Hypogonadotropic hypogonadism as a cause of NOA and its treatment

Abstract

Hypogonadotropic hypogonadism (HH) represents a relatively rare cause of nonobstructive azoospermia (NOA), but its knowledge is crucial for the clinical andrologists, as it represents a condition that can be corrected with medical therapy in 3 quarters of cases. There are forms of congenital HH, whether or not associated with an absent sense of smell (anosmic HH or Kallmann syndrome, and normosmic HH, respectively), and forms of acquired HH. In congenital HH, complete absence of pubertal development is characteristic. On the other hand, if the deficit occurs after the time of pubertal development, as in acquired HH patients, infertility and typical symptoms of late-onset hypogonadism are the main reasons for seeking medical assistance. Gonadotropin-releasing hormone (GnRH) or gonadotropin replacement therapy is the mainstay of drug therapy and offers excellent results, although a small but significant proportion of patients do not achieve sufficient responses.

INTRODUCTION

Adult testicular function is finely regulated by the hypothalamic–pituitary–gonadal (HPG) axis (Figure 1). The highest level of the HPG axis is occupied by the central nervous system (CNS), specifically by gonadotropin-releasing hormone (GnRH)-secreting neurons from the hypothalamus, which migrate from the medial olfactory placode of the forming nasal cavity during fetal development.1 From the hypothalamus, GnRH is transported into the pituitary portal system, where it encounters its receptor on the cells of the adenohypophysis, which instead develops from the oral ectoderm.2 Gonadotropic cells of the pituitary gland synthetize and secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in turn bind to their receptors at testicular level, representing key elements for initiation and maintenance of spermatogenesis.3 Fetal development of the structures involved in this system represents an extremely sensitive time for an individual’s future reproductive potential.

Figure 1.

The hypothalamic–pituitary–gonadal (HPG) axis. Gonadotropin-releasing hormone (GnRH) is released by the hypothalamus under the control of a hypothalamic neuronal network named “GnRH pulse generator”, of which activity is modulated by stimulatory and inhibitory signals, mainly represented by the kisspeptin–neurokinin–dynorphin (KNDy) system. GnRH is secreted in regulated pulses into the hypophyseal portal system and induces the synthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH acts on testicular Leydig cells stimulating testosterone production, which in turn regulates the release of GnRH and gonadotropin through negative feedback. A blockage at the level of the pituitary gland or higher prevents the release of gonadotropins, resulting in reduced testosterone production, a condition called hypogonadotropic hypogonadism. On the other hand, primary testicular insufficiency is accompanied by reduced testosterone production, resulting in reduced feedback on pituitary and hypothalamus and, consequently, increased gonadotropin release. This condition is called hypergonadotropic hypogonadism.

Guidance of the GnRH neuron migration process from the olfactory placode is controlled by several molecular signals. Among them, anosmin is a well-known adhesion molecule encoded by anosmin 1 (ANOS1) gene (formerly known as Kallmann syndrome 1 [KAL1]), which is located on the Xp22.3 region. Anosmin regulates olfactory and GnRH migration pathway by interacting with fibroblast growth factor receptor 1 (FGFR1). Loss-of-function mutations in ANOS1 lead to Kallmann syndrome (KS), which is a combination of hypogonadotropic hypogonadism (HH) and deficient sense of smell (anosmia).1

Notably, KS is very heterogeneous in its genetic origin. X-linked forms depend on mutation of ANOS1, of which more than 150 variants have been described, but it is estimated that only about 10% of individuals with KS have a pathogenic variant of ANOS1.1,4 In this regard, mutations in the FGFR1 gene (formerly Kallmann syndrome 2 [KAL2]) are another important cause of KS and are associated with autosomal dominant inheritance. Interestingly, FGFR1 mutations are associated with variable penetrance of GnRH deficiency (ranging from complete HH to partial puberty) and nonreproductive disorders, including encephalocraniocutaneous lipomatosis, Hartsfield syndrome, and osteoglophonic dysplasia.4

In addition to KS, there are genetic forms of HH in which, however, the sense of smell is normal, called normosmic HH. Normosmic HH is genetically heterogeneous as well, and two-thirds of cases involve the GNRHR (encoding for GnRH receptor) and TACR3 genes (encoding for the receptor for the tachykinin neurokinin 3 or neurokinin B [NKB]). Interestingly, in most cases of normosmic HH, GnRH production is normal, since reports of mutations in the GNRH1 gene (encoding the preprotein which after proteolytic splicing releases the active decapeptide GnRH) are extremely rare.5

