Keywords

Introduction

Male infertility (and hypogonadism) may be caused by a genetic defect, which should be investigated by cytogenetic or molecular genetic analysis. The two most severe clinical phenotypes in infertile males, which are identifiable by semen analysis, are oligo-(astheno-terato-)zoospermia (OAT) and azoospermia. While OAT describes reduced sperm number, altered morphology, and reduced motility, azoospermia is defined as the absence of spermatozoa in the ejaculate. Azoospermia is identified in ~15% of infertile men and can be classified into obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). OA is mainly caused by the physical blockage of the male excurrent ductal system and affected patients display normal gonadotropin and androgen levels, and normal spermatogenesis, because neither the endocrine system nor spermatogenesis is affected. OAT and NOA, which comprise the majority of azoospermic men (>80%), are frequent symptoms of primary testicular failure with elevated LH, FSH levels, and small testes and rarely of secondary testicular failure (Congenital Hypogonadotropic Hypogonadism (CHH)) with decreased LH and FSH and small testes. In contrast to OA, which may be caused by urogenital infections or mutations in the CFTR gene, the etiology of OAT and NOA remains largely unclear.

Currently, a specific genetic cause can be demonstrated in only about 4% of unselected infertile men, while this rate increases to about 20% in patients presenting with azoospermia (Table 1). Among the best known and most frequent genetic causes of male infertility are structural and numerical chromosomal aberrations (e.g., Klinefelter syndrome), microdeletions of the AZF (“AZoospermia Factor”) regions on the long arm of the Y chromosome, and mutations of the CFTR gene. Any of these causes can be identified by well-established genetic tests and form the widely applied clinical routine analyses. In addition, several genes involved in the migration and function of GnRH neurons have been discovered that can be mutated in patients with CHH with or without anosmia. Other genetic causes of male infertility comprise disorders of androgen action, genetic syndromes including infertility as a symptom, and specific defects of sperm morphology and function. Furthermore, mutations and polymorphisms of various genes have been found to be associated with unspecific spermatogenic failure/male infertility, but none of these have been introduced into the clinical workup of the infertile male so far.

Table 1 Genetic causes identified by routine analyses (patients of the Centre of Reproductive Medicine and Andrology, Münster)
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Overall, genetic causes increase in prevalence in men with lower sperm counts, i.e., increasingly severe spermatogenic failure. Therefore, the main indications for genetic testing in male infertility are severe oligozoospermia and azoospermia found in routine semen analysis. While an evidence-based cut-off for sperm counts justifying genetic tests does not exist, for clinical routine a threshold of a total sperm count below 10–15 million seems adequate. In cases of CHH and specific sperm defects, mutation screening of the associated genes is indicated.

Many forms of male infertility due to severe oligozoospermia or even azoospermia can be overcome by in vitro fertilization (IVF) combined with intracytoplasmic sperm injection (ICSI). Even though the detection of a genetic alteration will in most cases not substantially change the treatment, the clinical value lies in (1) establishing a definitive causal diagnosis, (2) the prognostic value comprising chances of testicular biopsy and pregnancy, and (3) assessing the risks for the offspring in case of successful treatment. Expert genetic counseling should accompany every (positive) genetic test.

Chromosomal Aberrations

Numerical and structural chromosomal aberrations (translocations [Fig. 1], inversions, deletions, duplications, etc.) can be discovered by conventional cytogenetic analysis (e.g., Giemsa/Trypsin staining on metaphases derived from peripheral blood lymphocytes), molecular cytogenetic methods (e.g., fluorescence in situ hybridization [FISH], or comparative genomic hybridization [CGH]). Overall, the prevalence of chromosomal aberrations in infertile men is around 10–15 times higher compared with the general population and ranges between 2% and 16% with an increasing frequency of abnormalities of the autosomes with decreasing sperm count. In contrast, sex chromosome abnormalities – most importantly Klinefelter syndrome (described separately below) – constitute the majority of pathological findings in men with azoospermia. The causative role of autosomal aberrations in relation to the men’s infertility can usually not be established in an individual case, but there is growing evidence that the abnormal chromosome(s) leads to disturbances of meiosis and thereby to oligo- or azoospermia. Structural aberrations of the Y chromosome are often associated with large or complex AZF deletions and are addressed in a specific paragraph below.

Fig. 1
figure 1

Examples of a balanced translocation between chromosomes 1 and 13, karyotype 46,XY,t(1;13) (a) and the most common Robertsonian translocation involving chromosomes 13 and 14, karyotype 45,XY,der(13;14)(q10;q10) (b)

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In the majority of cases, the infertile patient is an otherwise healthy male (aside from syndromic cases, see below) and the chromosomal anomaly does not imply any other risks for the man himself. However, men carrying, e.g., a translocation have a markedly increased risk to induce a pregnancy (e.g., by ICSI) ending in miscarriage/stillbirth or fathering a child with varying degrees of mental and/or physical retardation in case of an unbalanced karyotype in the offspring. Therefore, chromosomal analysis is indicated in every infertile man with (severe) oligo- or azoospermia. At least in case of an abnormal karyotype, accompanying genetic counseling should be offered which will include discussion of specific risks, the possibility of preimplantation and prenatal genetic diagnostics, and – for certain aberrations – the probability of other family members being carriers of the same aberration.

