deletion length, changes to copy number, and penetrance. The consequence of this is a wide variation in spermatogenic failure ranging from mild oligospermia to overt NOA.88 However, despite the logical risk of spermatogenic failure, a recent meta-analysis by Bansal et al.89 found that the true incidence of NOA was low and gr/gr mutations were more prone to generating oligospermia, especially among Caucasian populations.

Changes to the structure of the chromosomes inarguably play a major role in the genetics of male infertility and spermatogenic failure. Yet, other defects to the Y chromosome can occur on a macroscopic scale, beyond just microdeletions. Of all the macroscopic structural changes, Robertsonian translocations are the best studied and most frequently observed. Although an uncommon cause of azoospermia overall, the rates present in men with azoospermia are approximately 0.01%.90 Robertsonian and reciprocal translocations can occur when two or more acrocentric chromosomes exchange material. This can at times lead to gross structural changes to the Y chromosome occurring due to a miss-fusion of chromosomes at their acrosome. Uneven acrosomal fusion, therefore, leaves an unequal amount of genetic material on the short arm versus the long arm, ultimately truncating or eliminating the AZF or sex-determining region of the Y (SRY) gene regions, thereby causing azoospermic infertility.90,91 Importantly, both types of translocations produce genetically unbalanced gametes that are potentially heritable and transmissible to aneuploid offspring, a vital point to address during reproductive and genetic counseling.

Disruptions in the X chromosome can also lead to complete spermatogenic failure and NOA. Mutations and disruption in the androgen receptor (AR) genes cause a broad range of disorders of sexual development grouped under the umbrella term androgen insensitivity syndrome. There is a wide phenotypic variation ranging from female appearance to phenotypically normal males with infertility depending on the breadth of the AR defects. In a cohort of subfertile men, 492 were azoospermic, with AR mutations being present in 6 of the men (1.2%).92 It has been theorized that up to 40% of men with oligo- or azoospermia may have some degree of androgen receptor anomalies, a finding that may have some truth given there are at least 1029 AR mutations reported in the literature to date.93

Epigenetics

One of the current frontiers of genetic medicine is the interplay of epigenetics in the etiopathology of disease. There is emerging evidence that epigenetic modification may play a large role in NOA and potentially idiopathic infertility. A study looking at NOA and epigenetics found that in 502 cases of azoospermia, 58 NOA patients presented with significant hypermethylation of the promoter region of the SRY-related HMG-box (SOX30) gene when compared to obstructive azoospermia and control patients.94 SOX30 was found to be associated with testicular volume, male gamete generation, and spermatogenesis. Furthermore, mouse knock-out models of SOX30 showed a complete loss of spermatozoa; however, restoration of the knocked-out gene cassette allowed for restoration of normal fertility in the next generation.94 These results may explain how downregulation with epigenetic histone modification can result in NOA. An understanding of epigenetics may also prove invaluable in explaining the most frustrating variety of NOA: idiopathic.

Idiopathic infertility and emerging studies in NOA

It is currently estimated that only around 30% of NOA cases can be categorized into the etiologies aforementioned. Patients fitting this definition of NOA have classically been referred to as “idiopathic NOA”. While a causative factor in many of these patients often remains unsolved, advances in molecular genetics are beginning to shine a light on this difficult area of male infertility. Recently, there has been a surge of interest in mRNA profiles in relation to idiopathic infertility. One study of mRNA sequencing of 31 men (4 controls and 27 with an unknown cause of NOA) looked at microarray data sets and found a significant differential expression in a total of 74 mRNA, 14 micro-mRNA, and 10 long noncoding mRNAs when compared to the fertile controls.95 This has been correlated with other research, indicating that dysregulation of micro-mRNA is pivotal to the development of sperm maturation arrest, therefore providing a mechanistic explanation for the role of mRNA profiles in transcription regulation of spermatogenesis.96

Recent advances in whole-exome sequencing and transcriptome analysis have shed light on additional novel genetic variants exhibiting associations with NOA and idiopathic infertility. Research into alternatively spliced mRNA (iso-mRNAs) profiles in testis with NOA has shown an important role in gene expression along the spermatogenesis pathway. Transcriptome analysis of testicular samples obtained from NOA patients demonstrated differential expression of iso-mRNAs versus controls. Using bioinformatic analysis, their promoter regions were found to be downregulated in areas associated with mitosis, meiosis, and RNA regulation.97 Several transcription factors including DNA repair protein RAD51 homolog-1 (RAD51), heat shock transcription factor-4 (HSF4), and specialty protein-1 (SP1) were also identified as being involved in the testicular spermatogenic regulatory axis, adding to our understanding of the molecular basis of normal spermatogenesis.

