Abstract
The appearance of Leydig cells within the testes of a 46XY fetus from 7–10 weeks gestation marks the onset of testosterone (T) secretion stimulated by placental gonadotrophin (hCG). T diverts development from female default towards external male phenotype. The testes then begin their hormonally induced descent, while Sertoli cell–derived anti-mullerian hormone (AMH) induces regression of internal female reproductive organs.
1 Developmental Physiology of the Male Reproductive Axis
1.1 Embryology and Developmental Physiology
The appearance of Leydig cells within the testes of a 46XY fetus from 7–10 weeks gestation marks the onset of testosterone (T) secretion stimulated by placental gonadotrophin (hCG). T diverts development from female default towards external male phenotype. The testes then begin their hormonally induced descent, while Sertoli cell–derived anti-mullerian hormone (AMH) induces regression of internal female reproductive organs.
In the third trimester, a diffuse network of approximately 2,000 specialised neurons in the mediobasal hypothalamus begins secreting coordinated pulses of gonadotrophin-releasing hormone (GnRH). GnRH secretion is the central neuroendocrine regulator of reproduction, stimulating pituitary gonadotrophs to secrete luteinising- (LH) and follicle-stimulating hormones (FSH) (Figure 13.1). Gonadotrophins spur T secretion, causing penile growth and completion of testicular descent (1). GnRH-stimulated gonadotrophin secretion is critical for testicular development, T production and, in postnatal life, spermatogenesis (2).
Fig. 13.1 Simplified schematic depicting the hypothalamic-pituitary-gonadal (HPG) axis and its modulation by homeostatic and environmental cues.
(A) Hypothalamic GnRH neurons (in blue) receive multiple inputs including stimulation from the KNDy neurons that receive permissive signals from leptin secreted by white adipose tissue. Both internal and external stressors in the form of glucocorticoids, prolactin, inflammatory cytokines and opiates can inhibit GnRH secretion, principally via inhibitory inputs to hypothalamic KNDy neurons.
(B) Pulsatile GnRH secretion into the hypophyseal portal circulation stimulates gonadotrophs in the anterior pituitary to release LH and FSH.
(C) The gonadotrophins circulate peripherally, stimulating the testes to produce hormones (T), regulatory peptides (inhibin B) and gametes (sperm). The HPG axis functions via feed-forward and feedback loops. T (particularly its aromatised form E2) provides negative feedback on pituitary gonadotrophs and hypothalamic KNDy neurons. Inhibin B is a major regulator of FSH.
Stimulatory effects are depicted by (+) and green arrows and inhibitory effects by (–) and red arrows.
GnRH (gonadotrophin-releasing hormone); Kp (Kisspeptin); NKB (Neurokinin B); KNDy (Kp-NKB-dynorphin); WAT (white adipose tissue); LH (luteinising hormone); FSH (follicle stimulating hormone).
Around 80% of GnRH neurons originate within the embryonic olfactory placode, migrating during the first trimester to the hypothalamic arcuate nucleus along terminal-vomeronasal nerve fibres, with the rest having a neural crest origin (3). Developmental disorders disrupting the GnRH neural network result in congenital hypogonadotrophic hypogonadism (CHH), a rare condition characterised by absent or partial puberty and infertility (4).
1.2 Significance of Minipuberty in Boys
The hypothalamic-pituitary-gonadal (HPG) axis remains active for 4–6 months postnatally, when LH, FSH and T concentrations approach adult levels (5). This period of ‘minipuberty’ represents a key proliferative window for testicular germ cells and immature Sertoli cells although spermatogenesis is not initiated because Sertoli cells do not express the androgen receptor until the age of five (6).
Minipuberty is characteristically absent in congenital GnRH deficiency and combined pituitary hormone deficiency (CPHD) (Box 13.1), creating a brief diagnostic window-of-opportunity shortly after birth. For neonates with cryptorchidism ± micropenis, a single serum sample taken 4–8 weeks postnatally can pinpoint CHH more accurately than dynamic tests performed in later childhood or adolescence (4).
Healthy neonate males attain near adult levels of serum LH, FSH and T during minipuberty (4–8 weeks of life). Thus, male neonates exhibiting ‘red flag’ features prompting suspicion of CHH (e.g. cryptorchidism, micropenis, cleft lip/palate, hearing loss, absent erections on nappy-change, and/or family history of CHH) can be diagnosed by demonstrating low levels of LH, FSH and T.
Minipuberty is critical for testicular development and descent, and testicular maldescent can have far reaching negative consequences on future fertility potential. The high prevalence of cryptorchidism exhibited by CHH boys underscores the crucial role of minipuberty in the final stages of testicular descent and in anchoring the testes securely within the scrotum. Sexual (penile) development occurs much earlier in fetal development; thus, hypospadias is not seen in CHH.