Klinefelter Syndrome

Harry F. Klinefelter was the first to describe men with a syndrome comprising “gynecomastia, aspermatogenesis without Leydigism, and increased excretion of follicle stimulating hormone” in 1942. The cause for the syndrome was later found in 1959 by Patricia Jacobs as a supernumerary X chromosome resulting in the karyotype 47,XXY (Fig. 2).

Fig. 2
figure 2

Typical 47,XXY karyogram of a man with Klinefelter syndrome

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Klinefelter syndrome (KS) is established as the most common chromosome aneuploidy with a prevalence of about 1–2 per 1000 men. About 80–90% of KS men bear the “original” karyotype of 47,XXY, while the remaining exhibit (in decreasing frequency) varying mosaicism (e.g., 47,XXY/46,XY) and carry additional sex chromosomes (48,XXXY; 48,XXYY; 49,XXXXY) or structurally abnormal X chromosomes. The numerical chromosome aberration in KS men arises from nondisjunction either during the meiotic divisions occurring in gametogenesis of the parents or in postzygotic mitotic cell divisions during early embryogenesis (Fig. 3). Contrary to autosomal trisomies in which paternal nondisjunctions overall account for only about 10% of all cases, in KS the supernumerary X-chromosome is of paternal origin in about 50% of patients.

Fig. 3
figure 3

Different parental origins of KS by nondisjunction (depicted by flash) in maternal meiosis I (a), maternal meiosis II (b), during one of the first postzygotic divisions (c), and paternal meiosis I (d) (Tüttelmann and Gromoll 2010)

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Men with KS are common among infertile patients: the prevalence increases from around 3% in unselected to around 15% in azoospermic patients making KS the most frequent genetic cause of azoospermia. Affected men may be identified by the leading clinical features of reduced testicular volume (usually <6 ml bi-testicular volume), azoospermia (only very rarely KS patients have some few sperm in their ejaculate), and markedly increased gonadotropin levels (luteinizing hormone (LH) and follicle stimulating hormone (FSH)). In addition, patients may have low serum testosterone concentrations (<12 nmol/l) and varying symptoms of hypogonadism (e.g., undervirilized body constitution and/or gynecomastia). However, although KS is regularly associated with infertility due to azoospermia and hypergonadotropic hypogonadism, the clinical picture of KS patients may range from severe signs of androgen deficiency, or even a lack of spontaneous puberty, to normally virilized males that only consult because of their infertility. This variability is most likely explaining why supposedly only 10% of KS men are diagnosed until puberty and only ~25% during their lifetime. In contrast to previous thinking, KS patient’s intelligence lies within the normal range, but on average 10 IQ points lower than in age-matched men.

The vast majority of KS patients is infertile due to azoospermia, even if case reports of spontaneous conceptions have been published. However, with the introduction of ICSI, Klinefelter men have a chance to become fathers. Recent studies report high success rates of around 50% to yield spermatozoa for ICSI by performing a testicular biopsy and testicular sperm extraction (TESE). The best results can be achieved with special microsurgical techniques locating focal spermatogenesis (mTESE). Some authors report a decreasing success rate with increasing age, and previous testosterone substitution may have a negative effect. Therefore, the possibility of testicular biopsy/TESE and cryopreservation of tissue should be discussed upon first diagnosis of KS even without current wish for conception. According to the published TESE procedures and ICSI cycles, the pregnancy and live birth rates seem to be comparable to ICSI because of other indications. The outcome of children of KS fathers is overall reassuring without a significantly increased risk of chromosomal aberrations or birth defects.

The important question of how spermatogenesis is generally disturbed in KS and how it may still work in some testicular foci was long a topic of debate. While first the rare completion of meiosis of 47,XXY spermatogonia was postulated based on indirect clues, more recent analyses showed that all meiotic spermatocytes in KS men were euploid 46,XY. Thus, some spermatogonia in KS patients may have never carried a supernumerary X chromosome or may have lost their supernumerary X chromosome during fetal, neonatal, or pubertal development. Independent of the mechanism, some spermatogonia seem to contain a normal set of chromosomes (46,XY) and these are able to proceed through meiosis, establishing a testicular (tissue-specific) mosaicism. What role a disturbed testicular environment involving somatic 47,XXY Sertoli and Leydig cells plays in the disturbed spermatogenesis remains to be elucidated.

