Non-syndromic monogenic male infertility

Abstract

Infertility is a widespread clinical problem affecting 8-12% of couples worldwide. Of these, about 30% are diagnosed with idiopathic infertility since no causative factor is found. Overall 40-50% of cases are due to male reproductive defects. Numerical or structural chromosome abnormalities have long been associated with male infertility. Monogenic mutations have only recently been addressed in the pathogenesis of this condition. Mutations of specific genes involved in meiosis, mitosis or spermiohistogenesis result in spermatogenic failure, leading to the following anomalies: insufficient (oligozoospermia) or no (azoospermia) sperm production, limited progressive and/or total sperm motility (asthenozoospermia), altered sperm morphology (teratozoospermia), or combinations thereof. Androgen insensitivity, causing hormonal and sexual impairment in males with normal karyotype, also affects male fertility. The genetic causes of non-syndromic monogenic of male infertility are summarized in this article and a gene panel is proposed. (www.actabiomedica.it)

Introduction

Infertility is defined as failure to achieve a successful pregnancy after 12 months or more of regular unprotected intercourse (1). Its prevalence is not negligible, since about 48.5 million couples worldwide do not reach pregnancy after 5 years (2). Overall, about 50% of cases are due to a male factor infertility (3). Genetic causes have been estimated to exist in about 15% of infertile patients, especially in those with azoospermia or severe oligozoospermia (3).

Genetic causes of male infertility can be classified as pre-testicular (affecting hypothalamic-pituitary function), testicular (causing dysfunction at testicular level) and post-testicular (leading to obstruction or interfering with ejaculation of sperm). Other causes include androgen resistance and disorders of sexual development. Genetic causes of male infertility are outlined in Table 1.

Table 1.

Main causes of genetic forms of male infertility.

Pre-testicular causes	Normosmic hypogonadotropic hypogonadism Anosmic hypogonadotropic hypogonadism (Kallmann syndrome) Prader-Willy syndrome Laurence-Moon-Biedl syndrome Others
Testicular forms	Klinefelter syndrome Numerical chromosomal abnormalities Y chromosome microdeletions Chromosomal translocations Down syndrome Myotonic dystrophy (Steinert syndrome)
Post-testicular causes	Kartagener syndrome Congenital bilateral deferent duct agenesis Young syndrome
Others	Androgen resistance Disorders of sexual development

Despite a proper diagnostic work-up, the etiology of male infertility remains elusive in up to 75% of cases (4). In recent years, much effort has been made to investigate new candidate genes responsible for male infertility caused by single-gene mutations (5,6). Several genes involved in meiotic and mitotic divisions and in spermiohistogenesis have been examined as potential targets. They may play a role in the pathogenesis of defects of sperm number (oligozoospermia or azoospermia), motility (asthenozoospermia) or morphology (teratozoospermia) (7).

In this review we describe genes belonging to the panels developed by us for the diagnosis of monogenic spermatogenic failure and androgen insensitivity syndrome.

Genes involved in sperm number defects

Sperm number defects include azoospermia and oligozoospermia. Azoospermia is the absence of spermatozoa in semen. It affects 1% of the male population and accounts for 20% of all cases of male infertility (8). In about the 40% of patients, spermatogenesis occurs in a regular way but sperm emission is impaired by seminal duct obstruction (obstructive azoospermia) (9); in the other cases, azoospermia is due to spermatogenic failure (non-obstructive azoospermia) (10). Genetic causes of azoospermia include chromosome anomalies (numerical or structural aberrations of autosomal or sexual chromosomes) that affect 5% of all infertile males and 16% of males with azoospermia or oligozoospermia (11). In 5-15% of cases, azoospermia or oligozoospermia is associated with Y chromosome microdeletions; 6-8% of cases with obstructive azoospermia are associated with mutations in the cystic fibrosis transmembrane receptor gene (CFTR) that causes congenital bilateral absence of the vas deferens (11). Point mutations that cause azoospermia were recently found in the following genes: NR5A1, SYCP3, ZMYND15, TAF4B, TEX11 NANOS1, PLK4, MEIOB, SYCE1, USP9Y, SOHLH1, TEX15, HSF2 and KLHL10 (12-19) (Table 2).

Table 2.

