Human Genetics (2021) 140:203–215 https://doi.org/10.1007/s00439-019-02101-w REVIEW The X chromosome and male infertility Matthias Vockel1 · Antoni Riera‑Escamilla2 · Frank Tüttelmann1 · Csilla Krausz2,3 Received: 24 November 2019 / Accepted: 10 December 2019 / Published online: 24 December 2019 © The Author(s) 2019 Abstract The X chromosome is a key player in germ cell development, as has been highlighted for males in previous studies revealing that the mammalian X chromosome is enriched in genes expressed in early spermatogenesis. In this review, we focus on the X chromosome’s unique biology as associated with human male infertility. Male infertility is most commonly caused by spermatogenic defects to which X chromosome dosage is closely linked; for example, any supernumerary X chromosome as in Klinefelter syndrome will lead to male infertility. Furthermore, because males normally only have a single X chromosome and because X-linked genetic anomalies are generally only present in a single copy in males, any loss-of-function muta- tions in single-copy X-chromosomal genes cannot be compensated by a normal allele. These features make X-linked genes particularly attractive for studying male spermatogenic failure. However, to date, only very few genetic causes have been identified as being definitively responsible for male infertility in humans. Although genetic studies of germ cell-enriched X-chromosomal genes in mice suggest a role of certain human orthologs in infertile men, these genes in mice and humans have striking evolutionary differences. Furthermore, the complexity and highly repetitive structure of the X chromosome hin- der the mutational analysis of X-linked genes in humans. Therefore, we conclude that additional methodological approaches are urgently warranted to advance our understanding of the genetics of X-linked male infertility. Introduction quantitative alterations of spermatogenesis; (iii) qualitative alterations of spermatogenesis; and (iv) ductal obstruction/ Infertility is a frequent condition affecting one out of six dysfunction (Tournaye et al. 2016). Known genetic fac- couples in Western countries (Krausz and Riera-Escamilla tors are present in each of these etiological categories, and, 2018). Male factors contribute to infertility in about 50% depending on the severity of the spermatogenic impairment, of cases and can be classified into four major etiological they account for about 10–20% of cases. With the advent of categories: (i) hypothalamic–pituitary axis dysfunction; (ii) large-scale sequencing strategies (especially exome analysis by next-generation sequencing, or NGS), more and more Matthias Vockel and Antoni Riera-Escamilla are co-first authors. novel genes have been reported to be linked with male infer- tility. However, the majority of these genes still lack a defini- Frank Tüttelmann and Csilla Krausz are co-last authors. tive gene–disease relationship (Oud et al. 2019). Further, about 40% of quantitative spermatogenic impairment cases * Frank Tüttelmann frank.tuettelmann@ukmuenster.de cannot be causally diagnosed and are instead referred to as unexplained or idiopathic infertility; a large proportion of * Csilla Krausz csilla.krausz@unifit.it these cases are likely caused by yet unknown genetic factors. Both sex chromosomes in males (X and Y) are enriched 1 Institute of Human Genetics, University of Münster, in genes specifically or overexpressed in the testis (Skaletsky Vesaliusweg 12‑14, 48149 Münster, Germany et al. 2003; Mueller et al. 2008). Moreover, in males, de 2 Andrology Department, Fundació Puigvert, Universitat novo or rare mutations in these chromosomes may have a Autònoma de Barcelona, Instituto de Investigaciones direct phenotypic effect because there is no second com- Biomédicas Sant Pau (IIB- Sant Pau), Barcelona, Catalonia, Spain pensatory X or Y allele. The role of Y chromosome-linked 3 deletions in male infertility has been well understood since Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, Centre of Excellence DeNothe, 1996 (Vogt et al. 1996). Here, the removal of genes belong- University of Florence, Viale PIeraccini, 6, 50139 Florence, ing to the so-called azoospermia factor (AZF) region causes Italy 13 Vol.:(0123456789) 204 Human Genetics (2021) 140:203–215 azoospermia or oligozoospermia (Krausz et al. 2014; Krausz multigene family is the large superfamily called cancer testis and Casamonti 2017). While screening for Y chromosome antigens (CTA), in which members are arranged into com- deletions is a fundamental part of the diagnostic workup of plex direct and palindromic repeats. As has been estimated, severe oligo/azoospermic men, only two X chromosome- CTA families account for around 10% of X-linked genes linked genes are routinely tested in selected pathological (Simpson et al. 2005; Almeida et al. 2009), and they can be conditions (ANOS1 and AR). Thanks to high-throughput divided into the MAGE multigene family and the NY-ESO-1 approaches such as whole exome sequencing (WES) and family in region Xq24-q28 and the SSX family and GAGE/ comparative genomic hybridization (CGH) arrays, novel PAGE/XAGE families in region Xp11 (Zendman et al. 2003). X-linked gene