Recently, the potential role of novel molecules, such as kisspeptin, in regulating hypothalamic GnRH release emerged. Kisspeptin, indeed, is a neuropeptide that binds to its receptor, KISS1 receptor (KISS1R; first known as G-protein-coupled receptor 54 [GPR45]), which is located in the hypothalamus, stimulating GnRH release.6 Furthermore, KISS1R is expressed by gonadotropic cells as well, so that kisspeptin can also directly stimulate the pituitary to release LH and, in a lower measure, FSH.1 Interestingly, NKB and dynorphin are two neuropeptides of which expression frequently overlaps with kisspeptin production in the arcuate nucleus. In recent years, a population of neurons cosecreting kisspeptin, NKB, and dynorphin (so-called kisspeptin, neurokinin B, and dynorphin [KNDy] neurons) has been identified as the generator of GnRH pulse in humans.7 For this purpose, as stated before, mutations in TACR3 gene, encoding for NKB, represent one of the main causes of normosmic HH.5

Regarding the action of gonadotropins at the testicular level, FSH and LH play a key supporting function toward spermatogenesis. The main target of FSH is Sertoli cells, which express the FSH receptor (FSHR) and produce fundamental regulators of spermatogenesis, such as inhibins1 and androgen-binding globulin (ABG).8 In contrast with initial reports suggesting that subjects with FSHR-inactivating mutations still retained potential fertility,9 more recent studies have shown that complete absence of FSH resulted in azoospermia.10,11,12 This was then explained by further investigations showing that mutated FSHR still retains a minimal degree of activity,13 whereas mutations in FSHβ gene lead to complete FSH deficiency resulting in blockage of the interaction between FSH and FSHR.10 Animal studies confirmed that FSH-FSHR binding is critical for spermatogonial and spermatocyte proliferation.8 However, surprisingly, knockout mice for FSHβ gene show less pronounced impairment of gonadal development compared with FSHR knockout mice, and both are found to be fertile.14 In clinical practice, isolated FSH deficiency is a rare condition (real prevalence is unknown, but literature is limited to a few case reports) characterized by normal testosterone levels and azoospermia that could be reversed by administration of exogenous FSH.15 Accordingly, a recent case report described a man with FSH deficiency who was totally asymptomatic except for azoospermia and whose fertility was restored by administration of human menopausal gonadotropin (hMG).16

Regarding LH, it stimulates testosterone production by Leydig cells, which in turn exerts a paracrine action on Sertoli cells.1 Testosterone, indeed, is needed for the masculinization of internal and external genital tract and exerts fundamental effects on spermatogenesis. It binds to the androgen receptor (AR), which is expressed by Sertoli cells, and intratesticular testosterone concentration has been shown to reach 50–100 times the circulating testosterone levels.8 Exogenous testosterone administration leads to dose-dependent depression of HPG axis by negative feedback on the hypothalamus and the pituitary, with suppression of GnRH and consequently gonadotropin release. For this reason, testosterone replacement therapy (TRT) may restore circulating testosterone levels and correct sexual symptoms in men with HH, but it is not effective in stimulating spermatogenesis.17 For this reason, exogenous GnRH or gonadotropin treatment represents a rational choice in the treatment of men with HH with desire for fatherhood. Since the measurement of intratesticular testosterone concentration requires testicular aspiration that would be invasive and impractical, surrogate serum markers of Leydig cell activity such as insulin-like factor 3 (INSL3) and 17-hydroxyprogesterone (17-OH-Pg) have been proposed, but larger studies are needed before their introduction into clinical practice.8 Additional hormone markers, such as anti-Müllerian hormone (AMH) and inhibin B, may provide noninvasive evaluation of the spermatogenesis status. They are both secreted by immature Sertoli cells upon FSH stimulation, and AMH is downregulated by testosterone, so that AMH is high during early puberty, but normally decreases as androgen concentrations rise.18,19 A critical time for the maturation of the male gonad in the postnatal period is minipuberty. Although poorly evident on a clinical level, in fact, during the first few months of life, the HPG axis goes through a phase of momentary activation, which leads to an increase in the number of Sertoli cells and their maturation, evidenced by increased circulating levels of AMH.3 It is hypothesized that the failure of minipuberty may account for the different reproductive prognosis of congenital or acquired forms of HH.

