Genetic Causes of Qualitative Sperm Defects: A Narrative Review of Clinical Evidence.

Several genes are implicated in spermatogenesis and fertility regulation, and these genes are presently being analysed in clinical practice due to their involvement in male factor infertility (MFI). However, there are still few genetic analyses that are currently recommended for use in clinical practice. In this manuscript, we reviewed the genetic causes of qualitative sperm defects. We distinguished between alterations causing reduced sperm motility (asthenozoospermia) and alterations causing changes in the typical morphology of sperm (teratozoospermia). In detail, the genetic causes of reduced sperm motility may be found in the alteration of genes associated with sperm mitochondrial DNA, mitochondrial proteins, ion transport and channels, and flagellar proteins. On the other hand, the genetic causes of changes in typical sperm morphology are related to conditions with a strong genetic basis, such as macrozoospermia, globozoospermia, and acephalic spermatozoa syndrome. We tried to distinguish alterations approved for routine clinical application from those still unsupported by adequate clinical studies. The most important aspect of the study was related to the correct identification of subjects to be tested and the correct application of genetic tests based on clear clinical data. The correct application of available genetic tests in a scenario where reduced sperm motility and changes in sperm morphology have been observed enables the delivery of a defined diagnosis and plays an important role in clinical decision-making. Finally, clarifying the genetic causes of MFI might, in future, contribute to reducing the proportion of so-called idiopathic MFI, which might indeed be defined as a subtype of MFI whose cause has not yet been revealed.

A cryptozoospermic infertile male with Y chromosome AZFc microdeletion and low FSH levels due to a simultaneous polymorphism in the FSHB gene: a case report.

Genetic causes account for 10-15% of male factor infertility, making the genetic investigation an essential and useful tool, mainly in azoospermic and severely oligozoospermic men. In these patients, the most frequent findings are chromosomal abnormalities and Y chromosome long arm microdeletions, which cause a primary severe spermatogenic impairment with classically increased levels of FSH. On the other hand, polymorphisms in the FSH receptor (FSHR) and FSH beta chain (FSHB) genes have been associated with different FSH plasma levels, due to variations in the receptor sensitivity (FSHR) or in the production of FSH from the pituitary gland (FSHB). Here, we describe an unusual patient with a combined genetic alteration (classic AZFc deletion of the Y chromosome and TT homozygosity for the -211G>T polymorphism in the FSHB gene (rs10835638)), presenting with cryptozoospermia, severe hypospermatogenesis, and normal LH and testosterone plasma concentrations, but low FSH levels. The patient partially benefitted from treatment with FSH (150 IU three times/week for 6 months) which allowed him to cryopreserve enough motile spermatozoa to be used for intracytoplasmic sperm injection. According to our knowledge, this is the first report of an infertile man with AZFc microdeletion with low FSH plasma concentrations related to homozygosity for the -211G>T polymorphism in the FSHB gene.

Study of novel androgen receptor V770 variant in androgen insensitivity syndrome patients reveals the transitional state of the androgen receptor ligand binding domain homodimer.

We report the discovery of the androgen receptor missense mutation V770D, that was found in two sisters suffering from complete androgen insensitivity. Experimental validation of AR V770 variants demonstrated that AR V770D was transcriptionally inactive due to the inability to dimerize and a reduced ligand binding affinity. The more conservative AR V770A variant showed a dimerization defect at low levels of DHT with a partial recovery of the transcriptional activity and of the receptor's ability to dimerize when increasing the DHT levels. With V770 located outside of the proposed LBD dimerization interface of the AR LBD homodimer crystal structure, the effects of the V770A mutation on AR dimerization were unexpected. We therefore explored whether the AR LBD dimerization interface would be better described by an alternative dimerization mode based on available human homodimeric LBD crystal structures of other nuclear receptors. Superposition of the monomeric AR LBD in the homodimeric crystal structures of GR, PR, ER, CAR, TRß, and HNF-4α showed that the GR-like LBD dimer model was energetically the most stable. Moreover, V770 was a key energy residue in the GR-like LBD dimer while it was not involved in the stabilization of the AR LBD homodimer according to the crystal structure. Additionally, the observation that 4 AIS mutations impacted the stability of the AR LBD dimer while 16 mutations affected the GR-like LBD dimer, suggested that the AR LBD dimer crystal is a snapshot of a breathing AR LBD homodimer that can transition into the GR-like LBD dimer model.

Mutational Screening of Androgen Receptor Gene in 8224 Men of Infertile Couples.