mutations (e.g., TEX11 and ADGRG2) have Most of the CTA genes are expressed during spermatogen- been discovered as causative factors of different infertile esis, but their function in both germline tissues and tumors phenotypes. remains poorly understood (Fratta et al. 2011). In light of these findings, in this review we will discuss (i) It has been speculated that multicopy regions on the X X-linked genes and male fitness from an evolutionary point chromosome may have an important function for germ cell of view; (ii) the clinical consequences of X-linked aneuploi- survival, e.g., through dosage compensation. In this regard, dies and deletions; (iii) the role of X-linked gene mutations homologous repeats could self-pair during male meiosis in male infertility. such that they would potentially be protected from unpaired chromosome inactivation, as has been suggested for the Y chromosome (Skaletsky et al. 2003). However, a recent Structure and function of the X chromosome study implies that copy number expansions of palindro- mic genes might not be essential for spermatogenesis, as While both sex chromosomes are derived from a pair of reduced ampliconic gene expression following deletion of autosomes around 300 million years ago, most ancestral large X-linked palindromes did not reduce fertility in male functional elements have been only conserved on the X mice (Kruger et al. 2018). chromosome (Ross et al. 2005). The sex chromosome’s size also differs dramatically, and while the X chromosome has remained relatively stable in magnitude with around X chromosome‑linked genes and male 155 Mb, the Y chromosome decreased to around 55 Mb. The fitness: an evolutionary perspective X chromosome contains about 800 (human) to 850 (murine) protein-coding genes (Mueller et al. 2013), for which a dis- More than 50 years ago, it was postulated that the X chro- proportionately high number of inherited diseases were mosome is in an evolutionary stable state, and X-linked documented. The unique gene composition of the X chro- genes should differ little between placental mammals mosome also allows balanced gene dosage compensation (Ohno 1967). This hypothesis, called ‘Ohno’s law’, is through a well-regulated spatial and temporal expression of supported by several comparative mammalian studies X-linked genes (Deng et al. 2014). (e.g., Murphy et al. 1999; Kuroiwa et al. 2001; Raudsepp The X chromosome was previously considered the most et al. 2004; Ross et al. 2005). Later though, it was shown stable chromosome of the genome (Ohno 1967) and was that X chromosomes have undergone rapid evolution- largely perceived as a “female” counterpart to the “male” ary changes, and accumulation of ampliconic genes has Y chromosome. But, to the contrary, it turns out that the X occurred individually during mammalian evolution (Bel- chromosome is in a current state of rapid evolution toward lott et al. 2010; Mueller et al. 2013). This comparison of a specialized role for sperm production. multicopy genes in different lineages has offered impor- The hemizygosity in males allows for unique evolutionary tant insights into the mammalian evolution of X-linked patterns of X-linked genes for male sexual fitness and can genes. While most of the single-copy genes on the mouse favor positive selection of advantageous variants (Vicoso and human X chromosomes are conserved between these and Charlesworth 2006). Accordingly, the X chromosome species, approximately 340 genes are not shared between is enriched for sex-biased (Khil et al. 2004) and spermatogo- the two species, and most of these unshared genes are nia-expressed (Wang et al. 2001) genes. However, X-linked found in ampliconic regions (Fig. 1). About 10% of the genes are thought to be silenced during male meiosis by a human and 16% of mouse X-linked genes, respectively, mechanism known as meiotic sex chromosome inactivation have been independently acquired since their divergence (Turner 2007). To compensate for this inactivation, large from a common ancestor. In contrast to the shared, single- gene families with multiple copies have accumulated on the copy genes, these ampliconic genes are notable excep- X chromosome (Warburton et al. 2004), and these homolo- tions to the postulated Ohno’s law. Expression analyses gous palindromic repeats are highly expressed in spermatids further revealed that these ampliconic genes are active (Mueller et al. 2008). An example of an X-chromosomal almost exclusively in testicular germ cells, but not in 13 Human Genetics (2021) 140:203–215 205 Fig. 1  Evolution of mammalian X chromosomes. Contrary to (n = 800 human, 853 mouse) and predominantly testis-expressed Ohno’s law, the gene content of the X chromosomes differs signifi- genes (expression patterns of eight human tissues and three mouse cantly within the mammalian lineage. a Schematics of the variability tissues) in humans and mice. Bar graphs: percentages of protein-cod- of X-linked genes between rodents and primates. Colors denote the ing X-linked genes categorized as single copy (SC, blue), multicopy divergent evolutionary origins of X‑linked genes. Gray: conserved (MC, yellow) or ampliconic (AMP, red). Pie charts: percentages of