HYPOGONADOTROPIC HYPOGONADISM

Epidemiology

HH is a clinical syndrome in which gonadal failure is related to deficient GnRH or gonadotropin secretion,20 and it is also referred to as secondary (or central) hypogonadism. On the other hand, the forms of gonadal insufficiency in which the testis is primarily involved are called primary hypogonadism (or hypergonadotropic hypogonadism).21 HH is a rare cause of infertility, being involved in only 1%–2% of cases of male infertility22 and in 5% of nonobstructive azoospermia (NOA) cases.23 It is estimated that the prevalence of congenital HH is around 1:4000–1:15 000 in general population, with a marked male preponderance (approximately 4:1),20 whereas the incidence of acquired forms of HH ranges widely according to their causes (e.g., pituitary tumors/surgery and brain trauma), reaching 4:1000 patient-years.24 Notably, severity of semen impairment strictly depends on the age of onset of HH. Subjects with complete testosterone deficiency and prepubertal-onset hypogonadism (such as those with congenital HH), indeed, will not spontaneously undergo testicular growth and sperm production, with consequent NOA in all cases. On the opposite, most patients with adult-onset testosterone deficiency due to pituitary disease show normal semen quality, with only a slight decrease in progressively motile spermatozoa compared to healthy men, suggesting that spermatogenesis may be more robust than Leydig cell function.25 Nevertheless, over time, persistent low levels of gonadotropins may lead to testicular atrophy and NOA.26

Notably, the knowledge of pathogenesis of HH-related infertility is crucial for the clinical andrologists, as it is one of the few causes of male infertility that may be corrected by medical treatment.

Causes

From an etiological point of view, an initial distinction between the forms of HH can be made between congenital and acquired causes (Table 1), which has important clinical implications as well. Congenital HH (also referred to as “idiopathic” HH) can be classified into two main groups: impaired GnRH secretion and abnormal sense of smell (KS), and normosmic HH (in which secretion or action of GnRH is impaired, but sense of smell is unaffected).20 They are both genetically heterogeneous, with approximately 50 genes identified so far, 20 of which are associated with KS, whereas 50% of all cases remain unexplained. The first and most frequent mutations identified in patients with KS are in the ANOS1 and FGFR1 genes, followed by mutations in fibroblast growth factor 8 (FGF8), chromodomain-helicase-DNA-binding protein 7 (CHD7), prokineticin 1/prokineticin 2 (PROK1/PROK2), and GNRHR genes.27 On the other hand, the genetic causes of normosmic HH should be sought in the genes that regulate GnRH secretion (KISS1, KISS1R, TAC3, TAC3R, and GNRH1) or action (nuclear receptor subfamily 0 group B member 1 [NROB1], formerly known as dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome 1 [DAX1], and GNRHR). In addition, HH may occur as a part of multiple pituitary hormone deficiency, which is the result of defective development of pituitary gland, with combined hormone deficiencies that may develop over a variable period of time during life.20 Notably, adult-onset male idiopathic HH, which is characterized by normal puberty but subsequent GnRH deficiency, also exists, albeit being very rare.28

Table 1.

Causes of hypogonadotropic hypogonadism

Type of HH	Cause
Congenital HH	Isolated GnRH deficiency
	Kallmann syndrome
	Normosmic HH
	Congenital hypopituitarism
	Genetic syndromes associated with HH
	Laurence–Moon–Biedl
	Bardet–Biedl
	Prader–Willi
Acquired HH	Organic causes
	Pituitary/hypothalamic diseases
	Tumors
	Trauma
	Infiltrative diseases
	Inflammatory diseases
	Immunotherapy
	Functional causes
	Aging
	Medications
	Obesity
	Systemic diseases
	Anabolic androgenic steroids abuse