XX-Male Syndrome

The XX-male syndrome is characterized by the combination of male external genitalia, testicular differentiation of the gonads, and a 46,XX karyotype by conventional cytogenetic analysis. This sex-chromosome aneuploidy is much rarer than KS with a prevalence of 1:9000 to 1:20,000. In about 80% of XX-males, material of the Y chromosome is usually translocated to an X chromosome (Fig. 4). Translocation of a DNA segment that contains the testis-determining gene (SRY = Sex Determining Region Y) from the Y to the X chromosome takes place during paternal meiosis. The presence of the SRY gene is sufficient to cause the initially indifferent gonad to develop into a testis. This X;Y translocation cannot be detected using standard karyotyping. Thus, molecular cytogenetic analysis using a specific probe for the SRY locus should be carried out in all cases of XX males. The breakpoints and consecutively the size and content of the translocation seem to influence the severity of the phenotype. There are very few reports on Y;autosomal translocations including the SRY gene leading to XX males.

Fig. 4
figure 4

Fluorescence in situ hybridization highlighting the X-centromere (green) and the SRY-locus (red). (a) Normal constellation with one X chromosome and SRY on the Y chromosome. (b) XX-male with translocation of SRY to one of the X chromosomes.

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XX-males are often clinically compared with KS and, indeed, most SRY-positive XX-males are quite similar to KS patients concerning the hallmarks of small testes, azoospermia, and hypergonadotropic hypogonadism. However, 46,XX males present with significantly shorter stature than KS patients and also shorter-than-average stature compared with healthy men. The mean height is comparable with females, which is in line with the recent view that the number and constitution of sex-chromosomes (most importantly copies of the SHOX gene) largely determines final height. The incidence of cryptorchidism is significantly higher than that in KS and about every second XX-male develops gynecomastia. XX-males seem to have normal intelligence; however, exact data are lacking. The testicular histology of postpubertal SRY-positive XX-males shows atrophy and hyalinization of the seminiferous tubules devoid of germ cells and no chance for testicular sperm extraction.

In SRY-negative XX-males (about 20% of XX-males), mutations of SOX9 and RSPO1 have been described to be causing the syndrome. However, mutations in these genes are very rare and, thus, other candidate genes are likely responsible for the sex reversal. Overall, the mechanism underlying the majority of SRY-negative XX cases currently remains unclear. SRY-negative XX-males are generally even less virilized than SRY-positive ones and often show additional malformations of the genital organs such as bifid scrotum or hypospadia.

XYY Syndrome

Most 47,XYY males have no health problems distinct from those of normal 46,XY males. Usually the finding is incidental, occurring when karyotyping has been undertaken for unrelated issues, but may be also a finding during workup for infertility. The prevalence among unselected newborns is reported to be around 1:1000.

Men with 47,XYY syndrome exhibit serum testosterone and gonadotropin levels as well as testicular volumes comparable to those of normal healthy men. Most men with 47,XYY-syndrome also have normal fertility. Onset of puberty may be delayed and adult height is in excess of the male population mean. The intelligence quotient lies within the normal range, but men score an average of 10 points less than age-matched peers. Behavioral problems are more common in 47,XYY males. However, in contrast to outdated views, aggressive or violent behavior is exceptional.

Most 47,XYY men do not need any specific therapy and men who achieve fatherhood can expect chromosomally normal offspring probably with the same likelihood as normal men.

Y-chromosomal Deletions

The long arm of the Y chromosome contains three partially overlapping but discrete regions that are essential for normal spermatogenesis. The loss (deletion) of any of these submicroscopic regions, designated as Y-chromosomal AZF (“AZoospermia Factor”) microdeletions, regularly leads to infertility due to severe oligo- or azoospermia. The prevalence of AZF deletions lies between 5% and 10% in azoospermic men and between 2% and 5% in men with severe oligozoospermia (<5 million/ml sperm concentration). It is well established that microdeletions of the Y chromosome occur in infertile men but not in control men, although the frequency differs remarkably between countries, possibly depending on the selection criteria of the patients and on the ethnic background. Thus, patients with nonobstructive azoospermia or severe oligozoospermia should be investigated for the presence of AZF deletions , which represent one of the few, well-recognized genetic causes of spermatogenetic failure resulting in male infertility (Krausz et al. 2014). The molecular diagnosis of Y-chromosomal microdeletions is relatively easy, justifying its popularity, which now makes it one of the most frequently performed diagnostic tests in molecular genetics (see Chap. 17, “Genetic Analysis in Male Infertility”).

