Sequencing of a 'mouse azoospermia' gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest.

STUDY QUESTION: What is the diagnostic potential of next generation sequencing (NGS) based on a 'mouse azoospermia' gene panel in human non-obstructive azoospermia (NOA)? SUMMARY ANSWER: The diagnostic performance of sequencing a gene panel based on genes associated with mouse azoospermia was relatively successful in idiopathic NOA patients and allowed the discovery of two novel genes involved in NOA due to meiotic arrest. WHAT IS KNOWN ALREADY: NOA is a largely heterogeneous clinical entity, which includes different histological pictures. In a large proportion of NOA, the aetiology remains unknown (idiopathic NOA) and yet, unknown genetic factors are likely to play be involved. The mouse is the most broadly used mammalian model for studying human disease because of its usefulness for genetic manipulation and its genetic and physiological similarities to man. Mouse azoospermia models are available in the Mouse Genome Informatics database (MGI: http://www.informatics.jax.org/). STUDY DESIGN, SIZE, DURATION: The first step was to design of a 'mouse azoospermia' gene panel through the consultation of MGI. The second step was NGS analysis of 175 genes in a group of highly selected NOA patients (n = 33). The third step was characterization of the discovered gene defects in human testis tissue, through meiotic studies using surplus testicular biopsy material from the carriers of the RNF212 and STAG3 pathogenic variants. The final step was RNF212 and STAG3 expression analysis in a collection of testis biopsies. PARTICIPANTS/MATERIALS, SETTING, METHODS: From a total of 1300 infertile patients, 33 idiopathic NOA patients were analysed in this study, including 31 unrelated men and 2 brothers from a consanguineous family. The testis histology of the 31 unrelated NOA patients was as follows: 20 Sertoli cell-only syndrome (SCOS), 11 spermatogenic arrest (6 spermatogonial arrest and 5 spermatocytic arrest). The two brothers were affected by spermatocytic arrest. DNA extracted from blood was used for NGS on Illumina NextSeq500 platform. Generated sequence data was filtered for rare and potentially pathogenic variants. Functional studies in surplus testicular tissue from the carriers included the investigation of meiotic entry, XY body formation and metaphases by performing fluorescent immunohistochemical staining and immunocytochemistry. mRNA expression analysis through RT-qPCR of RNF212 and STAG3 was carried out in a collection of testis biopsies with different histology. MAIN RESULTS AND THE ROLE OF CHANCE: Our approach was relatively successful, leading to the genetic diagnosis of one sporadic NOA patient and two NOA brothers. This relatively high diagnostic performance is likely to be related to the stringent patient selection criteria i.e. all known causes of azoospermia were excluded and to the relatively high number of patients with rare testis histology (spermatocytic arrest). All three mutation carriers presented meiotic arrest, leading to the genetic diagnosis of three out of seven cases with this specific testicular phenotype. For the first time, we report biallelic variants in STAG3, in one sporadic patient, and a homozygous RNF212 variant, in the two brothers, as the genetic cause of NOA. Meiotic studies allowed the detection of the functional consequences of the mutations and provided information on the role of STAG3 and RNF212 in human male meiosis. LIMITATIONS, REASONS FOR CAUTION: All genes, with the exception of 5 out of 175, included in the panel cause azoospermia in mice only in the homozygous or hemizygous state. Consequently, apart from the five known dominant genes, heterozygous variants (except compound heterozygosity) in the remaining genes were not taken into consideration as causes of NOA. We identified the genetic cause in approximately half of the patients with spermatocytic arrest. The low number of analysed patients can be considered as a limitation, but it is a very rare testis phenotype. Due to the low frequency of this specific phenotype among infertile men, our finding may be considered of low clinical impact. However, at an individual level, it does have relevance for prognostic purposes prior testicular sperm extraction. WIDER IMPLICATIONS OF THE FINDINGS: Our study represents an additional step towards elucidating the genetic bases of early spermatogenic failure, since we discovered two new genes involved in human male meiotic arrest. We propose the inclusion of RNF212 and STAG3 in a future male infertility diagnostic gene panel. Based on the associated testis phenotype, the identification of pathogenic mutations in these genes also confers a negative predictive value for testicular sperm retrieval. Our meiotic studies provide novel insights into the role of these proteins in human male meiosis. Mutations in STAG3 were first described as a cause of female infertility and ovarian cancer, and Rnf212 knock out in mice leads to male and female infertility. Hence, our results stimulate further research on shared genetic factors causing infertility in both sexes and indicate that genetic counselling should involve not only male but also female relatives of NOA patients. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Spanish Ministry of Health Instituto Carlos III-FIS (grant number: FIS/FEDER-PI14/01250; PI17/01822) awarded to CK and AR-E, and by the European Commission, Reproductive Biology Early Research Training (REPROTRAIN, EU-FP7-PEOPLE-2011-ITN289880), awarded to CK, WB, and AE-M. The authors have no conflict of interest.