ancestral regions (corresponds to the long arm of the human X, and X-linked genes that are shared (sh, light green) in both species or are this region is rearranged in mice); Blue: acquired gene content in independently acquired (ia, dark blue) after the divergence of humans mammals (short arm of human X, region rearranged in mice); green and mice. Most ampliconic X-linked genes have been independently and orange: independently acquired ampliconic genes in humans acquired in primates and rodents and are predominantly expressed and mice, respectively (positions correspond to arbitrary genomic in testes (data adopted from Bellott et al. 2010; Mueller et al. 2013; locations). b Depicted diagrams show a comparison of all X-linked Deng et al. 2014) other male tissues or female tissues. As such, they are The consequences of too much and too little likely to contribute to sperm production (Mueller et al. X chromosome 2013). Although the physiological functions of most inde- pendently acquired, ampliconic genes are still unclear, Too much X: Klinefelter syndrome their rapid evolution suggests a strong positive selection that may have contributed to speciation. Klinefelter syndrome (KS) is a chromosomal condition It is possible that these genes have significant roles in that was first described in 1942 (Klinefelter et al. 1942). diseases related to infertility, but the use of model sys- It was not until 1959 when Jacobs and Strong (1959) tems for these genes is complicated because they are not determined that it was caused by the presence of an extra conserved in mice. This caveat seems to apply to all sex- X chromosome. Various genotypes are associated with linked genes, as a recent study has suggested that sex bias this condition, the most common being 47,XXY in up to in overall gene expression also arose recently in mam- 80–90% of cases, while in approximately 10% of cases malian evolution and, hence, could be species specific, the genotype is a mosaic form of 47,XXY/46,XY. Other which might prevent results on molecular variation from rare variants are 48,XXXY; 48,XXYY; 49,XXXXY; being translated from animal models to human reproduc- 49,XXXYY; and 49,XXYYY and are associated with tion research (Naqvi et al. 2019). Furthermore, sex chro- more severe phenotypes (Visootsak et al. 2001; Tartaglia mosomes evolve functional specialization by accumula- et al. 2008; Ottesen et al. 2010). KS shows an estimated tion of sexually antagonistic as well as sex-biased genes frequency of 1:600 in the general population, whereas in (Vicoso and Charlesworth 2006; Parsch and Ellegren patients with non-obstructive azoospermia it is as high 2013). The significant enrichment for testis-expressed as 15%. In adults, the diagnosis is mainly made because genes on the X chromosome and the differential expres- of infertility and/or sexual dysfunction. The reproductive sion of sex-biased genes imply that sexual conflict has phenotype of these individuals includes small, firm testes profound consequences on gene content evolution of X with hyalinization of seminiferous tubules with consequent chromosomes in mammals (Gurbich and Bachtrog 2008; spermatogenic failure. In fact, over 90% of KS patients are Larson et al. 2018). 13 206 Human Genetics (2021) 140:203–215 azoospermic, and the remaining show crypto/severe oligo- Regarding dosage dysregulation, women have two X zoospermia. Despite that spontaneous puberty is observed chromosomes, while men only have one. Thus, to compen- in these patients; the majority of them present signs of sate for the difference in gene dosage, in females one of the androgen deficiency, which range from hypogonadism two X chromosomes is inactivated. However, a small portion with gynecomastia and eunuchoid proportions to variable of X-chromosomal genes (~ 10–15%) escape inactivation, levels of undervirilization. among it the PAR genes. In this regard, the overdosage of Natural pregnancies have been reported mostly in mosaic SHOX has been demonstrated to be associated with the typi- KS cases. For the majority of non-mosaic 47,XXY KS men, cal excess height of KS patients. Apart from SHOX, other conception of a biological child is only possible through tes- PAR genes such as CD99 and CSF2RA were found differen- ticular sperm extraction (TESE), either conventional TESE tially expressed in the KS group vs. male controls (Zitzmann or microTESE combined with intracytoplasmic sperm injec- et al. 2015). The overexpression of these two genes could tion (ICSI) (Corona et al. 2017). The success rate of sperm contribute to the development of some of the typical co- retrieval varies in different laboratories, but according to a morbidities of KS [overexpression of CD99 was associated recent meta-analysis the mean sperm retrieval rate (SRR) with inflammatory cytokines, whereas CSF2RA with insulin is about 44% (Corona et al. 2017). Although an accurate resistance, waist circumference, and concentrations of the prediction of SRR is not possible, age seems to be a relevant procoagulatory substance PAI-1 and cytokines (Zitzmann prognostic parameter, and KS patients under 30 years old et al. 2015)]. show a higher success rate (Rohayem et al. 