Classical AZF Deletions

The portion of the male-specific region of the Y chromosome (MSY) affected by deletions was completely sequenced in 2003, allowing the molecular mechanism of microdeletions to be identified as homologous recombination between identical retroviral or palindromic sequences. The breakpoints of deletions are well characterized and five main microdeletion patterns have been identified, named AZFa, AZFb (P5-proximal P1), AZFbc (with two variants differing in the proximal breakpoint: P5/distal P1 and P4/distal P1), and AZFc (b2/b4) (Fig. 5). The vast majority of clinically recognized deletions (over 80%) comprise the AZFc region. The AZFb and AZFc regions are partially overlapping and together comprise 24 genes, most of which are present in multiple copies for a total of 46 copies. The complete deletion of AZFb removes 6.2 Mb (including 32 copies of genes and noncoding transcription units) and results from homologous recombination between the palindromes P5/proximal P1. The AZFc region includes 12 genes and transcription units, each present in a variable number of copies making a total of 32 copies. The classical complete deletion of AZFc (b2/b4 deletion) removes 3.5 Mb, corresponding to 21 copies of genes and transcription units. Combined AZFb and AZFc deletions occur by two major mechanisms involving homologous recombination between P5/distal P1 (7.7 Mb and 42 copies removed) or between P4/distal P1 (7.0 Mb, 38 copies removed). Interestingly, it currently remains unclear if any single gene of the respective regions or the deletions by itself (as a microdeletion syndrome) are responsible for the infertility.

Fig. 5
figure 5

Schematic representation of the Y chromosome and the current microdeletion model. Repetitive sequences (color coded palindromes) explain the origin of deletions in the AZFbc region by homologous recombination between identical sequences. The location of the STS primers suggested by the present guidelines is indicated by dashed lines

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Clinically, patients carrying an AZF deletion present with severely disturbed spermatogenesis; testicular endocrine function may or may not be present as in other cases of spermatogenetic failure. In azoospermic men, the presence of a complete deletion of AZFa seems to be associated with uniform germ cell aplasia (complete Sertoli-cell-only syndrome, SCOS), while a histological picture of SCOS or spermatogenic arrest seems common in men carrying complete AZFb or AZFbc deletions. However, in exceptional cases, complete AZFb-deletions seem compatible with finding, albeit very few, spermatozoa. Overall, the chances for successful sperm retrieval in carriers of complete AZFa as well as AZFb and AZFbc deletions have still to be considered “virtually zero.” In contrast, men carrying complete AZFc deletions may have a milder phenotype with about 50% having severe oligozoospermia – though mostly cryptozoospermia with very few sperm in the ejaculate, rarely sperm concentration of up to 1 million/ml and only hardly ever higher sperm counts. Repeated semen analysis might be useful in such patients, since spermatozoa may occasionally appear in the ejaculate and be used for ICSI. The other half of AZFc deleted men has azoospermia with a varying histological picture ranging from complete or focal SCOS to spermatogenic arrest or mixed atrophy with qualitatively intact but quantitatively severely reduced spermatogenesis. In general, TESE is possible in patients with AZFc deletions with a probability to recover sperm of about 50%. A progressive deterioration of spermatogenesis in adult patients with AZFc deletions has been proposed but never demonstrated.

There are no clinical parameters beyond azoospermia or severe oligozoospermia which can be used to predict the presence of a microdeletion of the Y chromosome and, accordingly, all men with very low sperm counts or azoospermia should be screened for AZF deletions. A positive result of the analysis provides a causal explanation for the patient’s disturbed spermatogenesis. Beyond this, the test also has prognostic value, as TESE may be possible in about 50% of men with AZFc deletion and every son of such a patient will carry the paternal Y chromosomal microdeletion and thereby inherit the disturbed fertility. Hence, genetic counseling is indicated for all carriers of Y chromosomal microdeletions.

The diagnosis of microdeletions is usually performed by PCR amplification of selected regions of the long arm of the Y chromosome. Lack of amplification suggests the presence of a microdeletion which, however, must be confirmed by a separate PCR based on different primers (“extension analysis”). Laboratory guidelines issued on behalf of the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN) are available (Krausz et al. 2014). It is estimated that the proposed basic protocol for routine microdeletion screening is sufficient to detect over 95% of clinically relevant deletions, although very rare exceptions of partial deletions within the above-mentioned regions might occur. These partial deletions, however, are of unclear pathogenic significance and their characterization is still experimental. An external quality assessment scheme is currently offered jointly by the EAA and the EMQN (www.emqn.org).

Partial AZF Deletions

Smaller deletions removing only part of the AZFc region have also been identified. These comprise the so-called gr/gr, b1/b3, b2/3 deletions, and others. These partial deletions arise by the same mechanism as classical, complete AZF deletions (homologous recombination) and have been extensively studied in large groups of men in different countries. Some of these partial AZF deletions have only been associated with infertility on a specific Y background (haplogroup) common, e.g., in Asia. In contrast, the gr/gr deletion, named after the fluorescent probes (“green” and “red”) used when first described, is significantly associated with infertility, especially oligozoospermia, in many populations. However, in specific Y haplogroups (such as D2b, Q3, and Q1) common in Japan and certain areas of China, the deletion is fixed and apparently does not have negative effects on spermatogenesis. Overall, gr/gr deletions are found in about 7% of infertile men but also in 4% of the controls with normal sperm counts. Several meta-analyses have reported significant odd’s ratios, reporting on average 2–2.5 fold increased risks of reduced sperm output/infertility. Thus, gr/gr deletions represent a significant risk factor for male infertility, but its clinical significance is still a matter of debate, because carriers may exhibit highly variable phenotypes ranging from azoo- to normozoospermia. Concerns have been raised that a gr/gr (partial AZFc) deletion may expand to a complete AZFc deletion in the next generation and gr/gr deletions have also been reported as risk factor for testicular cancer. Currently, however, no general agreement to advise routine testing has been reached.