CONTEXT: Mutations in the androgen receptor (AR) gene might be associated with infertility mainly because they cause various degrees of androgen insensitivity. OBJECTIVE: The aim of the study was to evaluate the frequency and type of AR variants in a large cohort of infertile males. METHODS: A total of 8224 males of Italian idiopathic infertile couples were referred to the University Hospital of Padova. The main outcome measures were mutational screening of AR, computational, and functional analyses. RESULTS: We found 131 patients (1.6%) harboring 45 variants in AR gene, of which 18 were novel missense AR variants. Patients with AR gene variants had lower sperm count (P = .048), higher testosterone (T) concentration (P < .0001), and higher androgen sensitivity index (ASI) (luteinizing hormone × T, P < .001) than patients without variants. Statistical analyses found T ≥ 15.38 nmol/L and ASI ≥ 180 IU × nmol/L2 as the threshold values to discriminate with good accuracy patients with AR variants. Patients with oligozoospermia and T ≥ 15.38 nmol/L had a 9-fold increased risk of harboring mutations compared with patients with normal sperm count and T < 15.38 nmol/L (odds ratio 9.29, 95% CI 5.07-17.02). Using computational and functional approaches, we identified 2 novel variants, L595P and L791I, as potentially pathogenic. CONCLUSION: This is the largest study screening AR gene variants in men of idiopathic infertile couples. We found that the prevalence of variants increased to 3.4% in oligozoospermic subjects with T ≥ 15.38 nmol/L. Conversely, more than 80% of men with AR gene variants had low sperm count and high T levels. Based on our findings, we suggest AR sequencing as a routine genetic test in cases of idiopathic oligozoospermia with T ≥ 15.38 nmol/L.

A novel genetic variant in DNAI2 detected by custom gene panel in a newborn with Primary Ciliary Dyskinesia: case report.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a highly heterogeneous genetic disorder caused by defects in motile cilia. The hallmark features of PCD are the chronic infections of the respiratory tract, moreover, clinical manifestations include also laterality defects and risk of male infertility. Clinical phenotypes of PCD are the result of mutations in genes encoding components of axonema or factors involved in axonemal assembly. Recent studies have identified over 45 PCD-associated genes, therefore, molecular analysis represents a powerful diagnostic tool to confirm and uncover new genetic causes of this rare disease. CASE PRESENTATION: Here, we describe a female infant of Moroccan origin with normal pressure hydrocephalus (NPH) in addition to most common PCD symptoms. Transmission Electron Microscopy (TEM) and molecular tests, such as a Next generation Sequencing panel and a custom array CGH, were performed for diagnosis of PCD. TEM revealed outer dynein arm (ODA) defects, whilst molecular analyses detected a novel 6,9 kb microdeletion in DNAI2 gene. CONCLUSIONS: Since DNAI2 mutations are very rare, this case report contributes to better delineate the important role of DNAI2 as causative of PCD phenotype, suggesting, furthermore, that the variations in DNAI2 may be as a new genetic risk factor for NPH. Indeed, although the association of hydrocephalus with PCD has been well documented, however, only a small number of human patients show this defect. Furthermore, this study highlights the importance of high-throughput technologies in advancing our understanding of heterogeneous genetic disorders.

Comparison of NGS panel and Sanger sequencing for genotyping CAG repeats in the AR gene.

BACKGROUND: The androgen receptor (AR) is a nuclear receptor, encoded by the AR gene on the X chromosome. Within the first exon of the AR gene, two short tandem repeats (STR), CAG and GGC, are a source of polymorphism in the population. Therefore, high-throughput methods for screening AR, such as next-generation sequencing (NGS), are sought after; however, data generated by NGS are limited by the availability of bioinformatics tools. Here, we evaluated the accuracy of the bioinformatics tool HipSTR in detecting and quantify CAG repeats within the AR gene. METHOD: The AR gene of 228 infertile men was sequenced using NGSgene panel. Data generated were analyzed with HipSTR to detect CAG repeats. The accuracy was compared with the results obtained with Sanger. RESULTS: We found that HipSTR was more accurate than Sanger in genotyping normal karyotype men (46,XY), however, it was more likely to misidentify homozygote genotypes in men with Klinefelter syndrome (47,XXY). CONCLUSION: Our findings show that the bioinformatics tool HipSTR is 100% accurate in detecting and assessing AR CAG repeats in infertile men (46,XY) as well as in men with low-level mosaicism.

Is there any clinical relevant difference between non mosaic Klinefelter Syndrome patients with or without Androgen Receptor variations?