GnRH: gonadotropin-releasing hormone; HH: hypogonadotropic hypogonadism

On the other hand, most cases of acquired HH occur in the adulthood, when the body has been subjected to adequate levels of androgens during fetal and pubertal development, but children may be affected by acquired forms of HH as well (e.g., hypopituitarism due to craniopharyngiomas). In adult males, HPG axis suppression may derive from different causes that can be distinguished into organic and functional. This distinction is crucial from a clinical point of view, because in the case of functional hypogonadism, the HPG axis is structurally intact, and removal of the cause of the blockage may fully resolve the hypogonadism.29 In functional hypogonadism, aging, medications, excessive adiposity, and comorbid illness may concur to impair testicular function. Accordingly, lifestyle changes are the preferred choice of treatment, since weight loss, diet, and physical exercise are demonstrated to be beneficial on both testosterone levels and hypogonadal symptoms.30 Accordingly, the UK guidelines from the Society for Endocrinology suggest the use of exogenous testosterone for treatment of hypogonadal symptoms in men with functional hypogonadism only if resolution of hypogonadism cannot be anticipated within a reasonable timeframe.31 Similarly, the European guidelines from the European Academy of Andrology (EAA) recommend lifestyle changes in overweight and obese men with functional hypogonadism, and withdrawal/modification of drugs potentially interfering with testosterone production, when clinically permissible.32 Notably, it should be taken into account that drugs may impair testicular function by inducing functional blockage or by creating organic damage on the testis and that sometimes different causes of hypogonadism can coexist in the patients with different comorbidities. For example, a subject undergoing chemotherapy for a neoplastic disease might present at the same time with drug-induced testicular damage and HPG axis suppression related to deteriorating general condition (e.g., neoplastic cachexia) and concomitant medication (e.g., high-dose steroids), with variable gonadotropin levels. This would make the differential diagnosis between the components of hypogonadism challenging. Medications or drugs that result in exclusively (or almost exclusively) functional suppression include opioids and narcotics, high-dose glucocorticoids, androgen deprivation therapy, antidopaminergic antipsychotics, cannabinoids, and estrogens.31 Of note, anabolic-androgenic steroids (AAS) determine a blockade of GnRH and gonadotropin release by feedback that is potentially reversible with discontinuation of abuse. By the way, the results of a recent meta-analysis suggest that, despite a trend toward normalization of pituitary function, circulating testosterone levels remain lower than baseline up to 16 weeks after discontinuation, with long-term effects of testicular atrophy and altered seminal quality.33

Organic causes of HH, on the other hand, include pituitary and hypothalamic tumors, trauma, surgery, and granulomatous diseases involving the pituitary, stalk section, and infiltrative diseases (sarcoidosis, histiocytosis, and iron overload/hemochromatosis).29,31 Among parasellar tumors, prolactinomas are the most frequently encountered in adult subjects, followed by Rathke’s cleft cysts, gliomas, and germinomas, whereas craniopharyngiomas typically occur during childhood.31 Prolactin-secretin adenomas, together with nonfunctioning pituitary adenomas, represent the most frequently encountered pituitary adenomas.34 Notably, macroprolactinomas could lead to both functional and organic HH, since hyperprolactinemia decreases GnRH pulsatility, leading to decreased gonadotropin secretion, and compression of the gonadotropic cells of the pituitary gland by the mass effect of the adenoma can impair the production and secretion of FSH and LH. Similarly, large adrenocorticotropic hormone (ACTH)- and growth hormone (GH)-secreting tumors may show a similar behavior in patients with Cushing’s disease and acromegaly, respectively.35 Moreover, recent advances in immunocytochemistry and molecular techniques demonstrated that 80%–90% of clinically silent pituitary adenomas express intact gonadotropins or their subunits, which could disrupt the normal pulsatility of LH and FSH, leading to testosterone deficiency and impaired spermatogenesis.34

Another frequent cause of acquired pituitary gland dysfunction is traumatic brain injury (TBI), with the prevalence of anterior hypopituitarism ranging from 16.8% to 35.3% after mild-to-severe trauma36 and a predicted prevalence of post-TBI hypogonadism of 16% in long-term survivors.37

Finally, hypophysitis is an emerging cause of hypopituitarism that should be kept in mind. Despite previous reports estimating a prevalence of 1 in 9 million individuals, recent studies suggest an increasing incidence in the last few years.38,39 In addition, a novel classification of different hypophysitis types has been proposed as follows: lymphocytic hypophysitis (2/3 of cases), granulomatous hypophysitis (20% of cases), histiocytosis, immunoglobulin G4 (IgG4)-related hypophysitis, and immunotherapy-related hypophysitis. In particular, the latter must be taken into account in subjects with cancer pathology treated with the new immunotherapy drugs directed against the cytotoxic T-lymphocyte protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed death ligand 1 (PD-L1), such as ipilimumab, pembrolizumab, and nivolumab.40

Therefore, symptoms suggestive of intracranial masses (e.g., headache and visual impairment), signs of hypophyseal hormone overproduction, history of previous head trauma, and information about medications or drug abuse should be accurately sought to define the cause of hypogonadism.