Structural Aberrations of the Y Chromosome Including Complex AZF Deletions

Larger and/or complex Y chromosomal deletions can be caused by structural Y-chromosomal rearrangements (Fig. 6). These are usually detected by cytogenetic and molecular cytogenetic analyses (see above), but may also be detected upon AZF deletion screening. A deletion of the whole AZF region (comprising AZFa, b, and c) is possible in case of a derivative Y chromosome consisting of two short arms leading to an isochromosome Yp. Comparably, a deletion of AZFb and AZFc can be caused by an isodicentric Y chromosome. Likewise, ring chromosomes may cause loss of larger parts of the Y chromosome. Furthermore, complex AZF deletions may indicate an unbalanced translocation between the Y chromosome and other chromosomes. All of these larger rearrangements have more severe implications for the offspring than the classical AZF deletions. For example, translocations between Y chromosome/autosome lead to a high risk for unbalanced chromosomal aberrations and larger structural aberrations of the Y chromosome increase the risk for mosaicism of cells with or without monosomy of the X chromosome in the offspring (45,X/46,XY). The latter may cause a broad spectrum of gonadal dysgenesis and ambiguous external genitalia, i.e., disorders of sexual development (DSD).

Fig. 6
figure 6

Examples of structural Y chromosomal aberrations detected in infertile males: Two differently sized Y ring chromosomes (a, b) and an isodicentric chromosome Yp that is accompanied by a deletion of AZFbc (c)

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Other Submicroscopic Chromosomal Aberrations

Since the advent of genome-wide technologies (array-Comparative Genomic Hybridization, array-CGH) to identify submicroscopic deletions and duplication, these are analyzed on a large scale in many diseases and are termed Copy Number Variants (CNVs). It immediately became clear that CNVs add to the variation in our genome and the majority has probably no relevance for disease. However, many novel microdeletion syndromes, many of which are associated with mental retardation and malformations, have now been described.

While the submicroscopic Y-chromosomal deletions in the AZF region described above have been analyzed in infertile men routinely for many years, other microdeletions (and -duplications) are currently only studied in research settings. To date, only few genome-wide studies have been conducted in infertile men (Tüttelmann et al. 2011) and some others have focused specifically on the X-chromosome (Lo Giacco et al. 2014). Probably the most important and consistent finding of these studies is an increased “burden” of microdeletions in infertile (oligo- or azoospermic) compared with fertile (or normozoospermic) men, i.e., infertile men carry significantly more microdeletions than controls. In conjunction with the notion that infertile men also seem to carry more nucleotide variations (SNPs) than controls, this may allude to an association between infertility and an increased genome instability. Nevertheless, to date no specific CNV has been identified (and replicated in an independent study) that explains spermatogenic failure in carriers. Thus, no testing for any CNV can currently be advised to be tested in clinical routine. In contrast, high-resolution detection of CNVs in a research setting may help identify novel genes in which mutations (deletions as well as point mutations) lead to male infertility, for which the gene TEX11 is a recent example (see below).

Single-Gene Defects

Mutations of some (few) genes have been well established as causes for specific diseases associated with male infertility. However, the large majority of infertile men does not fall into this category and causative mutations in men with isolated spermatogenic failure have long been sought for.

Congenital Absence of the Vas Deference (CBAVD)

Patients with obstructive azoospermia are candidates for genetic testing of mutations of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Homozygous or compound heterozygous mutations of this gene are well known to cause autosomal-recessive cystic fibrosis (CF) which comprises severe lung and pancreas problems as leading symptoms. Nearly all men with full clinical CF are also affected by Congenital Absence of the Vas Deference (CBAVD) and, thus, exhibit obstructive azoospermia and are infertile. However, CBAVD can also occur independently of full CF and is one form of the so-called CF-related disorders. Aside from the effects on other organs (lung, pancreas, etc.), the disruption of CFTR protein function leads to a congenital malformation of the Wolffian ducts, which are the precursors of the vas deferens, epididymis, and seminal vesicles during fetal development. Therefore, hypo- or aplasia of the epididymis or seminal vesicles can accompany the absence of the vas deferens.