Klinefelter Syndrome (KS) is the most common chromosomal disorder in men leading to non-obstructive azoospermia. Spermatozoa can be found by TESE in about 50% of adults with KS despite severe testicular degeneration. We evaluated AR variations and polymorphism length in 135 non-mosaic KS patients, aimed to find possible correlation with clinical features, sex hormones and sperm retrieval. Among 135 KS patients we found AR variations in eight subjects (5.9%). All variations but one caused a single amino acid substitution. Four variations P392S, Q58L, L548F, A475V found in six patients had been previously described to be associated with different degrees of androgen insensitivity. Moreover we observed in two patients Y359F and D732D novel variations representing respectively a missense variation and a synonymous variation not leading to amino acid substitution. All the Klinefelter patients with AR gene variations were azoospermic. Spermatozoa were retrieved with TESE for two men (40%), sperm retrieval was unsuccessful in other 3 patients. This is the only study reporting AR variations in KS patients. Relevant clinical differences not emerged between AR mutated and not AR mutated KS patients, but does each variation play an important role in the trasmission to the offspring obtained by ART in this patients?

Mutational screening of NR5A1 gene encoding steroidogenic factor 1 in cryptorchidism and male factor infertility and functional analysis of seven undescribed mutations.

OBJECTIVE: To study the role of NR5A1 in cryptorchidism and male factor infertility. Mutations in NR5A1 have been initially associated with primary adrenal insufficiency and 46,XY gonadal dysgenesis and more recently with less severe phenotypes, including preliminary descriptions in severe forms of male factor infertility. Far less clear is the possible involvement of NR5A1 mutations in cryptorchidism. DESIGN: Retrospective cross-sectional cohort study and functional analysis of mutant proteins. SETTING: University department. PATIENT(S): Nine hundred fifty-nine subjects, including children with cryptorchidism and adults with different semen phenotypes associated or not associated with a history of cryptorchidism. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Mutation screening of NR5A1 by sequencing all exons. Functional analysis of mutant proteins by transactivation assays of CYP11A1 and CYP17A1 promoters. RESULT(S): We identified seven undescribed and one previously described missense mutation in subjects with severe spermatogenic impairment, without (4/236, 1.7%) and with (3/85, 3.5%) a history of cryptorchidism. Newborns with cryptorchidism carry NR5A1 mutations at low frequency (0.7%), whereas no mutations were found in milder forms of infertility and normozoospermia, irrespective of the presence of cryptorchidism. The mutant proteins showed impaired transactivation of gonadal promoters. A single nucleotide polymorphism (rs1110061; c.437 G→C; p.Gly146Ala) was also associated with more severe forms of spermatogenic impairment with cryptorchidism. CONCLUSION(S): This study, combined with what is already known about NR5A1-associated phenotypes, suggests considering mutations in this gene as a novel genetic cause of more severe forms of male factor infertility, especially when associated with a history of cryptorchidism.

Identification of a novel mutation in exon 1 of androgen receptor gene in an azoospermic patient with mild androgen insensitivity syndrome: case report and literature review.

OBJECTIVE: To report a case of an azoospermic subject with mild androgen insensitivity syndrome (MAIS) and review the relevant literature. DESIGN: Case report. SETTING: Academic research hospital. PATIENT(S): A 49-year-old man with undermasculinized features and a history of cryptorchidism and azoospermia. INTERVENTION(S): Hormonal evaluation and genetic testing of the androgen receptor gene (AR). MAIN OUTCOME MEASURE(S): Hormonal levels and sequence chromatogram of the proband and his mother. RESULT(S): We found total T in the normal range and high levels of gonadotropins. Karyotype was 46,XY. Genetic testing identified a novel mutation of exon 1 of AR, which resulted in an alanine to serine substitution in the transactivation domain at codon 240 (A240S). Fourteen other mutations of exon 1 of AR have been associated with MAIS to date. CONCLUSION(S): The novel mutation A240S of AR is involved in MAIS, a syndrome associated with azoospermia.

Toward a pharmacogenetic approach to male infertility: polymorphism of follicle-stimulating hormone beta-subunit promoter.