2015). Hence, Finally, regarding epigenetic events, the supernumerary X an early diagnosis of this syndrome is clearly relevant, chromosome in KS has an interesting effect on DNA meth- since it would allow both the preservation of fertility and ylation. Methylome studies in blood and brain samples have the introduction of preventive therapies to avoid or to better shown differences between KS patients and 46,XY males control associated co-morbidities. In fact, besides reproduc- (Viana et al. 2014; Sharma et al. 2015; Wan et al. 2015; tive/sexual problems, KS patients may present a series of Skakkebaek et al. 2018). Unfortunately, only few overlap- co-morbidities such as metabolic syndrome, autoimmune ping differentially methylated regions (DMRs) have been diseases, osteoporosis, breast and germ cell tumors, venous observed between studies, which could be in part explained thromboembolisms and cognitive/psychiatric disturbances by differences in the type of sample analyzed (blood vs (Gravholt et al. 2018 and references therein). brain) and the resolution of the arrays (Illumina 27 K vs Illu- What underlies the heterogeneous phenotypic presenta- mina450K) (Skakkebaek et al. 2018 and references therein). tion is still not clear, but is probably related to various fac- Gene set enrichment analysis of the observed DMRs showed tors (Tüttelmann and Gromoll 2010; Zitzmann et al. 2015). an overrepresentation of terms relevant to the KS pheno- For instance, the proposed phenotypic modulators include type such as diabetes, obesity, height, coronary and arterial parental origin of the extra X chromosome, the androgen disease, hypercholesterolemia, gingivitis and periodontitis, receptor CAG repeat length, gene dosage dysregulation, bone mineral density, cancer (breast, prostate, colon, pan- and/or epigenetic events. First, the parental origin of the creas, leukemia, lymphoma), connective tissue diseases, supernumerary X chromosome has only been addressed by infection and inflammation and others (Skakkebaek et al. few studies reporting a potential effect on impaired speech/ 2018). However, it is still unknown whether these epigenetic motor development (Stemkens et al. 2006), later onset of alterations are responsible for the KS co-morbidities through puberty (Wikstrom et al. 2006) and schizotypal traits (Bruin- altering gene expression regulation. In fact, only a moder- ing et al. 2010). While impaired speech/motor developmen- ate correlation between epigenetic alterations and some of tal problems and later onset of puberty were significantly the phenotype-associated gene expression levels (BOLA1, associated with paternal origin of the X chromosome, sig- DDX43, ZBTB44 and KDM5C) has been observed (Skakke- nificantly higher scores on schizotypal traits were associated baek et al. 2018). Further studies on different tissue samples with maternal origin. are needed to elucidate the biological basis for the heteroge- Next, regarding the CAG repeats in the androgen recep- neous phenotypic presentation of KS. tor (AR) gene, the number modulates its transcriptional activity in vitro (Gao et al. 1996), and an increased num- Too little X: X‑linked deletions ber of repeats was positively correlated with height, arm span and both arm and leg length in KS patients (Zitzmann Copy number variants (CNVs) are a type of structural vari- et al. 2004; Bojesen et al. 2011; Chang et al. 2015). On the ant involving alterations in the number of copies of specific other hand, data on the (CAG)n polymorphism in relation regions of DNA (often defined as above 1 kb in length) in to testicular volume, gynecomastia, lipid metabolism and comparison with a reference genome (Feuk et al. 2006). The bone-related parameters are still controversial (Gravholt functional consequence of a CNV can be difficult to predict et al. 2018 and references therein). and depends on the exact position of the CNV breakpoint, 13 Human Genetics (2021) 140:203–215 207 i.e., the region where a fragment was inserted (gain/duplica- patients carried the deletion, whereas it was not found in tion) or lost (loss/deletion). CNVs may affect gene function any of the 838 controls from Sichuan Province in south- directly or indirectly. A direct effect through the involve- western China (Shen et al. 2017). On the contrary, Ma et al. ment of coding sequences may be the consequence of the (2017) reported the prevalence of the CNV67 to be about CNV (i) altering the copy number of dosage-sensitive genes; 3% in fertile controls, but the controls had unknown sperm (ii) disrupting the gene-coding sequence, as partial gain or counts and belonged to a specific geographic area (the Guan- deletion of coding sequences can produce different alleles, dong population). The exact mechanism by which CNV67 including both loss and gain of function; or (iii) produc- may alter spermatogenesis is still unknown. Studies have ing chimeric proteins when CNV breakpoints lie within two suggested that the CNV67 might cause a partial deletion of different genes, leading to the fusion of two partial coding the proximal copy of the MAGEA9B gene, which is a CTA regions. An indirect effect may occur when CNVs lie in non- specifically expressed in the testis