Both CF and isolated CBAVD are caused by an overlapping but also distinct spectrum of CFTR mutations which is highly dependent on the ethnic origin of the patient. CF is regularly caused by two mutations completely abolishing or severely impairing CFTR protein function, with a deletion of 3 bp in exon 10, leading to the loss of the amino acid phenylalanine (F508del) being the most common mutation in nearly all populations. In contrast, isolated CBAVD is mostly caused by a combination of one severe and a second milder CFTR mutation that only partially disturbs CFTR protein function (e.g., most commonly F508del/R117H).

In contrast to men with Y-chromosomal microdeletions, patients with CBAVD exhibit distinct clinical features: azoospermia in conjunction with decreased seminal volume, pH, and markers of epididymis (α-glucosidase) and seminal vesicles (fructose) in the presence of normal LH, FSH, and testosterone and normal testicular volume. In the majority of men with CBAVD, normal spermatogenesis will be found upon testicular biopsy with histological evaluation/TESE, which also confirms the diagnosis of obstructive azoospermia, and thus these men have a high chance to conceive a child by ICSI. However, the risk for the child having CF is increased in comparison with the general population and can be estimated depending on the CFTR carrier status of the female partner because CF is inherited in an autosomal recessive manner. The carrier rate (heterozygote frequency) may be as high as 5% in the Caucasian population. Hence, CFTR mutational analysis is usually not only indicated in the men with CBAVD but also his partner before testicular biopsy/TESE and IVF therapy. Current recommendations include a two-step approach to genetic testing of suspected CBAVD cases: first, frequent mutations are analyzed (usually by a commercial kit) and afterwards full sequence analysis is performed if only one or no mutations were found. Depending on the extent of analytic technologies used, the detection rate of two mutated CFTR alleles in men with CBAVD has been reported to be around 80% and in about an additional 10% only one mutated allele can be identified (Ratbi et al. 2007). CFTR mutations have also been described in men with unilateral absence of the vas deference (CUAVD) and in patients with oligo-/azoospermia without clinical features of obstruction, but the published data are not consistent.

Very recently, mutations of the X-linked gene ADGRG2 have been described to also cause CBAVD (Patat et al. 2016). Truncating mutations of ADGRG2 were identified in about 15% of men with CBAVD in whom CFTR analysis was negative. Although this is currently the only study on this gene, sequence analysis of ADGRG2 will very likely be embedded in the routine genetic analyses of men with CBAVD soon, because it will allow for appropriate genetic counseling with regard to the X-linked transmission of the molecular defect.

Hypogonadotropic Hypogonadism

Congenital Hypogonadotropic Hypogonadism (CHH, also “Idiopathic” or “Isolated” HH (IHH)) is characterized by low gonadotropin levels leading to a lack of sex steroid production and consequently no or incomplete pubertal development and anovulation in females/no spermatogenesis in males. Circulating LH and FSH levels are either undetectable or very low, resulting from a defect in the normal pulsatile secretion pattern of GnRH from the hypothalamus or impaired GnRH action. Clinically, CHH can present in conjunction with many other symptoms including cleft lip and/or palate, dental agenesis, renal agenesis, digit malformations, and synkinesia. A distinctive feature is a normal sense of smell (in about 40% of cases) or an impaired sense of smell (anosmia, in about 60% of cases), the latter being the hallmark of Kallmann syndrome.

The primary anatomic defect of Kallmann syndrome , identified in 1989, is the agenesis of the bulbus olfactorius with associated failure of the GnRH neurons to migrate from the olfactory epithelium to the hypothalamus. The first gene identified to be responsible for this phenotype was denominated KAL1 (the official gene symbol is ANOS1) that encodes the extracellular matrix protein anosmin 1. Successively, many more genes were found to be associated with CHH with or without anosmia (Table 2), such as the GnRH receptor gene (GNRHR), the fibroblast growth factor receptor 1 gene (FGFR1) (involved in the formation of the olfactory bulb and responsible for the autosomal dominant form of the Kallmann syndrome), the KISS1 receptor gene (KISS1R, formerly G protein-coupled receptor 54, GPR54), the prokineticin 2 gene (PROK2) and its receptor (PROKR2), and the fibroblast growth factor 8 gene (FGF8). Mutations of GNRHR, GNRH1, KISS1R, TAC3, and TACR3 genes are found in CHH patients without anosmia, while mutations of KAL1, PROK2, and PROKR2 are usually associated with reduction of the sense of smell suggesting a role for these genes in olfactory bulb formation. All other currently known genes (FGFR1, CHD7, FGF8, NSMF, WDR11, HS6ST1, and SEMA3A) are variably associated with impaired sense of smell.