OBJECTIVE: To verify in another population (Italians) whether a single-nucleotide polymorphism in the FSHB gene promoter previously associated with serum FSH levels in Estonians is indeed associated with sperm count and FSH plasma levels, and especially to verify whether it could be a pharmacogenetic tool for the treatment of male infertility with FSH. DESIGN: Cross-sectional and prospective study. SETTING: Infertility center at a university hospital. PATIENT(S): Five hundred fourteen subjects with nonobstructive azoospermia and oligozoospermia and 248 subjects with normozoospermia. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen parameters, reproductive hormone levels, and FSHB -211 G/T polymorphism (rs10835638). RESULT(S): FSHB -211 TT genotype was associated with significantly lower FSH levels (mean ± SD: 3.3 ± 2.5 IU/L vs. 9.1 ± 8.9 IU/L in GG homozygotes). TT homozygotes were seen in 25% of subjects with azoo-oligozoospermia and low FSH levels (≤1.5 IU/L). We did not observe this genotype in men with high FSH levels (>8 IU/L) or in men with normozoospermia. Treatment with FSH induced a significantly higher improvement in sperm count and quality in TT homozygotes regarding carriers of the G allele. CONCLUSION(S): FSHB -211 TT genotype might represent a novel treatable form of male infertility characterized by severe spermatogenic impairment and low or inappropriately normal FSH plasma levels. This genetic marker could represent a valid pharmacogenetic approach for identification of potential responders to FSH treatment.

Bone mass in subjects with Klinefelter syndrome: role of testosterone levels and androgen receptor gene CAG polymorphism.

CONTEXT: Klinefelter syndrome (KS) is a chromosomal alteration characterized by supernumerary X-chromosome(s), primary hypogonadism, decreased pubertal peak bone mineral density (BMD), and accelerated bone loss during adulthood. Decreased bone mass has been traditionally related to low testosterone levels. However, testosterone replacement therapy does not necessarily increase bone mass in these patients, and low BMD can be observed also in patients with normal testosterone levels. The androgen receptor (AR) gene CAG polymorphism seems to modulate the sensitivity to testosterone and previous studies have related it to some clinical aspects of KS, to include BMD, gynecomastia, testes and prostate volume, and hemoglobin concentration. OBJECTIVE: To analyze the relation between bone mass, testosterone, and AR CAG polymorphism in men with KS. DESIGN: Cross-sectional cohort study. SETTING: University department. PATIENTS: One hundred twelve consecutive treatment-naïve 47,XXY Klinefelter patients (mean age 33.5 ± 4.7 yr) and 51 age-matched normal male controls. MAIN OUTCOME MEASURES: Dual-energy x-ray absorptiometry, CAG repeat length polymorphism, X-chromosome inactivation, and testosterone levels. RESULTS: Forty-nine of 112 KS subjects (42.5%) had low bone mass (osteopenia or osteoporosis). Lumbar and/or femoral T-scores were lower in KS patients compared with controls. No significant relationship was observed between testosterone levels and bone parameters, and the prevalence of osteopenia/osteoporosis was similar in subjects with normal and low testosterone levels (43.7% and 40.5%, respectively). The mean CAG repeat length calculated after X-chromosome inactivation analysis showed no differences between patients with normal and low bone mass. CONCLUSIONS: Testosterone levels and AR CAG polymorphism are not associated with bone mass phenotype in KS.

Mutations in INSL3 and RXFP2 genes in cryptorchid boys.

Mutations in the INSL3 and RXFP2 genes have been associated with human cryptorchidism but with contrasting data. We analyzed the frequency of mutations in these genes in 600 newborns with cryptorchidism (396 unilateral and 204 bilateral) and 300 noncryptorchid subjects. We found five RXFP2 mutations in five bilateral cryptorchid boys, one INSL3 mutation in a unilateral cryptorchid boy, and one INSL3 mutation in a boy with unilateral cryptorchidism at birth and spontaneous descent during the first month of life. Overall, the frequency of INSL3 and RXFP2 mutations was therefore 7/600 at birth (1.2%) and 7/303 (2.3%) in persistent cryptorchid boys, with a higher prevalence of bilateral forms (5/120, 4.2%). No mutations were found in controls. This study confirmed the association between INSL3 and RXFP2 gene mutations and human cryptorchidism.

Relationship between vascular damage degrees and endothelial progenitor cells in patients with erectile dysfunction: effect of vardenafil administration and PDE5 expression in the bone marrow.

OBJECTIVES: To evaluate the levels of circulating progenitor cells (PCs) and the effect of a single dose of vardenafil 20mg on the number of these cells in men with erectile dysfunction (ED) and various degree of vascular injury at the carotid artery level. METHODS: Sixty-eight patients with ED and various degree of carotid damage, and 25 controls were enrolled. Patients were divided into three groups according to their intima media thickness (IMT) status (normal, mild increase, or plaque). All subjects received vardenafil 20mg, and evaluation of the number of circulating PCs was performed at baseline and 4h after vardenafil administration. An RNA expression analysis of phosphodiesterase type 5 (PDE5) on bone marrow was also performed. RESULTS: We found a significant reduction of circulating PCs in ED patients with respect to controls and a reduction in PC counts in patients with mild IMT increase or plaque, but not in those with normal IMT. Four hours after vardenafil administration we observed an increase in the number of PCs in all patients and controls. Reverse transcriptase-polymerase chain reaction analysis showed that human bone marrow expresses PDE5 messenger RNA. CONCLUSIONS: Patients with ED and a low number of circulating PCs may be considered at increased risk for an endothelial dysfunction. An impaired response to vardenafil stimulus may be proposed as a surrogate marker of a patient's endothelial regenerative ability.