and in some tumors (Shen coding regions and disrupt the function of genes through the et al. 2017). In summary, further studies in multiple popu- deletion or transposition of critical regulatory elements, such lations including fertile controls with known sperm counts as promoters, enhancers and silencers (Hurles et al. 2008). are needed to elucidate the clinical relevance of this CNV. A number of CNVs generate alleles with a pathogenic effect in andrology; the best examples of such a clear-cut effect are the Y-chromosomal AZF deletions (Krausz et al. X chromosome‑linked gene mutations 2014; Krausz and Casamonti 2017). Comparative genomic in male infertility hybridization (CGH) arrays (whole genome or X chromo- some specific) have been employed to analyze patients with Many X-linked genes are promising candidates as causes for quantitative spermatogenic disturbances in five laboratories infertility owing to their testicular expression and expected (Tüttelmann et al. 2011; Krausz et al. 2012; Stouffs et al. involvement in spermatogenesis. The advances in high- 2012; Lopes et al. 2013; Yatsenko et al. 2015), and four of throughput sequencing approaches have allowed for the them provide information about X-linked CNVs (Tüttelmann identification of additional genetic factors in azoospermic et al. 2011; Krausz et al. 2012; Lopes et al. 2013; Yatsenko patients either by targeted sequencing or exome sequenc- et al. 2015). Comparing the raw data of these studies, only ing. However, most studies have not been replicated so far, few partially overlapping CNVs have been observed, which and the functional roles of most genes are still ill-defined. could be in part explained by differences in the resolution Furthermore, the repetitive nature of the X chromosome can of the arrays (Krausz et al. 2015). The only consistent find- complicate the detection of deleterious variants. Although ing of the available studies is that there is a significantly several candidate genes have been postulated, the number of higher ‘burden’ of deletions, particularly on the X chromo- promising or definitively causative genes for male infertility some, in infertile men versus normozoospermic controls. remains low when assessed in a standardized way (Smith The observed higher burden of X-linked deletions has been et al. 2017), as has recently been done by Oud and collogues hypothesized to be an expression of a generalized genomic (Oud et al. 2019); see Table 1. instability, which may also lead to general health problems. This hypothesis would be in line with the epidemiological observations showing a link between altered spermatogenic Non‑obstructive azoospermia (NOA) function and a higher incidence of morbidity (including can- cer) and lower life expectancy (Jensen et al. 2009; Salonia AR et al. 2009; Eisenberg et al. 2014). Besides the CNV burden, CGH arrays have allowed The androgen receptor (AR) remains the best studied researchers to identify a few X-linked deletions with a pos- X-linked gene so far in the etiology of male infertility. The sible role in male infertility (Krausz et al. 2015 and ref- AR is critical for diverse developmental processes and gen- erences therein). Only one potential clinically relevant X erally functions as a DNA-binding transcription factor. Upon chromosome-linked deletion has been analyzed in several binding of steroid hormone ligands in the cytoplasm, the studies. CNV67 was first described as patient specific in receptor translocates into the nucleus and then stimulates 1.1% (7/627) of infertile men from Spain and Italy (Lo the expression of androgen-responsive genes. The phenotype Giacco et al. 2014). A subsequent study in a Mediterranean of patients with mutations in AR is highly variable and var- population confirmed the role of the CNV67 in male infer- ies from complete androgen insensitivity syndrome (CAIS) tility, since it was found in 1.2% of infertile men but not in with sex reversal to mild symptoms (Brinkmann 2001). normozoospermic controls (Pinho et al. 2019). The CNV67 Patients with mild androgen insensitivity syndrome (MAIS) has also been analyzed in the Chinese population with con- in particular can have normal genitalia and isolated male troversial results. One study reported that 6/884 infertile infertility. Variants detected in infertile patients are mainly 13 208 Human Genetics (2021) 140:203–215 Table 1  Overview of disease-associated X-linked genes with testis-specific expression in mice and humans Gene Semen/testicular pheno- Proposed functions Mouse phenotype Publications Level of evidence type Phenotype: isolated spermatogenic failure AR Oligozoospermia, azoo- Hormone activated tran- Infertile; partial androgen Hiort et al. (2000), Ferlin Definitive spermia scription factor insensitivity syndrome et al. (2006) TEX11 Non-obstructive azoo- Meiotic DNA recombina- Infertile; meiotic arrest Yang et al. (2015a), Yat- Strong spermia, meiotic arrest tion senko et al. (2015) MAGEB4 Non-obstructive azoo- Germ cell-specific mitosis No mouse model Okutman et al. (2017) Limited spermia HAUS7 Severe oligozoospermia Meiotic chromosome No mouse model Li et al. (2018) Limited alignment USP26 Oligozoospermia, azoo- Deubiquitinating