Association of serum testosterone levels and testicular volume in adult patients.

A retrospective observational study was undertaken to gain new insight into the relationship between total testicular volume and levels of serum testosterone, luteinising hormone, follicle-stimulating hormone, prolactin and clinical variables. A total of 312 men with sexual dysfunction or infertility were divided into groups A and B (156 each) on the basis of basal plasma testosterone ≤5 nmol/L of ≥12 nmol/L respectively. Group A was subclassified in A1 (primary hypogonadism) and A2 (secondary hypogonadism). There were significant differences in total testicular volume between group A (15.33 ± 11.94 ml) and group B (36.74 ± 6.9; p < .001) and also between subgroup A1 (11.07 ± 8.49 ml) and subgroup A2 (23.62 ± 13.04 ml; p < .001). Only 13.5% of patients in group B had a total testicular volume <30 ml. Differences in all studied parameters were found between group A and group B. There were no variations when comparing age, body mass index and testosterone in groups A1 and A2 . The use of total testicular volume and body mass index together for predicting testosterone levels yields a sensitivity and specificity of 85.3% and 86.5% respectively. Logistic regression analysis, univariate and multivariate models, using the measurement of total testicular volume resulted in a high capacity to predict testosterone levels.

Recurrent X chromosome-linked deletions: discovery of new genetic factors in male infertility.

BACKGROUND: The role of X-linked genes and copy-number variations (CNVs) in male infertility remains poorly explored. Our previous array-CGH analyses showed three recurrent deletions in Xq exclusively (CNV67) and prevalently (CNV64, CNV69) found in patients. Molecular and clinical characterisation of these CNVs was performed in this study. METHODS: 627 idiopathic infertile patients and 628 controls were tested for each deletion with PCR+/-. We used PCR+/- to map deletion junctions and long-range PCR and direct sequencing to define breakpoints. RESULTS: CNV64 was found in 5.7% of patients and in 3.1% of controls (p=0.013; OR=1.89; 95% CI 1.1 to 3.3) and CNV69 was found in 3.5% of patients and 1.6% of controls (p=0.023; OR=2.204; 95% CI 1.05 to 4.62). For CNV69 we identified two breakpoints, types A and B, with the latter being significantly more frequent in patients than controls (p=0.011; OR=9.19; 95% CI 1.16 to 72.8). CNV67 was detected exclusively in patients (1.1%) and was maternally transmitted. The semen phenotype of one carrier (11-041) versus his normozoospermic non-carrier brother strongly indicates a pathogenic effect of the deletion on spermatogenesis. MAGEA9, an ampliconic gene reported as independently acquired on the human X chromosome with exclusive physiological expression in the testis, is likely to be involved in CNV67. CONCLUSIONS: We provide the first evidence for X chromosome-linked recurrent deletions associated with spermatogenic impairment. CNV67, specific to spermatogenic anomaly and with a frequency of 1.1% in oligo/azoospermic men, resembles the AZF regions on the Y chromosome with potential clinical implications.

Y-chromosome microdeletions are not associated with SHOX haploinsufficiency.