Suppression of minipuberty in primates has been shown to impair subsequent testicular maturation at puberty.
Even with gonadotrophin replacement or GnRH pump therapy, men with severe GnRH deficiency (lacking minipuberty) typically do not achieve normal testicular volumes and semen quality.
1.3 Puberty and Spermatogenesis
The HPG axis is quiescent during childhood, although subtle testicular development continues. Pubertal-onset is marked by sleep-entrained GnRH pulses, with nocturnal low-frequency GnRH pulses initially favouring FSH secretion over LH. The resulting rises in serum gonadotrophin and T levels progressively extend to the waking hours, culminating in reproductive maturity. Normal spermatogenesis requires the coordinated actions of both FSH and endogenously secreted T on testicular germs cells and seminiferous tubules (Box 13.2).
Whilst T secretion is essential for spermatogenesis, exogenous T treatment is noted as a cause of low sperm count in medical textbooks, websites and product inserts, which can cause confusion.
Gonadotrophins are needed for spermatogenesis (FSH as well as LH to stimulate T secretion by the Leydig cells). T exerts central negative feedback, so when exogenous T treatment is started, endogenous LH and FSH are suppressed and, consequently, so is sperm production.
This adverse effect only arises with T (or anabolic steroid) use under the following circumstances:
In men who were eugonadal at baseline with intact spermatogenesis, e.g.
athletes or body-builders wishing to enhance their performance and/or appearance
normal men misdiagnosed with hypogonadism, e.g. following one-off non-fasting/post-prandial low serum T level, or as a result of a screening questionnaire without biochemical confirmation.
In hypogonadal men with partial gonadotrophin deficiency, e.g. pituitary tumour.
In the above circumstances, a rise in serum T with exogenous therapy causes inhibition of pituitary gonadotrophin secretion and, consequently, a paradoxical fall in intratesticular T levels with consequent loss of (any residual) spermatogenesis.
Broadly, LH stimulates interstitial Leydig cells to mature and secrete T. The resulting local micromolar concentrations of T exert a paracrine action on the seminiferous tubules to induce and maintain spermatogenesis in concert with FSH. T is also secreted into the testicular vein, whence it circulates at nanomolar concentrations to exert classic endocrine actions on body tissues. FSH is essential for development of the tubular compartment where spermatogenesis occurs, stimulating the proliferation of immature Sertoli cells, which secrete inhibin B and AMH and determine final seminiferous tubule length. With seminiferous tubules accounting for approximately 90% of testicular volume (TV), TV is thus a key indicator of fertility potential (6).
With the onset of puberty, rising intratesticular T ends the proliferative phase; Sertoli cells mature, sex cords lumenate to become tubules and spermatogenesis is initiated. Intratesticular T levels are around 30-times higher than serum concentrations, which is a paracrine requisite for spermatogenesis. Thus, FSH combined with hCG-induced T synthesis induces spermatogenesis in men with CHH, while FSH combined with exogenous T does not (7).
1.4 Disrupted GnRH Secretion/Action and Congenital Hypogonadotrophic Hypogonadism
Figure 13.1 illustrates physiological control of the adult male HPG axis, with the hypothalamus integrating internal-homeostatic and external-environmental signals to modulate secretion of GnRH and, consequently, gonadotrophin-mediated testicular function. The HPG axis is sexually dimorphic in three respects. First, the male axis lacks a sex steroid–mediated positive feedback pathway (via paraventricular kisspeptin neurons) on GnRH secretion. Second, obesity and corticosteroids both act to inhibit GnRH secretion, causing an HH-like biochemical picture, whereas in females they promote ovarian hyperandrogenism. Finally, the male axis appears more resistant to bioenergetic deficit that predisposes females to HH (‘hypothalamic amenorrhoea’).
Our understanding of these pathways has been greatly informed via clinical and genetic studies of patients with CHH, a rare disease affecting 1-in-4,000 men (4). The underlying gene defects comprise two broad clusters, namely (a) neuroendocrine regulation of GnRH secretion (resulting in a purely ‘reproductive’ phenotype) and (b) defective embryonic GnRH neuron migration/fate-specification, often associated with non-reproductive defects such as the absent sense of smell (anosmia) that defines Kallmann syndrome (KS) (8). HH is the only form of male infertility that is directly treatable with endocrine replacement therapy, without obligate use of assisted reproduction techniques (ART).
2 Differential Diagnosis, Classification and Fertility Prognosis of Male Hypogonadism
Male hypogonadism is typically associated with azoospermic infertility. The key distinction is between primary (hypergonadotrophic hypogonadism / primary testicular insufficiency) and secondary (hypogonadotrophic / central) hypogonadism (HH). (Box 13.3).