pepti- Fertile Paduch et al. (2005) Limited spermia dase RHOXF1/2 Oligozoospermia, severe Homeodomain transcrip- No mouse model Borgmann et al. (2016) No evidence tion factor TAF7L Non-obstructive azoo- TFIID transcription factor Complete spermatogen- Akinloye et al. (2007) No evidence spermia esis; abnormal sperm number/morphology Phenotype: obstructive azoospermia/CBAVD ADGRG2 Congenital bilateral G-protein-dependent fluid Infertile; obstructive Patat et al. (2016) Strong absence of the vas defer- homeostasis ens (CBAVD) CLDN2 Obstructive azoospermia BTB tight junction forma- Fertile Askari et al. (2019) Limiteda tion Phenotype: astheno-/teratozoospermia PIH1D3 Asthenozoospermia Flagella dynein assembly Infertile; mild PCD Paff et al. (2017), Olcese Strong et al. (2017) AKAP4 Asthenozoospermia, PKA binding in sperm Infertile; multiple Baccetti et al. (2005), Limited teratozoospermia flagellum morphological abnor- Visser et al. (2011) malities of the sperm flagella FATE1 Oligoasthenozoospermia SF1-dependent mitochon- No mouse model Olesen et al. (2003) No evidence drial regulation Phenotype: Kallmann syndrome ANOS1 Oligozoospermia, azoo- GnRH neuronal migration Infertile; hypogonado- Gonçalves et al. (2017) Definitive spermia tropic hypogonadism a Not in Oud et al. (2019) because published subsequently. Assessment according to same criteria by MV and FT independently missense mutations affecting the transactivation potential et al. 2004). The association between the AR-CAG repeat of the AR (Goglia et al. 2011; O’Hara and Smith 2015). and male infertility has been intensively studied, but effects However, of the great number of reported AR mutations seem to be small and are still debated. Longer CAG repeat (Gottlieb et al. 2012), only a small number have been clearly lengths may be a risk factor for male infertility, although associated with azoospermia or oligozoospermia. Thus, AR population studies with variation detection in healthy con- mutations can be considered a rare cause of male infertility trols argue against a strong association with unexplained (Hiort et al. 2000; Ferlin et al. 2006). male infertility, as concluded from large meta-analyses Furthermore, the AR gene contains a polymorphic CAG (Davis-Dao et al. 2007; Xiao et al. 2016). The transacti- repeat in exon 1. Repeats longer than 35 are associated with vation domain of the AR comprises a further polyglycine spinal and bulbar muscular atrophy (SBMA) and prostate tract (10–30 in length) encoded by a repeat consisting of cancer (McCrea et al. 2016; Cortes and La Spada 2018). GGN trinucleotides. However, the functional effect of the Besides, the length of the polymorphic CAG repeat encod- polyglycine tract remains controversial (Jääskeläinen 2012; ing a polyglutamine stretch (8–35 in length) in the AR N-ter- Grigorova et al. 2017). minal transactivation domain has been linked to male infer- Apart from AR, additional X-chromosomal genes have tility. The number of CAG repeats is inversely correlated been postulated to be causative for male infertility, including with transcriptional activity in vitro (Tut et al. 1997; Ferlin TEX11, MAGEB4, RHOX and TAF7L. 13 Human Genetics (2021) 140:203–215 209 TEX11 and nonsense USP26 variants were also detected in fertile controls (Ravel et al. 2006; Christensen et al. 2008; Shi TEX11 encodes a protein that regulates DNA recombina- et al. 2011), and the functional impact of missense variants tion and formation of chromosome synapses and crossovers on enzyme activity appears to be low (Zhang et al. 2015; (Adelman and Petrini 2008). The gene is highly expressed Liu et al. 2018). In vivo analysis of knock-out mice sug- in late spermatocytes and in spermatids in mice (Wang et al. gests that Usp26 is dispensable for mouse gametogenesis 2001), and disruption of Tex11 in knock-out mice causes (Felipe-Medina et al. 2019). Hence, convincing evidence meiotic arrest and results in azoospermia (Yang et al. 2008). for an association of USP26 variants with male infertility is Recently, an intragenic TEX11 deletion removing exons currently lacking. 10–12 was identified by high-resolution X-specific array- CGH in two infertile patients with NOA due to meiotic RHOX arrest and mixed testicular atrophy, respectively (Yatsenko et al. 2015). Further analysis by targeted sequencing identi- RHOX genes belong to a large multicopy homeobox gene fied further TEX11 variants of which loss-of-function (LoF) cluster (Maclean et al. 2005) and are expressed in male mutations were associated with meiotic arrest, while the germ cells (Song et al. 2013). In severe oligozoospermic role of missense variants remains less clear (Yatsenko et al. patients, variants were described in the genes RHOXF1 2015; Yang et al. 2015a). Together, these data provide strong and RHOXF2 (Borgmann et al. 2016). However, it remains evidence that LoF variants in TEX11 cause non-obstructive unclear whether variants in a single RHOX gene could be azoospermia due to meiotic arrest also in human males. causative for NOA, as other members of this large gene clus- ter might have redundant roles (MacLean and Wilkinson MAGEB4 2010). Further exploration of these ampliconic regions is