Clinical features of congenital HH

Signs and symptoms of hypogonadism rely on the age of onset, the severity, and the underlying cause of the testosterone deficiency.41 In congenital hypogonadism, prenatal or prepubertal onset leads to delayed/incomplete sexual development, with failure of puberty being the most obvious clinical sign, but not the earliest. Indeed, cryptorchidism and micropenis may be observed at birth and they should rise suspicion for congenital hypogonadism. In such cases, measurement of gonadotropin and testosterone levels during the first weeks of life could be informative about Leydig cell activity, since physiological peak levels of LH, testosterone, and INSL3 are reached during the 3^rd month. After that, they begin to decline to be undetectable by the 6^th month of life. On the other hand, AMH and inhibin B progressively rise and remain stable during childhood, allowing the functional status of Sertoli cells to be assessed at later stages.41

Delayed puberty often represents the first and main clinical manifestation of milder forms of congenital HH. Once excluded functional causes of hypogonadism such as chronic disease, medications, inadequate nutritional status, or psychosocial conditions, distinction from constitutional delay of growth and puberty (CDGP) represents the main challenge when gonadotropin levels are low. A family history of CDGP in parents or siblings, delayed bone age, and normal sense of smell suggests CDGP, with additional support provided by normal levels of inhibin B.41 However, in many cases, the clinical presentation overlaps between congenital HH and CDGP, and the “gold standard” to distinguish between these conditions is clinical observation until the age of 18 years. To identify children with congenital HH at an earlier age, different second-line investigations have been proposed, including GnRH/GnRH agonist stimulation test, but significant overlap between diagnostic thresholds exists. FSH-stimulated inhibin B concentrations (cut-off value <116 pmol l⁻¹) and kisspeptin-stimulated LH concentrations (cut-off value ≤0.4 mIU ml⁻¹) recently demonstrated promising results, but the need for larger studies to validate the thresholds, potential issues regarding availability, and costs of the tests limit the introduction of these investigations into current clinical practice.42

Clinical features of adult-onset HH

In men with acquired HH (and in the rare cases of adult-onset congenital HH), signs and symptoms of testosterone deficiency arise when puberty has already occurred, and the cause of hypogonadism could become apparent by investigating the medical history of the patient (i.e., brain trauma, infiltrative or metabolic disorders, and parasellar tumors). According to the latest international guidelines,31,43 diagnosis of male hypogonadism requires a combination of clinical features and low testosterone levels. Highly suggestive for testosterone deficiency are sexual (reduced libido and erectile dysfunction), reproductive (impaired sperm quality), vasomotor (hot flushes), hematological (low hematocrit), and skeletal (low bone mineral density with higher risk for frailty fractures) features.31 Decrease in testicular volume and glandular gynecomastia could be present as well.21 In addition, disturbances of mood and sleep, together with reduced muscle mass and increased body fat, are less specific but also described in men with hypogonadism.31

Regarding testosterone levels, testosterone concentrations in serum should be measured in the morning (between 7:00 a.m. and 11:00 a.m.), and at least in two different days, in the fasting state, because testosterone production exhibit significant diurnal and day-to-day variations and can be suppressed by food intake.41,43 Circulating total testosterone (TT) is 58% weakly bound to albumin and 40% tightly bound to SHBG, whereas only 0.5%–2.0% circulates free of binding to plasma proteins (free testosterone [FT]). Since testosterone can easily release from albumin, FT plus albumin-bound testosterone are also referred as “bioavailable testosterone”.21 Mass spectrometry is the gold standard of testosterone assays, but immunoassays are more suitable for clinical practice and provide acceptable results,41 with certification by an accuracy-based standardization or quality control program being advisable.43 In men with TT concentrations near the lower limit of the normal range (320 ng dl⁻¹ or 11 nmol l⁻¹), or in those with conditions associated with decreased (obesity, diabetes mellitus, glucocorticoids, nephrotic syndrome, hypothyroidism, and acromegaly) or increased SHBG concentrations (aging, human immunodeficiency virus [HIV] infection, chronic liver disease, hyperthyroidism, anticonvulsants, and estrogens), FT levels should be assessed. Notably, most of the commercially available immunoassays do not provide accurate results, and direct determination of FT levels should rely on equilibrium dialysis assays, but they do not yet find wide distribution. Alternatively, calculations using TT, SHBG, and albumin concentrations (such as the Vermeulen’s formula) may provide an accurate estimate of FT levels, with FT <64 pg ml⁻¹ (220 pmol l⁻¹) consistent with the diagnosis of hypogonadism.43 Differences between clinical presentations of congenital and acquired HH are shown in Figure 2 and Table 2.