Box 13.3 Classification of male hypogonadism
Primary testicular insufficiency = Hypergonadotrophic hypogonadism = Intrinsic dysfunction of testis
↑ LH, ↑↑ FSH levels.
Apart from acute castration, it is typically progressive with a post-pubertal onset.
Spermatogenesis is typically lost before onset of overt hypogonadism/decline in T secretion.
Common causes: Klinefelter syndrome (47XXY), age-related, post-orchitis, external beam radiation therapy, chemotherapy with alkylating agents and, increasingly, castration for gender reassignment.
Fertility treatment: sperm/tissue storage, micro-testicular sperm extraction (mTESE) with intracytoplasmic sperm injection (ICSI), donor sperm.
Secondary / Central: Hypogonadotrophic hypogonadism = Deficient gonadotrophin stimulation
↓ LH and FSH levels, or ‘inappropriately normal’ or low serum T.
Onset: Congenital forms present as pubertal failure; acquired forms (post-puberty) have variable presentations.
Fertility: potentially restorable with gonadotrophin replacement (or pulsatile GnRH via pump unless there is primary hypopituitarism).
2.1 Hypergonadotrophic Hypogonadism
This is diagnosed biochemically with elevated gonadotrophins (LH and FSH) in the setting of low serum T levels and is progressive, irreversible and usually unresponsive to pharmacologic (hormonal) fertility treatment. Sperm-retrieval can be considered if identified early (e.g. adolescents with Klinefelter syndrome) (9) and prospective transwomen should consider sperm cryopreservation.
2.2 Hypogonadotrophic Hypogonadism (HH)
Beware of patients labelled as ‘secondary hypogonadism’ or ‘HH’ with inadequate characterisation; an identical biochemical profile (low T with low-normal, gonadotrophins) is also observed in eugonadal men under certain conditions (Box 13.3). The diagnosis of HH should be based on fasting venepuncture 8–10 am in an otherwise healthy (no systemic diseases), well-rested man (not during phase of night shift-work) (10) – also ideal for screening-out broader hypopituitarism and metabolic syndrome. In CHH, pituitary function is otherwise normal and other relevant conditions are excluded (4) (Box 13.4).
Box 13.4 Important causes and rule-outs of HH
I. Apparent / artefactual HH
Physiological suppression of gonadotrophic axis due to chronic illness.
Inappropriate timing of measurement: afternoon clinic, or postprandial venepuncture.
Low level of sex hormone binding globulin (SHBG), such that calculated free testosterone men is normal range even with low total testosterone.
Inaccurate measurement: low-quality T immunoassay.
II. Organic diseases causing HH
Structural lesions of hypothamalus or pituitary gland.
Gonadotroph failure secondary to iron overload, e.g. genetic haemochromatosis.
GI malabsorption, eg. Coeliac disease, inflammatory bowel disease.
III. Functional / Reversible causes of HH
HH secondary to opiate use/ abuse.
HH secondary to post-anabolic steroid abuse
HH secondary to hyperprolactinaemia (tumour- or drug-induced).
Stress-induced HH related to high stress / low body mass index (BMI) / excessive exercise / and/or underlying eating disorder.
HH secondary glucocorticoid excess: exogenous or endogenous.
Further to correct interpretation of biochemistry, clinical features may frame the diagnosis within a defined medical syndrome, e.g. Kallmann’s (CHH + anosmia); adult-onset HH with structural pituitary disease (e.g. visual field disturbance); functional/reversible HH secondary to opiate intake, or anti-psychotic-induced hyperprolactinaemia (Box 13.4).
For a man started inappropriately on T following incorrect diagnosis of hypogonadism (e.g. as a result of ‘screening questionnaire’, afternoon non-fasted venepuncture, or in the context of chronic illness, stopping T will eventually permit reversion to previous fertility. Although clinical trial data are lacking, emerging data suggest possible benefits of oestrogen-antagonists (e.g. clomiphene citrate) and aromatase inhibitors (e.g. anastrazole) in improving sperm quality by boosting gonadotrophin secretion in men with functional HH, e.g. due to opiates, obesity or other chronic disease process. Organic HH is treatable with exogenous gonadotrophin therapy, although extended treatment periods (two years or longer) may be required for men with congenital disease (CHH). Whereas females achieve their lifetime supply of oocytes in utero, males require three distinct phases of testicular maturation to develop and sustain spermatogenesis, comprising the effects of placental hCG in utero, and of pituitary gonadotrophins during perinatal minipuberty and adolescent puberty.