Table 2 Selected genes mutated in Congenital hypogonadotropic hypogonadism and Kallmann syndrome
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Mutations of the above-mentioned genes have been described in several families and show variable penetrance, with cases of full clinical CHH, but also isolated anosmia or only delayed puberty within the same family. Interestingly, mutations of the FGF8 gene have been found also in cases of acquired (adult-onset) hypogonadotropic hypogonadism. The growing family of genes involved in GnRH neuron migration and function and the expanding phenotypic expression of mutations thereof suggest that the distinction between Kallmann syndrome , isolated anosmia, normosmic CHH, delayed puberty, and acquired HH is probably arbitrary. Indeed, these clinical pictures are often present in various combinations within families carrying the same mutation. In addition, it has been shown that CHH and Kallmann syndrome can spontaneously revert in about 10% of patients upon discontinuation of treatment.

The search for mutations of the genes associated with CHH (with or without anosmia) requires direct gene sequencing. Initially, to keep time and resources low, it was tried to limit the number of genes to be screened by careful definition of the full spectrum of clinical symptoms, identification of the familial cases, and definition of the pattern of inheritance (autosomal-dominant, autosomal-recessive, or X-linked). However, with the advent of next-generation sequencing (NGS) technology, targeted sequencing of a panel of all potential genes is now the more cost-efficient laboratory approach. Still, the clinical evaluation needs to go hand in hand with the genetic analyses, because often several variants are identified that would otherwise be difficult to interpret concerning their pathogenicity. Comprehensive sequencing of larger numbers of the above-mentioned genes (and not stopping when one mutation of one gene was identified) has led to the notion that CHH is in a not small fraction of cases caused by more than one mutation of one gene, i.e., establishing a oligogenic causes of the disease. Overall, causal mutations are still only found in about 40% of cases although more than 20 genes have now been identified to be associated with CHH (Vezzoli et al. 2016). Thus, numerous others so far unidentified genes or other mechanisms (e.g., epigenetic regulation) remain to be described in the pathogenesis of CHH.

Aside from “isolated” CHH, several syndromes are associated with hypogonadotropic hypogonadism. Examples are Prader-Willi syndrome (caused by a loss of paternal 15q11.2), combined pituitary hormone deficiency (caused by mutations of PROP1, HESX1, or LHX3), obesity syndromes (caused by mutation of PCSK1, LEP, or LEPR), Bardet-Biedl syndrome (caused by mutations in at least 16 genes), and X-linked adrenal hypoplasia congenital (caused by mutation of DAX1). Because of their additional, usually severe symptoms, these syndromes are usually diagnosed much earlier than isolated CHH and warrant the specific genetic analyses.

Disorders of Androgen Action

Mutations in the X-linked Androgen Receptor (AR) gene cause a wide spectrum of androgen insensitivity syndromes (AIS). Depending on the functional impact on the receptor, mutations may lead to complete androgen insensitivity (CAIS) with a female phenotype in karyotypic males, partial forms (PAIS) in patients with ambiguous genitalia, or mild forms (MAIS) in men with hypospadias, gynecomastia, and spermatogenic impairment. However, mutations in the AR gene seem to be a rare cause of isolated male infertility as only few cases have been described although rather large sequencing studies have been performed. Therefore, AR gene sequencing is currently not warranted in isolated male infertility, but only in patients with additional symptoms, e.g., severe hypospadias.

The AR gene contains a CAG-repeat in exon 1 encoding a polyglutamine stretch in the AR protein. If expanded to or above 38 CAG-repeats, this expansion causes X-linked recessive Spinal and Bulbar Muscular Atrophy (SBMA, also known as Kennedy’s disease). SBMA occurs only in males and is characterized by a gradually progressive neuromuscular disorder in which degeneration of lower motor neurons results in muscle weakness, muscle atrophy, and fasciculations. Affected individuals additionally often show gynecomastia, testicular atrophy, and reduced fertility as a result of mild androgen insensitivity.

According to in vitro as well as a number of clinical studies, the CAG-repeat in the AR gene also modulates the activity of the receptor protein in the normal range (9–34 repeats, median ~21/22 repeats in the European population and significantly different, e.g., in Africans [shorter] and Asians [longer]). Overall, longer CAG-repeats encode a less active androgen receptor, i.e., the same amount of circulating testosterone will have less effect. Therefore, determining the specific length of the AR-CAG-repeat in hypogonadal men receiving testosterone substitution may help titrate the individual dosage in a pharmacogenetic approach (Zitzmann 2009). In addition, men with symptoms of hypogonadism and borderline testosterone serum levels may be eligible for testosterone treatment if they carry a long AR-CAG-repeat (leading to a less active receptor).

Many studies have sought for an association between the AR-CAG-repeat length and male infertility. However, although meta-analysis has shown a significant difference between CAG-repeat length of infertile patients and controls, this difference comes down to about one half CAG-repeat between these two groups, which has no clinical implication at all (Davis-Dao et al. 2007). Thus, the analysis of the AR-CAG-repeat in infertile men is not warranted.