Figure 2.

Clinical features of acquired versus congenital hypogonadotropic hypogonadism.

Table 2.

Clinical presentations of congenital and acquired hypogonadism

Clinical features	Prepubertal-onset hypogonadism	Postpubertal-onset hypogonadism
Genitalia	Small testes (<4 ml) and microphallus	Normal or slightly decreased testicular volume
Secondary sexual characteristics	Scarce pubic and axillary hair, no chest hair	Normally distributed but sparsely present body hair
Body proportions	Enucoid habitus	Regular body proportions
Body composition	Reduced male musculature	Increased body fat and decreased muscle mass
Breast tissue	Gynecomastia	Gynecomastia
Larynx	High-pitched voice	Normal voice
Sexual function	No sexual drives	Low libido and erectile dysfunction

Treatment

Treatment of NOA in men with HH requires replacement of gonadotropins, which can be obtained directly by the administration of FSH and LH or indirectly with GnRH. Notably, prepubertal-onset hypogonadism requires both FSH and LH, whereas in postpubertal hypogonadism, the administration of LH alone could be sufficient. On the other hand, GnRH could represent the most physiological way of stimulating the HPG axis, with pulsatile administration of 5–20 µg every 2 h delivered via an infusion pump, but intact pituitary function is required.22

In subjects with congenital HH, gonadotropin or TRT may be used to similarly induce the development of secondary sexual characteristics and promote the progression of puberty, but gonadotropins are required to initiate and maintain spermatogenesis. Several formulations of gonadotropins and different sperm-induction regimens are currently available. Human chorionic gonadotropin (hCG), purified from the urine of pregnant women, is generally used to stimulate Leydig cells as a surrogate of LH. Interestingly, in vitro models show that hCG and LH activate differently signal transduction by the same receptor; however, this results in an equal activity on testosterone production in vivo.34 Recombinant hCG (rhCG) is also available, but the experience for the treatment of male infertility is limited.44 hCG is usually dosed at 1000–3000 IU for 2–3 times weekly to achieve eugonadal status.22 Regarding FSH, menotropin (or human menopausal gonadotropin [hMG]), urinary FSH, and recombinant human FSH (rhFSH) are all available and can be used for induction of spermatogenesis.45 No evident difference in terms of successful achievement of spermatogenesis for different FSH preparations exists.44

For both gonadotropin and GnRH replacement therapy, a success rate of about 75% is expected after variable time span of treatment,44 which reaches 88% after 18–24 months of treatment according to a recent report.46 Depending on the severity of the hypogonadism and the time of the onset, different regimens may be chosen as follows.

Firstly, monotherapy with hCG is the regimen of choice for men with adult-onset acquired HH who underwent regular pubertal development. hCG is typically initiated at 3000–5000 IU per week (in 2 or 3 injections) and gradually titrated to reach optimal levels of serum testosterone every 4–6 weeks. Common side effects include erythrocytosis and gynecomastia. As testes enlarge during treatment, optimal sperm count is expected to be achieved in about 6 months. If sperm count remains low, FSH may be added to maximalize spermatogenesis.20 This is supported by the results of a recent meta-analysis which demonstrated that combination of hCG and FSH is more effective than hCG alone in terms of cumulative sperm concentration achieved.44

Secondly, combined gonadotropin treatment (hCG plus FSH) is recommended in subjects with prepubertal-onset HH. Classically, FSH is administered at the starting dose of 75 IU for 3 times per week, with titration up to 300 IU for 3 times per week to achieve serum FSH of 4–8 IU l⁻¹. In normal pubertal development, Sertoli cells and seminiferous tubules proliferate under the stimulation of FSH, and when intratesticular testosterone starts to increase, they undergo terminal differentiation. Therefore, FSH-priming prior to combination with hCG (sequential gonadotropin therapy) could prevent premature differentiation of Sertoli and germ cells, leading to better results in terms of sperm quality.20 For this reason, sequential treatment approach could be preferable for men with prepubertal testes (volume <4 ml), and some authors suggested that it may be the treatment of choice for those men with a history of maldescended testes.47 Since this regimen is safe, well tolerated, and free from significant side effects, it represents a promising option that would deserve further investigation. On the opposite, hCG monotherapy could induce detrimental effects on spermatogenesis, leading to premature maturation and consequent depauperation of the germ cell pool, and should be avoided in subjects with prepubertal-onset HH.