Genes associated with spermatogenic failure

Gene	Inheritance	OMIM gene	OMIM phenotype	OMIM or HGMD phenotype ID	Spermatogenic defect
NR5A1	AR	184757	SPGF8	613957	AZS/OZS
SYCP3	AD	604759	SPGF4	270960	AZS/OZS
ZMYND15	AR	614312	SPGF14	615842	AZS/OZS
TAF4B	AR	601689	SPGF13	615841	AZS/OZS
TEX11	XLR	300311	SPGFX2	309120	AZS
NANOS1	AD	608226	SPGF12	615413	AZS/OZS/OZS+ASTHZ+TZS
PLK4	AD	605031	/	1556988045	AZS
MEIOB	AR	617670	SPGF22	617706	AZS
SYCE1	AR	611486	SPGF15	616950	AZS
USP9Y	YL	400005	SPGFY2	400042	AZS
SOHLH1	AD	610224	SPGF32	618115	AZS
TEX15	AR	605795	SPGF25	617960	AZS/OZS
HSF2	AD	140581	/	702994563	AZS
KLHL10	AD	608778	SPGF11	615081	OZS; TZS; AZS
AURKC	AR	603495	SPGF5	243060	TZS (macrozoospermia)
DPY19L2	AR	613893	SPGF9	613958	TZS (globozoospermia)
SPATA16	AR	609856	SPGF6	102530	TZS (globozoospermia)
PICK1	AR	605926	/	247048065	TZS (globozoospermia)
BRDT	AR	602144	SPGF21	617644	ASS
SUN5	AR	613942	SPGF16	617187	ASS
SLC26A8	AD	608480	SPGF3	606766	AZS
CATSPER1	AR	606389	SPGF7	612997	AZS
SEPT12	AD	611562	SPGF10	614822	AZS; OZS+ASTHZ+TZS
CFAP43	AR	617558	SPGF19	617592	MMAF
CFAP44	AR	617559	SPGF20	617593	MMAF
DNAH1	AR	603332	SPGF18	617576	MMAF
PLCZ1	AR	608075	SPGF17	617214	OAF

SPGF = spermatogenic failure; OZS = oligozoospermia; AZS = azoospermia; ASTHZ = asthenozoospermia; TZS = teratozoospermia; OZS+ASTHZ+TZS = oligoasthenoteratozoospermia; ASS = acephalic spermatozoa syndrome; MMAF = multiple morphological abnormalities of the flagellum; OAF = oocyte activation failure; AR = autosomal recessive; AD = autosomal dominant; XLR = X-linked recessive; YL = Y-linked; HGMD = Human Gene Mutation Database (https://portal.biobase-international.com/hgmd/pro/)

Frameshift mutations in ZMYND15 cause the SPGF14 phenotype. The protein encoded by this gene is involved in temporally normal haploid gene expression during spermatogenesis (13).

A homozygous mutation in SYCE1 is associated with the SPGF15 phenotype. This gene encodes a member of the synaptonemal complex, a structure that physically links homologous chromosomes during meiosis I (14).

Mutations in TEX11 have been associated with meiotic arrest and azoospermia with a frequency of 1-15% in the azoospermic males. TEX11-encoded protein regulates the coupling of homologous chromosomes in double-strand DNA repair through formation of the synaptonemal complex and the chiasma during the crossover process (20). A similar role is performed by SYCP3 that has also been found mutated in sterile men (16).

SOHLH1 is mutated in some cases of azoospermia and encodes a testicular transcription factor essential for spermatogenesis (21).

A mutation in NR5A1, encoding steroidogenic factor 1, has been reported in a Pakistani patient with meiotic arrest and normal levels of follicle-stimulating and luteinizing hormones, and testosterone (22).

Finally, a patient with spermatogenesis blocked at the spermatocyte stage had a dominant negative mutation in HSF2, encoding heat shock transcription factor 2 (23).

Genes involved in defects of sperm morphology

Teratozoospermia is a heterogeneous group of disorders. Sperm morphological evaluation considers the main functional regions (head, body and tail), which may have anomalies in shape and size. Phenotype may involve a single type of malformation in a single patient or different types of malformation in the same patient (24). Recent studies in consanguineous families and small phenotypically homogeneous cohorts has made it possible to identify autosomal recessive cases of teratozoospermia.

Macrozoospermia is a rare condition observed in <1% of infertile men. It is characterized by a high percentage of sperm with large irregularly-shaped heads, multiple flagella and abnormal acrosome. Macrozoospermia is generally associated with severe oligoasthenozoospermia and a high rate of sperm chromosomal anomalies. Homozygous mutations in AURKC are the major cause of macrozoospermia. AURKC is highly expressed in male germline cells and is involved in chromosomal segregation and cytokinesis (25).

Globozoospermia is a rare condition characterized by round sperm lacking acrosomes. Genes reported mutated in patients with this condition are SPATA16 and DPY19L2. SPATA16 encodes a protein specific to Golgi apparatus, highly expressed in the testes. A genetic study in a single consanguineous family with male infertility due to globozoospermia revealed a homozygous variant in the three affected siblings (26). Subsequent studies identified mutations in DPY19L2 in 66.7% of 54 probands with globozoospermia (25). The protein encoded by DPY19L2 is necessary for elongation of the sperm head and acrosome formation during spermatogenesis (27).