Among the most studied genetic variants in NOA is the testis-expressed gene (TEX) family, which is X-linked and is important to proper meiotic recombination. An early study by Yatsenko et al.98 demonstrated TEX mutations in up to 2.4% of men with NOA, but absent in controls. A more recent study of 235 men with idiopathic NOA and maturation arrest found 5 novel genes including adenosine deaminase domain containing 2 (ADAD2), telomere repeat binding bouquet formation protein 1 (TERB1), shortage in chiasmata 1 (SHOC1), mutS protein homolog 4 (MSH4), and RAD21 cohesin complex component like 1 (RAD21L1) were all associated with NOA. An additional 7 variants in known genes, including testis-expressed protein 14 (TEX14), double-sex and mab-3-related transcription factor-1 (DMRT1), testis-expressed protein 11 (TEX11), synaptonemal complex central element protein-1 (SYCE1), meiosis-specific with OB-fold (MEIOB), stromal antigen-3 (STAG3), and meiotic double-stranded break formation protein 1 (MEI1), responsible for NOA were found on exome sequencing in this sterile cohort.99 While this mandates further basic and clinical investigation, novel genetics such as this could serve as a kind of diagnostic genetic panel in men with NOA and/or idiopathic infertility in the future.

CELLULAR AND HISTOLOGICAL FEATURES OF NOA

Although the interplay of RNA profiles in relation to histologic changes is still an evolving field of NOA research, much more is known of the histologic patterns with respect to the likelihood of finding sperm present on biopsy or TESE, and the relative incidence of NOA. Spermatogenesis often exists along a spectrum, one which was initially categorized by Johnsen in the 1970s assessing tubules and ranking them 1–10 based on how advanced the germ cells were under light microscopy.100 This gave rise to the histopathologic definitions and classifications most reproductive physicians are familiar with. In the most extreme cases, only Sertoli cells can be identified on light microscopy, a condition referred to as Sertoli cell-only syndrome (SCOS). Germ cells along various stages of development with decreased seminiferous epithelium indicate hypospermatogenesis. Broadly, as sperm development is stalled along its course of maturation (in the absence of fully developed sperm), the pathology is termed either early or late maturation arrest based on the timing of arrest during the meiotic cycle.

In the past, testicular biopsy was the only recourse for information important to treatment decisions, especially in the era before genetics and endocrine testing became prevalent. Yet, testicular histopathology has been shown many times to be an imperfect predictor of spermatogenesis. Studies have shown that men with NOA and no germ cells on testicular biopsy have been found to have focal areas of spermatogenesis present in other regions of the testicle.101 A recent study looking at the interplay of histologic subtypes on the testicular biopsies of 276 men with NOA found normal spermatogenesis in 5% of men, hypospermatogenesis in 16%, maturation arrest in 29%, and SCOS in 50%.102 Among these men, there was found to be a right-to-left testicular discrepancy in 30 patients with NOA, and there was no difference in predictive value based on age, testis volume, or overall Johnsen score. Only finding tubules with spermatozoa present was associated with significant Pearson correlation. This in conjunction with similar data in the field connotes the inability of histology as an accurate predictor of spermatogenesis in men with NOA and more research is needed in this difficult area of infertility.

Although histology itself is not universally predictive of sperm retrieval as aforementioned, there is still value in recognizing the various histologic patterns as they pertain to known etiologies of male infertility and NOA. In a study of 139 patients with SCOS, 33 patients (24%) had abnormal karyotypes, with KS patients accounting for 29 of the 33 patients.103 The second most common genetic disorders are AZF deletions; however, there are a host of additional mutations (single-nucleotide polymorphisms, epigenetic changes, and miRNA alterations) that can result in SCOS.104 Maturation arrest is also multifactorial, a study of 223 men with NOA by Tsai et al.105 found that the frequency of late maturation arrest (84.6%) was due to testicular insults such as varicoceles, trauma, and cryptorchidism. Early maturation arrest was found to be more associated with karyotype and Y-chromosomal genetic anomalies (42.8%).105 Similarly, hypospermatogenesis is a multifactorial phenomenon resulting from testicular insults and genetic anomalies. By looking at whole-genome DNA methylation and mRNA microarrays in testicular samples of NOA patients, candidate genes for methylation dysregulation were discovered including boule homolog (BOLL), RNA-binding protein (RBP), DEAD-box helicase 4 (DDX4), HORMA domain-containing protein (HORMAD1), and maelstrom spermatogenic transposon silencer (MAEL). Discovery of these novel genes adds further nuance to our understanding of the etiologic associations between NOA and testicular histopathology.106

CONCLUDING REMARKS

NOA remains one of the most challenging conditions to treat in andrology. Causes of NOA can be grouped into pretesticular and testicular etiologies. Pretesticular causes affect the HPT axis and result in decreased secretion of LH and FSH and ultimately under stimulation of the testes. Oftentimes, pretesticular causes can be reversed with medical management. Testicular causes of NOA usually exert a direct effect on the testicle or are caused by intrinsic testicular pathology and cannot be reversed. Surgical sperm retrieval may be an option in some cases. The genetics of male infertility is a rapidly evolving field and new genetic abnormalities leading to azoospermia are being identified. Experimental approaches are also being studied on how to treat NOA due to genetic causes.

AUTHOR CONTRIBUTIONS

LH contributed significantly to the conception and design of the study, acquisition of data, analysis and interpretation of manuscripts, and drafting the manuscript. AR contributed to the interpretation of available manuscripts and critically revised the manuscript for important intellectual content. SG provided guidance throughout the study, supervision of the research group, ensuring the accuracy and integrity of the work, and critically revising the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.