STUDY QUESTION: Are Y-chromosome microdeletions associated with SHOX haploinsufficiency, thus representing a risk of skeletal anomalies for the carriers and their male descendents? SUMMARY ANSWER: The present study shows that SHOX haploinsufficiency is unlikely to be associated with Y-chromosome microdeletions. WHAT IS KNOWN ALREADY: Y-chromosome microdeletions are not commonly known as a major molecular genetic cause of any pathological condition except spermatogenic failure. However, it has been recently proposed that they are associated not only with infertility but also with anomalies in the pseudoautosomal regions (PAR), among which SHOX haploinsufficiency stands out with a frequency of 5.4% in microdeletion carriers bearing a normal karyotype. This finding implies that sons fathered by men with Y-chromosome defects will not only exhibit fertility problems, but might also suffer from SHOX-related conditions. STUDY DESIGN: Five European laboratories (Florence, Münster, Barcelona, Padova and Ancona), routinely performing Y-chromosome microdeletion screening, were enrolled in a multicenter study. PARTICIPANTS/MATERIALS, SETTING, METHODS: PAR-linked and SHOX copy number variations (CNVs) were analyzed in 224 patients carrying Y-chromosome microdeletions and 112 controls with an intact Y chromosome, using customized X-chromosome-specific array-CGH platforms and/or qPCR assays for SHOX and SRY genes. MAIN RESULTS AND THE ROLE OF CHANCE: Our data show that 220 out of 224 (98.2%) microdeletion carriers had a normal SHOX copy number, as did all the controls. No SHOX deletions were found in any of the examined subjects (patients as well as controls), thus excluding an association with SHOX haploinsufficiency. SHOX duplications were detected in 1.78% of patients (n = 4), of whom two had an abnormal and two a normal karyotype. This might suggest that Y-chromosome microdeletions have a higher incidence for SHOX duplications, irrespective of the patient's karyotype. However, the only clinical condition observed in our four SHOX-duplicated patients was infertility. LIMITATIONS, REASONS FOR CAUTION: The number of controls analyzed is rather low to assess whether the SHOX duplications found in the two men with Y-chromosome microdeletions and a normal karyotype represent a neutral polymorphism or are actually associated with the presence of the microdeletion. WIDER IMPLICATIONS OF THE FINDINGS: Men suffering from infertility due to the presence of Y-chromosome microdeletions can resort to artificial reproductive technology (ART) to father their biological children. However, infertile couples must be aware of the risks implied and this makes genetic counseling a crucial step in the patient's management. This study does not confirm previous alarming data that showed an association between Y-chromosome microdeletions and SHOX haploinsufficiency. Our results imply that deletion carriers have no augmented risk of SHOX-related pathologies (short stature and skeletal anomalies) and indicate that there is no need for radical changes in genetic counseling of Yq microdeletion carriers attempting ART, since the only risk established so far for their male offspring remains impaired spermatogenesis. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Italian Ministry of University (grant PRIN 2010-2012 to C.K.), Tuscan Regional Health Research Program ('Progetto Salute 2009') to G.F., the Spanish Ministry of Health (grant FIS-11/02254) and the European Union 'Reprotrain' Marie Curie Network (project number: 289880 to C.K.). The authors declare that no conflicting interests exist.

Further insights into the role of T222P variant of RXFP2 in non-syndromic cryptorchidism in two Mediterranean populations.

The aetiopathogenesis of isolated cryptorchidism remains largely unknown. Mutation screenings in the most relevant candidate genes for testicular maldescent lead to controversial data in the literature. In particular, the role of the T222P genetic variant of the RXFP2 gene is still debated. Given the controversies, the aim of this study was to provide further data on this genetic variant in two Mediterranean populations. A total of 577 subjects from Spain and 550 from Italy (with and without a history of cryptorchidism) were analysed. The T222P substitution was found in both unilateral and bilateral cases and in a total of 12 controls. These data exclude a clear-cut cause-effect relationship between T222P variant and testicular maldescent. The T222P variant was found at a similar frequency in both cases and controls in the Spanish population, whereas in Italy, the frequency of T222P resulted significantly higher in the cryptorchid group (p = 0.031). The observed difference between the two countries and the highly variable phenotypic expression of the T222P variant may depend on the genetic background or on environmental conditions. The haplotype analysis of the RXFP2 gene in T222P carriers and their parents showed that this variant is linked to the previously inferred C-C-G-A-13 haplotype and consequently provides further support to the 'founder effect' hypothesis. In conclusion, our data indicate that T222P is a frequent variant in the Spanish population with no pathogenic effect. Although in Italy it seems to confer a mild risk (odds ratio = 3.17, 95% confidence interval: 1.07-9.34) to cryptorchidism, the screening for this variant for diagnostic purposes is not advised because of the relatively high frequency of control carriers (1.4% of Italian men without a history of cryptorchidism).