Male infertility and androgen receptor gene mutations: clinical features and identification of seven novel mutations.

OBJECTIVE: Androgens and a functioning androgen receptor (AR) are essential for development and maintenance of the male phenotype and spermatogenesis. Consistent with this, mutations in the AR gene cause a variety of defects related to androgen insensitivity, ranging from complete feminization to phenotypic males with infertility. The aim of his study was to analyse the prevalence of AR gene mutations in male infertility and to clarify the genotype-phenotype relation. DESIGN: Males with infertility were recruited consecutively at the Centre for Male Gamete Cryopreservation at the University of Padova from January 1996 to January 2005. PATIENTS: One thousand five hundred and seventeen men with < 10 million sperm/ml and 310 age-matched normozoospermic controls. METHODS: Screening for AR gene mutation was done by DHPLC and sequencing, and reproductive hormone concentrations were measured. RESULTS: We found 20 mutations in 26 of 1517 patients (1.7%) and no mutations in controls. A high number of mutations localized in exon 1 of the AR gene coding for the transactivation domain of the protein. Of 20 mutations, 7 represent novel mutations. With respect to men without AR mutations, subjects with AR mutations have lower ejaculate volume, higher testosterone levels, higher oestradiol levels, and higher androgen sensitivity index. However, the ranges for these variables were highly overlapping between men with and without AR gene mutations. Also clinical manifestations of AR mutations are not unique and 22 men had only spermatogenic impairment. CONCLUSIONS: AR gene mutations are quite frequent in unselected infertile men but no specific hormonal or clinical data could be used to preselect patients at risk of mutations.

Androgen receptor gene CAG and GGC repeat lengths in cryptorchidism.

OBJECTIVE: Cryptorchidism is the most common congenital birth defect in male children, and accumulating evidence suggests that genetic abnormalities may be associated with it. The androgen receptor has two polymorphic sites in exon 1, with different numbers of CAG and GGC repeats, resulting in variable lengths of polyglutamine and polyglycine stretches. Longer CAG repeats result in a reduced androgen receptor transcriptional activity, but the role of the GGC triplets is less clear. In this study we analysed CAG and GGC repeat lengths in men with a history of cryptorchidism, associated or not with impairment of sperm production, in comparison with normal fertile subjects. METHODS: We analysed CAG and GGC repeat lengths in a group of 105 ex-cryptorchid men in comparison with 115 fertile non-cryptorchid men. RESULTS: No difference was found between patients and controls in the mean and median values, and in distribution of CAG and GGC, when considered separately. However, the analysis of the joint distribution of CAG and GGC showed that some combinations are significantly more frequent in men with bilateral cryptorchidism (who frequently presented severe testiculopathies), in a manner similar to that found in idiopathic infertile subjects. CONCLUSIONS: Although further studies are needed to elucidate the possible role of specific CAG/GGC combinations as a causative factor, these data suggest a possible association between androgen receptor gene polymorphisms and cryptorchidism.

A novel circulating hormone of testis origin in humans.

Insulin-like factor 3 (INSL3) is a member of the relaxin-insulin family, and it is expressed in pre- and postnatal Leydig cells of the testis. This peptide affects testicular descent during embryonic development, and mutations in INSL3 gene or its receptor LGR8 (leucine-rich repeat-containing G protein-coupled receptor 8)/GREAT (G protein-coupled receptor affecting testicular descent) cause cryptorchidism in humans. The expression of LGR8/GREAT in different tissues and the production of INSL3 also by adult-type Leydig cells suggest additional roles of this hormonal system in adulthood. In this preliminary report we performed the first analysis in humans of INSL3 using a novel RIA kit to measure INSL3 concentrations in serum of normal men and with different testicular pathologies. The results show that INSL3 is circulating in adult men, and it is almost exclusively of testicular origin. Subjects with severe testicular damage, such as men with severe infertility, produce low amount of INSL3, and the concentrations of this hormone seem to reflect the functional status of the Leydig cells. In particular, INSL3 concentrations may be an even more sensitive marker of Leydig cell function than testosterone itself. Analysis of men treated with different combinations of hormones of the hypothalamus-pituitary-testis axis suggests that the production of INSL3 is related to LH in a manner similar to that of the LH-testosterone axis.