Large-scale analyses of the X chromosome in 2,354 infertile men discover recurrently affected genes associated with spermatogenic failure.

Although the evolutionary history of the X chromosome indicates its specialization in male fitness, its role in spermatogenesis has largely been unexplored. Currently only three X chromosome genes are considered of moderate-definitive diagnostic value. We aimed to provide a comprehensive analysis of all X chromosome-linked protein-coding genes in 2,354 azoospermic/cryptozoospermic men from four independent cohorts. Genomic data were analyzed and compared with data in normozoospermic control individuals and gnomAD. While updating the clinical significance of known genes, we propose 21 recurrently mutated genes strongly associated with and 34 moderately associated with azoospermia/cryptozoospermia not previously linked to male infertility (novel). The most frequently affected prioritized gene, RBBP7, was found mutated in ten men across all cohorts, and our functional studies in Drosophila support its role in germ stem cell maintenance. Collectively, our study represents a significant step towards the definition of the missing genetic etiology in idiopathic severe spermatogenic failure and significantly reduces the knowledge gap of X-linked genetic causes of azoospermia/cryptozoospermia contributing to the development of future diagnostic gene panels.

Actionable secondary findings following exome sequencing of 836 non-obstructive azoospermia cases and their value in patient management.

STUDY QUESTION: What is the load, distribution and added clinical value of secondary findings (SFs) identified in exome sequencing (ES) of patients with non-obstructive azoospermia (NOA)? SUMMARY ANSWER: One in 28 NOA cases carried an identifiable, medically actionable SF. WHAT IS KNOWN ALREADY: In addition to molecular diagnostics, ES allows assessment of clinically actionable disease-related gene variants that are not connected to the patient's primary diagnosis, but the knowledge of which may allow the prevention, delay or amelioration of late-onset monogenic conditions. Data on SFs in specific clinical patient groups, including reproductive failure, are currently limited. STUDY DESIGN, SIZE, DURATION: The study group was a retrospective cohort of patients with NOA recruited in 10 clinics across six countries and formed in the framework of the international GEMINI (The GEnetics of Male INfertility Initiative) study. PARTICIPANTS/MATERIALS, SETTING, METHODS: ES data of 836 patients with NOA were exploited to analyze SFs in 85 genes recommended by the American College of Medical Genetics and Genomics (ACMG), Geisinger's MyCode, and Clinical Genome Resource. The identified 6374 exonic variants were annotated with ANNOVAR and filtered for allele frequency, retaining 1381 rare or novel missense and loss-of-function variants. After automatic assessment of pathogenicity with ClinVar and InterVar, 87 variants were manually curated. The final list of confident disease-causing SFs was communicated to the corresponding GEMINI centers. When patient consent had been given, available family health history and non-andrological medical data were retrospectively assessed. MAIN RESULTS AND THE ROLE OF CHANCE: We found a 3.6% total frequency of SFs, 3.3% from the 59 ACMG SF v2.0 genes. One in 70 patients carried SFs in genes linked to familial cancer syndromes, whereas 1 in 60 cases was predisposed to congenital heart disease or other cardiovascular conditions. Retrospective assessment confirmed clinico-molecular diagnoses in several cases. Notably, 37% (11/30) of patients with SFs carried variants in genes linked to male infertility in mice, suggesting that some SFs may have a co-contributing role in spermatogenic impairment. Further studies are needed to determine whether these observations represent chance findings or the profile of SFs in NOA patients is indeed different from the general population. LIMITATIONS, REASONS FOR CAUTION: One limitation of our cohort was the low proportion of non-Caucasian ethnicities (9%). Additionally, as comprehensive clinical data were not available retrospectively for all men with SFs, we were not able to confirm a clinico-molecular diagnosis and assess the penetrance of the specific variants. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, this study analyzed medically actionable SFs in men with spermatogenic failure. With the evolving process to incorporate ES into routine andrology practice for molecular diagnostic purposes, additional assessment of SFs can inform about future significant health concerns for infertility patients. Timely detection of SFs and respective genetic counseling will broaden options for disease prevention and early treatment, as well as inform choices and opportunities regarding family planning. A notable fraction of SFs was detected in genes implicated in maintaining genome integrity, essential in both mitosis and meiosis. Thus, potential genetic pleiotropy may exist between certain adult-onset monogenic diseases and NOA. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Estonian Research Council grants IUT34-12 and PRG1021 (M.L. and M.P.); National Institutes of Health of the United States of America grant R01HD078641 (D.F.C., K.I.A. and P.N.S.); National Institutes of Health of the United States of America grant P50HD096723 (D.F.C. and P.N.S.); National Health and Medical Research Council of Australia grant APP1120356 (M.K.O'B., D.F.C. and K.I.A.); Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação grant POCI-01-0145-FEDER-007274 (A.M.L., F.C. and J.G.) and FCT: IF/01262/2014 (A.M.L.). J.G. was partially funded by FCT/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through the Centre for Toxicogenomics and Human Health-ToxOmics (grants UID/BIM/00009/2016 and UIDB/00009/2020). M.L.E. is a consultant for, and holds stock in, Roman, Sandstone, Dadi, Hannah, Underdog and has received funding from NIH/NICHD. Co-authors L.K., K.L., L.N., K.I.A., P.N.S., J.G., F.C., D.M.-M., K.A., K.A.J., M.K.O'B., A.M.L., D.F.C., M.P. and M.L. declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Efecto del tadalafilo 5mg/día en la hemodinámica peniana en pacientes con disfunción eréctil / Effect of tadalafil 5mg daily treatment on penile haemodynamics in patients with erectile dysfunction