necessary for a conclusive evaluation of pathogenicity. MAGEB4 is a type I melanoma antigen gene from the CTA family specifically expressed during germ cell differentiation TAF7L with a yet-unclear function, although a role in murine germ cell-specific mitosis has been postulated (Osterlund et al. The transcription factor TAF7L is expressed during meiosis 2000). This gene is part of a multicopy gene cluster of type with a dynamically regulated intracellular localization (Poin- I melanoma antigens (Lee and Potts 2017). In a single con- tud et al. 2003). Although Taf7l knock-out mice normally sanguineous family, a stop-loss variant was segregated with complete meiosis, they show reduced sperm production and azoospermia and oligozoospermia (Okutman et al. 2017). abnormal sperm morphology (Cheng et al. 2007). Variants The evidence that this gene plays a causative role in NOA in TAF7L were described as risk factors for spermatogenic is currently very limited, as (i) the functional relevance of failure (Akinloye et al. 2007), although most variants were the described stop-loss variant is still unclear; (ii) no other also found in fertile controls (Stouffs et al. 2006). Taken variants in MAGEB4 have been identified so far; and iii) the together, the disease association of TAF7L variants with role of other homologous MAGE family members remains azoospermia remains with no evidence. elusive. HAUS7 Obstructive azoospermia HAUS7 encodes a subunit of the human Augmin protein ADGRG2 complex (Goshima et al. 2008) with a proposed function in chromosome alignment during meiosis. While a single Obstructive azoospermia can be caused by congenital HAUS7 variant has been described in a familial case of bilateral absence of the vas deferens (CBAVD). In most severe oligozoospermia (Li et al. 2018), its role in NOA CBAVD cases, recessive mutations are identified in the pathology remains to be determined. cystic fibrosis transmembrane conductance regulator gene (CFTR), and CFTR analysis by sequencing and USP26 screening for large rearrangements allows for a mutation detection rate of about 87% in CBAVD patients (Ratbi USP26 is a deubiquitylating enzyme with a transcription et al. 2007). By exome sequencing of CFTR-negative pattern restricted to the male germline (Wang et al. 2001). CBAVD patients, LoF mutations were recently identified Several variants of unknown clinical significance were in the X-linked ADGRG2 gene (Patat et al. 2016), which reported in azoospermic patients (Paduch et al. 2005; Ma encodes an adhesion G protein-coupled receptor that is et al. 2016; Luddi et al. 2016). However, reported missense specifically expressed in the epididymis and efferent ducts 13 210 Human Genetics (2021) 140:203–215 (Obermann et al. 2003). Subsequently, further truncating to the female offspring in case their partner is a heterozy- and missense variants in ADGRG2 have been described gous carrier. (Yang et al. 2017; Yuan et al. 2019; Pagin et al. 2019). In accordance with human data, previous studies have already AKAP4 demonstrated that Adgrg2-knock-out male mice develop obstructive infertility (Davies et al. 2004). These studies AKAP4 encodes a major structural component of sperm established ADGRG2 as a novel gene responsible for a fibrous sheath (Turner et al. 1998). Intragenic deletions specific subtype of obstructive azoospermia. The molecu- of the AKAP3 and AKAP4 genes were first described in a lar mechanism of X-linked CBAVD remains uncertain, patient with total sperm immotility due to dysplasia of the although a regulation of fluid reabsorption by G-protein- fibrous sheath (DFS) (Baccetti et al. 2005). In 2011, an addi- dependent CFTR coupling and β-arrestin-dependent recep- tional patient with DFS was reported carrying a hemizygous tor internalization has been postulated (Zhang et al. 2018; missense variant in AKAP4 (Visser et al. 2011). The Akap4- Azimzadeh et al. 2019). defficient mice resemble the asthenozoospermic phenotype (Fang et al. 2019) observed in men. However, apart from these two case reports, AKAP4 mutations were not found in CLDN2 other cohorts of DFS patients, indicating currently limited evidence for this gene defect (Turner et al. 2001a, b; Yang Recently, a CLDN2 missense variant was described in a et al. 2015b; Pereira et al. 2015). family with several male relatives affected by obstructive azoospermia (Askari et al. 2019). The encoded protein FATE1 Claudin-2 is a paracellular transmembrane protein, and the authors proposed a gain-of-function effect on the FATE1 belongs to the CTA family, and in 2003 it was blood–epididymis barrier by the missense variant. How- sequenced in a cohort of 144 randomly selected infertile ever, Cldn2-knock-out mice are fertile, and CLDN2 was patients (Olesen et al. 2003). The authors reported two mis- shown to be expressed in leaky epithelia, in contrast to sense variants in two unrelated asthenozoospermic men. barrier-forming claudins like CLDN1/4 (Muto et al. 2010). However, for one of the carriers a family study revealed that It remains to be shown whether CLDN2 variants can be the variant was compatible with fertility; for the other one, responsible for obstructive azoospermia, e.g., by influenc- the