2.3 CHH: Reproductive Phenotype, Genetic Basis and Relationship to Cryptorchidism
CHH is clinically and genetically heterogeneous and the degree of GnRH deficiency influences the clinical presentation. Males typically present in adolescence or early adulthood with impairment of puberty (4) and undeveloped testes, but the most severe cases exhibit neonatal cryptorchidism ± micropenis, or absent pubertal development (TV ≤ 4 mL) in adolescence. Around one-third have partial GnRH deficiency, evidenced by some spontaneous testicular development (TV > 4 mL) (4).
Most cases are sporadic, in keeping with impairment of fertility, although a third display familial inheritance, comprising autosomal dominant, autosomal recessive, X-linked recessive and oligogenic forms (8). Some 30 loci have been identified to date, with mutations acting alone or in synergy, but still only accounting for around 50% of cases. For the majority of CHH-associated genes, full disease penetrance probably occurs only with homozygous, hemizygous or compound heterozygous mutations (4). Clinical phenotypes may sign-post molecular defects: mirror movements (synkinesia) or renal agenesis suggest KAL1; clefting and skeletal defects, including dental agenesis, suggest FGFR1, FGF8 or the FGF8 synexpression group, while deafness suggests CHD7, IL17RD or SOX10 mutations. Notably, men with KAL1 mutations (approximately 10% of male KS) respond least well to fertility treatment, even accounting for their higher prevalence of cryptorchidism (4).
Sons fathered by KAL1 males are unaffected and daughters are carriers, but given the sometimes complex genetic architecture of CHH the potential for transmission to offspring is usually less predictable. Men usually welcome the opportunity to have a family despite the risk of transmission, being confident they could ensure their child was diagnosed promptly and treated at an appropriate age, but genetic counselling should be offered to couples pursuing fertility treatment (4).
Numerous studies have highlighted the high prevalence of cryptorchidism among CHH men with TV ≤ 4 mL (i.e. severe GnRH deficiency) (11). Cryptorchidism is a key adverse prognostic factor for fertility in CHH, especially if bilateral, but is not per se diagnostic of CHH as it affects 2–5% of full-term neonates and resolves spontaneously in approximately 75%, typically within the first six months of life (12, 13). However, when testes remain undescended by one year, the impact on germ cell survival and long-term fertility can be dramatic, particularly with bilateral disease and higher-lying testes. Men with a history of bilateral cryptorchidism have lower serum inhibin B levels, smaller TV, lower sperm counts (14) and are six times more likely to be infertile compared to men with unilateral disease or normally descended testes (15).
Outcomes in boys who undergo orchidopexy later strongly favour earlier intervention and currently recommendations for timing is between 6 and 12 months of age (12, 13). However, for CHH infants lacking mini-puberty, combined gonadotrophin treatment to augment TV prior to orchidepexy would likely render surgery technically easier, or possibly even unnecessary, apart from having potential benefits on testicular and penile maturation (16).
3 Spermatogenesis Induction Protocols in Hypogonadotrophic Hypogonadism
HH men are typically azoospermic, but fall broadly into three prognostic categories in respect of predicted response to therapy. At the mildest end of the spectrum are men with adult-onset HH (e.g. secondary to pituitary adenoma), with the most unfavourable being those with severe CHH (or CPHD) and history of bilateral cryptorchidism. In between are the approximately one-third of CHH men with some spontaneous partial puberty at presentation (TV > 4 mL) (17).
Approaches to stimulate gonadal development and fertility include pulsatile GnRH therapy or subcutaneous gonadotrophins injections (4, 11). Although both approaches are equally effective in inducing spermatogenesis in the majority of CHH men (18), gonadotrophins are more readily available due to their obligate use in female superovulation protocols for ART and, importantly, are also effective in primary pituitary disease.
3.1 Predictors of Success for Fertility-Inducing Treatment in Men with CHH
Careful assessment of TV is key to estimating the pretreatment probability of successful spermatogenesis and of subsequent pregnancy (See Box 13.5) (4, 11, 17, 19). CHH men with absent pubertal development (baseline TV ≤ 4 mL) have impaired proliferation of immature Sertoli cells and germ cells – reflected biochemically in low serum inhibin B levels – and thus consistently poorer fertility outcomes (17). Optimal treatment for CHH during adolescence remains to be defined (i.e. gonadotrophins versus T) and prospective studies are needed, but exogenous androgen exposure is no longer believed to reduce the subsequent likelihood of attaining sperm thresholds for conception (18). However, high intratesticular T levels achieved during hCG-monotherapy can induce terminal differentiation of limited pre-proliferative Sertoli and germ cell populations.
Box 13.5 Predicting the success of fertility treatments in HH
| Positive predictors | Negative predictors |
|---|---|
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