Evaluation of 172 candidate polymorphisms for association with oligozoospermia or azoospermia in a large cohort of men of European descent.

BACKGROUND: In spite of tremendous efforts by a number of groups, the search for single nucleotide polymorphisms (SNPs) strongly associated with male factor infertility by means of gene re-sequencing studies has yielded few likely candidates. A recent pilot, genome-wide SNP association study (GWAS) identified a list of SNPs associated with oligozoospermia and azoospermia. This is an expanded follow-up study of the SNPs identified by the GWAS as well as other SNPs from previously published gene re-sequencing studies. METHODS: On the basis of the pilot GWAS and SNPs with published associations with male infertility, 172 SNPs were genotyped in men with idiopathic azoospermia or oligozoospermia using the Illumina BeadXpress platform. RESULTS: Several SNPs were identified or confirmed to be significantly associated with oligozoospermia and/or azoospermia. More importantly, this follow-up study indicates that, at least in Caucasian men, no single common SNP accounts for a significant proportion of spermatogenic failure cases. CONCLUSIONS: The associations reported in this study are promising, but much larger genome-wide studies will be necessary to confidently validate these SNPs and identify novel SNPs associated with male infertility.

Combination therapy for erectile dysfunction involving a PDE5 inhibitor and alprostadil.

The phosphodiesterase-5 inhibitors (PDE5Is) are the first-line treatment option for men with erectile dysfunction (ED), with alprostadil considered a second-line choice. Consideration has to be given to patients who fail these treatments and what their options are. This review evaluates the data on the combination of a PDE5I with alprostadil in patients who have previously failed therapy with either drug. A PubMed search was conducted and identified nine publications relating to combination treatment with alprostadil as intracavernosal, intraurethral or topical application. The results indicate that with all three formulations the combination therapy resulted in an improved outcome compared with either of the drugs as monotherapy. This was demonstrated by the increased total International Index of Erectile Function (IIEF) scores as well as IIEF erectile function domain scores. This finding was also valid for patients with post-prostatectomy ED. The associated side effects of the combined treatment did not result in treatment discontinuation. These findings suggest that combination therapy with a PDE5I and alprostadil might be considered a treatment option in patients who have previously had a poor response to either drug.

Adipocyte accumulation in corpus cavernosum: First clinical evidence and pathophysiological implications in erectile dysfunction. / Acumulación de adipocitos en el cuerpo cavernoso: primera evidencia clínica e implicaciones fisiopatológicas en la disfunción eréctil.

OBJECTIVES: Animal models have shown that erectile dysfunction is associated with adipocyte accumulation under tunica albugínea, which could be involved in venous leakage and loss of penile rigidity. In the current sudy, we compared the histology of the penile sub-albuginean region of drug-refractory erectile dysfunction patients undergoing penile prosthesis implantation with potent patients with Peyronie's disease undergoing curvature correction procedures. MATERIALS AND METHODS: Seventeen refractory erectile dysfunction patients and fourteen potent patients with Peyronie's disease were recruited. Sub-albuginean tissue samples were taken in each surgery. An expert uropathologist analysed each section. A bivariate analysis was performed. Multivariate logistic regression was used to calculate adjusted odds ratios; P value<.05 was considered significant. RESULTS: Eleven patients (11/17) in the case group presented cavernous fat cell accumulation, while only one patient (1/14) in the control group presented this finding (P<.05). Adjusted odds ratio for erectile dysfunction was 40.72; 95% CI 2.28-727.29 (P=.012). CONCLUSIONS: Different studies have shown that androgen disruption could be involved in penile structural changes, leading to trabecular smooth muscle apoptosis and trans or de-differentiation into adipocytes. This is the first prospective study in humans to report an association between erectile dysfunction and sub-albuginean adipocyte accumulation. Venous leakage secondary to this phenomenon could be a factor in the pathophysiology of erectile dysfunction, especially in patients that do not respond to medical therapy.