In a recent meta-analysis, Rastrelli et al.44 investigated the effects of gonadotropin-replacement therapy on spermatogenesis in men with HH. Sixty studies were retrieved, with 44 for gonadotropin and 16 for GnRH therapy, and sperm appearance (presence with at least one spermatozoon in semen) and sperm concentration were considered main outcomes. No significant differences emerged between the two different groups, with an overall success rate of 75% (95% confidence interval [CI]: 69%–81%) and 75% (95% CI: 60%–85%), and a mean sperm concentration after treatment of 5.92 × 10⁶ (95% CI 4.72 × 10⁶–7.13 × 10⁶) ml⁻¹ and 4.27 × 10⁶ (95% CI: 1.80 × 10⁶–6.74 × 10⁶) ml⁻¹ for gonadotropin and GnRH therapy, respectively. No difference was observed among different FSH formulations, but higher success rate was found for subjects using both hCG and FSH rather than hCG alone. Notably, only observational studies were included. This is because randomized placebo-controlled trials would require the administration of a placebo to patients with HH who desire paternity, representing an obvious ethical dilemma. By the way, no major publication bias emerged, and gonadotropin-replacement therapy demonstrated a high success rate in men with NOA due to HH.

Prognosis

As stated before, gonadotropin-replacement treatment could lead to appearance of at least one spermatozoon in the ejaculated in about 75% of treated patients. In addition, among responders, even if sperm concentration remains far below normal (5.92 × 10⁶ ml⁻¹ and 4.27 × 10⁶ ml⁻¹ for gonadotropin and GnRH therapy, respectively),44 paternity may be spontaneously obtained despite very low sperm concentration (even <1 × 10⁶ ml⁻¹).48 Although this is a good result, one out of four patients treated remains azoospermic despite adequate medical treatment.

According to distinct clinical, biochemical, and genetic features, Sykiotis et al.49 described three different groups of nonresponders to gonadotropin treatment.

(1) Group 1 included subjects with GnRH deficiency, pituitary resistance, and testicular failure (“triple defect”). This subset of patients presents with minimal but suboptimal gonadotropin response to GnRH administration (as for hypothalamic defect). In those subjects, LH levels remain inappropriately low despite very high doses of GnRH (up to 800 ng kg⁻¹ per pulse). In addition, after normalization of FSH and LH levels, they show persistent low testosterone levels (as for pituitary defect), and azoospermia (as for testicular defect). These patients may show high genetic and phenotypic variability, being either affected by KS or idiopathic HH

(2) Group 2 included subjects with GnRH deficiency and testicular resistance. In these patients, GnRH response stimulates testosterone production, but supraphysiological levels of LH and FSH are required to normalize testosterone levels, indicating that responsiveness of Leydig cells is partially compromised. A high prevalence of KS (90%) was observed in this group of patients

(3) Group 3 included subjects with GnRH deficiency and azoospermia. In this group, patients remain azoospermic despite normalization of testosterone, FSH, LH, and inhibin B levels, even after prolonged GnRH administration (up to 10 years). High genetic heterogeneity is usually observed in these patients.

Recently, larger testicular size at baseline was reported as a good predictor of spontaneous pregnancy after gonadotropin therapy, with further testicular volume increase during treatment being suggestive for successful spermatogenesis. On the opposite, undescended testes are predictive of poor response to gonadotropin therapy.47,50 Moreover, Rastrelli et al.44 observed that gonadotropins are more effective in patients with postpubertal-onset hypogonadism. This may be related to the absence of the physiological minipuberty that appears to be crucial for the proliferation of immature Sertoli cells. Interestingly, Sertoli and germ cells do not express androgen receptors until the age of 5 years, so their premature differentiation induced by intratesticular testosterone is prevented in the infancy.20 For this reason, postponing gonadotropin in the adult life could act outside the optimal therapeutic window, leading to suboptimal results.

Notably, Rastrelli et al.44 also reported that previous TRT does not affect the success rate of gonadotropin treatment. This could be related to the fact that exogenous testosterone does not raise intratesticular testosterone concentration, so that immature Sertoli and germ cells do not undergo premature maturation. For this reason, the use of exogenous testosterone to revert hypogonadal symptoms during FSH pretreatment in sequential gonadotropin therapy has been suggested,51 but data supporting the safety of this approach are lacking.