Specific Defects of Spermatozoa

Monomorphic sperm defects are rare but will be caused in the large majority by genetic defects. The two distinctive forms of morphological defects in which some genetic causes have been identified so far relate to the correct formation of the acrosome and the construction of the sperm tail. Globozoospermia is characterized by round headed spermatozoa lacking the acrosome. This rare form of teratozoospermia can be caused by mutations in the SPATA16 or DPY19L2 genes (both described as autosomal-recessive disorders) and probably many other so far unknown genetic defects.

Primary Ciliary Dyskinesia (PCD) is caused by abnormal ciliary structure and function resulting in highly variable symptoms like situs abnormalities (situs inversus), chronic otosinopulmonary disease, and also abnormal sperm motility. While “full” PCD is a severe, multiorgan disease often comprising male infertility due to reduced or absent sperm motility, milder forms may cause isolated sperm motility defects. PCD is a highly heterogeneous disease and can be caused by mutations in DNAH5, DNAH11, CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1, CCDC114, RSPH4A, DNAAF1, DNAAF2, and LRRC6. PCD is invariably inherited in an autosomal-recessive manner. So far, only the minority of PCD genes have been analyzed in isolated male infertility due to immotility of spermatozoa and, thus, the role of mutations in these genes in infertile men remains to be elucidated.

Single-Gene Defects in Isolated Severe Spermatogenic Failure

Over the last few years, many genes have been reported to be mutated in men with severely impaired sperm production exhibiting severe oligozoospermia, cryptozoospermia, or azoospermia without symptoms of obstruction (NOA). However, most of these genes have so far not been replicated in independent studies and their role currently remains unclear. Exceptions are NR5A1 and DMRT1 which are involved in gonadal development and in which mutations have been known for a longer time to cause gonadal dysgenesis and disorders of sexual development (DSD). Milder (missense) mutations in these two genes have now also been described in men with isolated spermatogenic failure, i.e., NOA or severe oligozoospermia. Mutations in the TEX11 have very recently been identified as first common X-linked cause of meiotic arrest (a specific form of NOA) in about 15% of men with this phenotype (Yatsenko et al. 2015).

Genetic Syndromes Associated with Male Infertility

Many complex genetic (syndromic) diseases are associated with some form of male infertility. These diseases, of which some are summarized in Table 3, are usually diagnosed early in life. If fertility evaluation is required, the preexisting diagnosis needs to be taken into account to prevent unnecessary examinations. Hemochromatosis and Myotonic Dystrophy Type 1 are mentioned specifically as both are rather common genetic diseases also causing male infertility.

Table 3 Selection of rare complex genetic syndromes that can include male infertility as symptom
Full size table

Autosomal-recessive hemochromatosis is caused by mutations in the HFE gene and characterized by inappropriately high absorption of iron by the gastrointestinal mucosa leading to excessive storage of iron in the liver, skin, pancreas, heart, joints, and testes. Untreated individuals may exhibit progressive increase in skin pigmentation, diabetes mellitus, congestive heart failure, and/or arrhythmias, arthritis, and hypogonadism including testosterone deficiency and oligo-/azoospermia.

Autosomal-dominant myotonic dystrophy type 1 is caused by an expansion of a CTG-repeat at the 3′-end of the DMPK gene. Myotonic dystrophy is a multisystem disorder that affects skeletal and smooth muscle as well as the eye, heart, endocrine system, and central nervous system. The clinical findings span a continuum from mild to severe and include male infertility due to testicular atrophy and oligo-/azoospermia.

Outlook

For many years, single candidate genes have been evaluated with the goal of identifying causal mutations for spermatogenic failure and usually genotyping SNPs or direct sequencing has been used. However, most of these approaches were not very successful most probably because (1) “male infertility” as well as “spermatogenic failure” is genetically highly heterogeneous and (2) selection of patient groups is often not stringent. Conversely, novel genetic technologies now allow unbiased approaches to decipher the underlying causes for specific forms of male infertility. These technologies comprise genome-wide association studies (GWAS), array-CGH, and whole-exome or even genome sequencing. These novel methodologies easily outperform the previous candidate gene approaches, which is illustrated by an increasing number of recent publications identifying genetic defects causing spermatogenic failure, of which some examples have been mentioned above. In the near future, these novel technologies will greatly increase the fraction of infertile men with a specific genetic diagnosis.

Summary

Depending on the clinical findings, the infertile male patient needs genetic evaluation: Karyotype analysis should be performed in patients with oligo- or azoospermia to rule out structural chromosomal abnormalities and Klinefelter syndrome. Severe oligozoospermia and azoospermia indicate Y chromosome microdeletion screening. Men with obstructive azoospermia need careful analysis of the CFTR-gene. Mutation screening should be performed in patients with CHH and other syndromic forms of male infertility. In the near future, men with severe spermatogenic failure (firstly NOA) may be evaluated by targeted sequencing of a panel of genes. All positive genetic analyses should be accompanied by expert genetic counseling.

Cross-References