Surgical treatment of Peyronie's disease with small intestinal submucosa graft patch.

The objective of the study was to report our results using a porcine small intestinal submucosa graft (Surgisis ES, Cook Medical) for tunica albuginea substitution after plaque incision. We retrospectively evaluated patients surgically treated at our institution for Peyronie's disease (PD) by means of plaque incision and porcine small intestinal submucosa grafting (Surgisis) between 2009 and 2013. At the same time a literature review was conducted, searching for similar reports and results. Forty-four patients were identified who had been diagnosed with PD between 2009 and the beginning of 2013, and had been treated with corporoplasty, plaque incision and grafting with Surgisis for a severe curvature of the penis. Curvature of the penis was dorsal in 40 patients (90%) and laterally on the right in 4 patients (10%). Mean duration of surgery was 165 min (range 90-200). Mean size of the graft was 6.5 cm(2) and the mean follow-up was 19.2 months (range 11-48). In patients with severe curvature of the penis due to PD and the need for corporoplasty with plaque incision and graft placement, Surgisis represents a good option with a low risk of complications, below the rate described with previously investigated graft tissues.

Comprehensive investigation in patients affected by sperm macrocephaly and globozoospermia.

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients' genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR-based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients' genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy; (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90-100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART.

[Surgical treatment with prosthesis implantation in 100 patients with erectile sexual dysfunction]. / Tratamiento quirúrgico con implante de prótesis en 100 pacientes con disfunción sexual eréctil.

We present our case study of 100 penile prostheses in patients suffering from erectile sexual dysfunction of different etiologies. We carry out a review of the different diagnostic stages and the different surgical approaches for prosthetic implant and their complications.

[Testicular microlithiasis: is there a relationship with malignant tumour?]. / Microlitiasis testicular: existe relación con patología maligna?

The aim of this revision is to assess the relationship between testicular microlithiasis and malignant tumours. We reviewed 98 charts of patients with pathological diagnosis of testicular malignant tumour. Diagnosis of testicular tumour was made by ultrasound, and 6.02% (6 pts.) presented testicular lithiasis, and this finding makes us conclude that this is not a characteristic feature of this entity. We also reviewed bibliography related to this theme, and conclude that the management of microlithiasis remains being controversial.

Sensory changes after surgical correction of penile curvature.

In 32 men (mean age 54.94 years, range 25-70 years), thresholds for penile thermal and vibratory sensation were recorded before and 3 months after surgery using a genital sensory analyser. The following significant changes were found postoperatively. The cold threshold decreased in the glans from 27.24 to 25.33 degrees C (P<0.001) in men older than 60 years, and in the ventral penile shaft from 28.54 to 25.46 degrees C (P<0.001) in men aged 35-59 years. The warm threshold increased in the glans from 38.31 to 40.61 degrees C (P=0.002) in men aged 35-59 years, and from 39.20 to 40.58 degrees C in men older than 60 years (P=0.023). The warm threshold also increased in the ventral penile shaft in men aged 35-59 years, from 36.62 to 37.82 degrees C (P=0.023). For all three age groups combined, the vibratory threshold changed in the glans from 2.33 to 2.93 mum (P<0.001). It is concluded that penile sensitivity changed significantly after surgery for penile curvature treatment, regardless of whether a ventral or dorsal surgical approach is used.

[Endoscopy surgery of utriculocele]. / Cirugía endoscópica del utriculocele.

Of 12 patients with utriculocele, 6 consulted for sterility and were submitted to endoscopic surgery. Transurethral resection of the utricular capsule removed distal obstruction of the seminal duct in all cases (increased ejaculate volume), in half of the cases sperm density and motility improved, and in 1 case pregnancy was achieved with insemination of capacitated semen (ICA).