To date, more than 20 cases of patients with acephalic sperm have been reported. Biallelic mutations in SUN5 can be found in 47% of cases (28), and a homozygous mutation in BRDT has been reported in a consanguineous family (27).

Genes currently associated with morphological sperm defects are: AURKC, ZPBP, DPY19L2, SPATA16, PICK1, BRDT and SUN5 (Table 2).

Genes involved in sperm motility defects

Asthenozoospermia is a condition leading to reduced sperm motility due to defects in the flagellum. The axoneme, outer dense fibers, mitochondria or fibrous sheath of the flagellum may be affected (30). Ultrastructural defects in 9+2 axoneme structure may involve the outer or inner dynein arms, central microtubules and radial spokes.

Biallelic mutations in DNAH1, that encodes heavy chain 1 of axonemal dynein expressed in the testis, cause a heterogeneous group of anomalies defined as multiple morphological anomalies of the flagellum (MMAF) (31,32). Mutations in DNAH1 are the major cause of MMAF and account for 28-44% of cases (33). In four out of 30 Chinese subjects with MMAF, Tang et al. identified mutations in either CFAP43 or CFAP44. The proteins encoded, CFAP43 and CFAP44, are cilium- and flagellum-associated, almost exclusively expressed in the testis.

The genes associated with sperm motility defects are: SLC26A8, CATSPER1, SEPT12, CFAP43, CFAP44, DNAH1 and PLCZ1 (Table 2).

We uses a multi-gene NGS panel to detect nucleotide variations in coding exons and flanking introns of the aforementioned genes (Table 2). Pathogenic variants may be missense, nonsense, splicing or small indels. Large deletions/duplications have been reported in USP9Y, DPY19L2, SPATA16, SUN5 and CFAP43. We use MLPA to detect duplications and deletions in the latter genes.

Androgen insensitivity syndrome

Androgens and their receptor are essential for the development and maintenance of the male phenotype and spermatogenesis. The gene AR encoding the androgen receptor is X-linked and the encoded protein is a nuclear receptor that recognizes the canonical androgen response elements on the genome (34).

Mutations in AR (OMIM gene ID: 313700) cause a spectrum of conditions defined as androgen insensitivity syndromes (OMIM phenotype ID: 300068), which are disorders of sexual development characterized by external female genitalia, ambiguous genitalia or virilization defects, 46, XY karyotype and little or no response to androgens. Androgen insensitivity syndromes (AIS) may be complete or partial. Patients with the complete form have a female phenotype with little or no pubic/axillary hair or secondary sexual features and a 46, XY karyotype (35).

Mutations can be found throughout the gene, but are more frequent in five exons that encode ligand-binding domains. The androgen insensitivity syndrome phenotype is due to loss-of-function mutations in AR, making target cells insensitive to testosterone or dihydrotestosterone. In 95% of cases, a mutation in the AR gene can be detected. In 30% of cases the mutations are de novo. The disorder is inherited with a X-linked recessive inheritance (36).

Clinical diagnosis is based on symptoms and biochemical features of 46, XY females. The typical hormonal profile of adults includes increased basal concentrations of luteinizing hormone and testosterone. Serum levels of anti-Mullerian hormone may be normal or elevated. Subjects with partial loss-of-function mutations in AR (partial AIS) can have infertility as first or only symptom. Partial androgen insensitivity syndrome may be suspected in infertile males with high plasma levels of testosterone and LH, although a precise threshold has not yet been established (36).

Interestingly, mutations in AR can be identified in 2-3% of cases of azoospermia and oligozoospermia (38). There may be a genotype-phenotype correlation in AIS patients, as reported in the Androgen Receptor Gene Mutations Database (39), whereas a correlation in partial AIS patients is less clear and the same mutations can express as different phenotypes (37). The AR gene may therefore be included among candidate genes in patients with apparently idiopathic azoospermia or oligozoospermia.

Conclusions

We created a NGS panel to detect nucleotide variations in coding exons and flanking regions of all the genes associated with infertility. When a suspect of male infertility is present we perform the analysis of all the genes present in this short article.

In order to have a high diagnostic yield, we developed a NGS test that reaches an analytical sensitivity (proportion of true positives) and an analytical specificity (proportion of true negatives) of ≥99% (coverage depth ≥10x).

Conflict of interest:

Each author declares that he or she has no commercial associations (e.g. consultancies, stock ownership, equity interest, patent/licensing arrangement etc.) that might pose a conflict of interest in connection with the submitted article