current bioinformatic tools predict the variant as benign. ing the paracellular water transport in the epididymis. Therefore, given that no more cases have been reported since 2003, it is highly unlikely that this gene plays an important role in male infertility. Teratozoospermia and asthenozoospermia Hypogonadotropic hypogonadism PIH1D3 ANOS1 PIH1D3 (also known as CXorf41) encodes a protein which plays a role in preassembly of both outer (ODA) ANOS1 (Xp22.31), previously called KAL1, encodes the and inner (IDA) dynein arms of cilia and sperm flagella anosmin-1 glycoprotein which is involved in the migration of (Dong et al. 2014; Paff et al. 2017; Olcese et al. 2017). GnRH neurons. ANOS1 point mutations and gene deletions Point mutations and complete PIH1D3 gene deletions are the most common cause of Kallmann syndrome, a rare have been reported in patients affected by primary ciliary endocrine disease characterized by both congenital hypog- dyskinesia (PCD) (Paff et al. 2017; Olcese et al. 2017). onadotropic hypogonadism (cHH) and hyposmia/anosmia. PCD is a multisystemic disorder characterized by chronic ANOS1 mutations and deletions may explain the phenotype respiratory tract infections, abnormally positioned internal in about 5–10% of cases (Balasubramanian and Crowley organs and asthenozoospermia due to motility defects of 1993); in addition to ANOS1, mutations in more than 20 cilia and flagella (Knowles et al. 2016). PCD can be caused autosomal genes can also lead to Kallmann syndrome. by a number of genes, and transmission can be autosomal The reproductive phenotype of patients affected by cHH dominant, recessive or X-linked, with PIH1D3 as the only may range from the complete form with absent puberty validated X-linked PCD gene. For PIH1D3 carriers, ICSI (usually associated with cryptorchidism, micropenis and is an option. However, it should be noted that they are at gynecomastia) to late-onset HH (Krausz et al. 2018 and risk for transmission of the entire spectrum of symptoms references therein). Other non-reproductive and non-olfac- tory alterations described in KAL1 mutation carriers are 13 Human Genetics (2021) 140:203–215 211 bimanual synkinesia (in about 80% of cases), unilateral studies based on massive parallel sequencing remains the renal dysgenesis (30%), arched palate, tooth agenesis, discovery and the demonstration of the pathogenic effect of hearing impairment, pes cavus and ptosis, among others mutations in multicopy spermatogenesis candidate genes. (Balasubramanian and Crowley 1993; Dode and Hardelin 2009). Spermatogenesis is inducible with the administra- Acknowledgements This work was in part supported by the Span- ish Ministry of Health Instituto Carlos III-FIS (Grant number: FIS/ tion of gonadotrophins, hence cHH patients may generate FEDER-PI14/01250; PI17/01822), the European Commission, Repro- their own biological children either through natural con- ductive Biology Early Research Training (REPROTRAIN, project ception or assisted reproductive techniques. Preimplanta- number: 289880) (both awarded to CK and AR-E), the Fondazione tion genetic testing in syndromic forms is advisable to Ente Cassa di Risparmio di Firenze (Grant to CK), and the German Research Foundation Clinical Research Unit ‘Male Germ Cells: from prevent the transmission of the mutation. Genes to Function’ (DFG CRU326, Grants to FT). The authors thank Celeste Brennecka for editorial assistance. Compliance with ethical standards Conclusions Conflict of interest The authors declare that they have no conflicts of Currently, validated X chromosome-linked monogenic interest. causes of male infertility are surprisingly rare. From a Open Access This article is licensed under a Creative Commons Attri- total of eight genes studied in men with idiopathic oligo/ bution 4.0 International License, which permits use, sharing, adapta- azoospermia, only AR and TEX11 reach sufficiently high tion, distribution and reproduction in any medium or format, as long clinical evidence for inclusion in diagnostic testing. On as you give appropriate credit to the original author(s) and the source, the other hand, targeted studies on highly specific infer- provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are tile phenotypes have successfully identified a novel gene included in the article’s Creative Commons licence, unless indicated for congenital absence of vas deferens (ADGRG2) and for otherwise in a credit line to the material. If material is not included in complete asthenozoospermia due to primary ciliary dys- the article’s Creative Commons licence and your intended use is not kinesia (PIH1D3). permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a Although the X chromosome contains several structural copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. elements which may predispose it to AZF-like deletions, only one deletion, CNV67, has been the object of multi- ple validation studies. This deletion affects MAGEA9, an ampliconic gene belonging to one of the CTA gene fami- References lies. Similar to CNV67, the majority of X-linked deletions and duplications affect or remove CTA genes. 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