Nevertheless, men who exhibit persistent azoospermia may have a good chance to obtain spermatozoa from the testis using microdissection testicular sperm extraction (micro-TESE), as recently observed by Chen et al.,52 who reported a high sperm retrieval rate (90%) in a small sample of men who had previously undergone an average 12.1 months of gonadotropin treatment. Hence, for men who do not respond sufficiently to medical therapy, assisted reproductive technology (ART) with sperm retrieval procedures should be recommended after a period of at least 6 months, with increased testicular volumes and normalization of hormones to be considered treatment endpoints prior to retrieval.22

CLINICAL CASES FROM DIAGNOSIS TO TREATMENT

Some emblematic clinical cases related to the treated conditions are described below. Patient data were selected from electronic records related to subjects who came to the outpatient clinics of the Endocrinology Clinic in Ancona, Italy, between 2019 and 2024. All patients provided written informed consent to publish their data anonymously. Approval from the ethics committee was waived as a policy of our institution for the publication of case reports.

Congenital HH

A 21-year-old male, with a well-known history of congenital HH, presented for fertility evaluation. Diagnosis was made at puberty, when he underwent clinical examination due to the absence of signs of pubertal development until the age of 16 years. At that time, gonadotropin and testosterone levels were very low, but GnRH and hCG stimulation tests showed normal response, suggesting intact pituitary and testicular function. The patient had a previous history of unilateral cryptorchidism surgically corrected when he was 2 years old. He reported normal sense of smell that was objectively confirmed by evaluation using a commercial tool (Sniffin’ Sticks). Genetic testing failed to show mutations of definite pathological significance in the genes most commonly involved in HH. A magnetic resonance imaging (MRI) of the brain confirmed normal pituitary and olfactory bulbs. According to patient’s preferences (who refused daily subcutaneous gonadotropin injections or a GnRH pump), TRT was initiated with a short course of transdermal testosterone followed by injectable testosterone (testosterone propionate, 100 mg monthly, progressively increased to 250 mg every 2–3 weeks). Recently, upon the request of his girlfriend, a 20-year-old female with regular ovulatory cycles, the patient underwent semen analysis that showed azoospermia, prompting the couple to seek for fertility counseling. Medical examination of the patient showed prepubertal scrotal testes (volume <4 ml bilaterally), and blood tests taken 4 weeks after testosterone withdrawal showed undetectable serum gonadotropins (LH <0.5 IU l⁻¹ and FSH <0.4 IU l⁻¹) and very low testosterone concentrations (0.8 nmol l⁻¹). Given his history of maldescended testis and low testicular volume, pretreatment with FSH therapy (rhFSH 150 IU for 3 days a week) was initiated, followed by hCG 1000 IU for 3 times a week after 2 months, gradually increased to 1500 IU for 3 times a week. After 6 months of combined gonadotropin treatment, repeated sperm analysis showed the appearance of spermatozoa in the semen, with sperm concentration of 2 × 10⁶ ml⁻¹. Given the young age of the couple, it was suggested to continue with the current therapy and regular, unprotected intercourse for at least 12 months before considering ART.

Acquired HH

A 33-year-old male presented with secondary male infertility. He already had a 4-year-old child from a previous relationship, but after almost 18 months of unprotected intercourse with his 28-year-old current partner, no pregnancy occurred. He also complained of low libido and decreasing erectile function and semen volume. Semen analysis showed azoospermia, whereas blood tests revealed undetectable serum LH levels (<0.5 IU l⁻¹) and very low FSH levels (1.5 IU l⁻¹) and testosterone concentrations (6.3 nmol l⁻¹). No previous signs suggestive for prepubertal-onset hypogonadism emerged from his medical history. In particular, testicles were in the scrotal position at birth, and secondary sexual characteristics were normal. Notably, testicular volume was slightly decreased (10 ml bilaterally), and the patients reported that he noted a progressive shrinking. A slight increase in serum prolactin levels was reported. Then, he underwent MRI of the brain that showed pituitary stalk enlargement in T1-weighted images, with uniform contrast enhancement, raising the suspicion of hypophysitis. Blood tests performed at completion documented intact remaining pituitary function. Conservative management was chosen, and no corticosteroids were administered. hCG was then prescribed at the doses of 1000 IU for 3 times a week, with additional FSH therapy (rhFSH 150 IU for 3 times a week) after 4 months due to persistent azoospermia despite normal testosterone levels. After 4 months further, a few spermatozoa were observed in the ejaculated, and the couple underwent ART.

AUTHOR CONTRIBUTIONS

GS conducted literature search and drafted the manuscript. GB and AK contributed to critical review and revision of the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.