Antecedentes y objetivo: Los inhibidores de la fosfodiesterasa 5 son los fármacos más utilizados para tratar a los pacientes con disfunción eréctil (DE). Los métodos para diagnosticar y evaluar la DE así como la respuesta al tratamiento son básicamente clínicos, es decir, la percepción del paciente sobre la calidad de su erección y la realización de cuestionarios validados. Actualmente aun es necesario identificar marcadores que puedan predecir la respuesta al tratamiento farmacológico. Este estudio pretende evaluar si la toma diaria de tadalafilo 5mg es capaz de inducir cambios en la hemodinámica peniana (midiendo la velocidad del pico sistólico (VPS) y la aceleración sistólica (AS) mediante ecografía doppler, sin utilizar alprostadilo) y estudiar si estos cambios tienen una correlación clínica. El objetivo es identificar un factor predictivo hemodinámico de respuesta al tratamiento farmacológico.Materiales y métodos: Se realizó un estudio prospectivo a 40 pacientes con DE a los que se inició tratamiento con tadalafilo 5mg/día. Estos respondieron a los cuestionarios IIEF-6, EHS, SEP-2 y SEP-3, y se les realizó una ecografía doppler peniana en 3 momentos distintos: antes de comenzar el tratamiento, a los 3 y a los 6 meses (estando previamente 30 días sin medicación).Resultados y conclusiones: El tratamiento durante 3 meses no modificó de forma significativa la VPS y la AS. Sí hubo una mejoría clínica tras 3 meses de tratamiento de acuerdo a los cuestionarios, que no se mantuvo a los 6 meses. No se pudo establecer una correlación clínico-radiológica ni encontramos un valor hemodinámico predictor de respuesta al tratamiento. (AU)

Introduction and objective: Phosphodiesterase-5 inhibitors (PDE-5) are the first-line treatment. ED diagnostic and evaluation methods are clinical: erection self-perception and evaluation questionnaires. It is necessary to identify predictive markers to choose the best drug and the best dose for each individual patient. This study seeks to evaluate the effect of tadalafil 5mg daily treatment on penile haemodynamics (penis in flaccidity, using doppler ultrasound, without using alprostadil) and to study if these changes are clinically associated.Material and methods: 40 patients who started treatment with tadalafil 5mg daily were consecutively selected in a prospective study. Each patient underwent a penile doppler ultrasound and completed questionnaires (IIEF-6, EHS, SEP-2, SEP-3) at three different times: before starting the treatment, at 3 months and at 6 months (within the last 30 days without treatment).Results and conclusions: Tadalafil 5mg daily treatment for 3 months did not result in significant modifications to VPS and AS. There was clinical improvement according to the questionnaires after three months of treatment, which was not maintained at 6 months. Neither a clinical-radiological correlation nor a haemodynamic value predicting response to treatment could be established. (AU)

[Effect of tadalafil 5mg daily treatment on penile haemodynamics in patients with erectile dysfunction]. / Efecto del tadalafilo 5mg/día en la hemodinámica peniana en pacientes con disfunción eréctil.

INTRODUCTION AND OBJECTIVE: Phosphodiesterase-5 inhibitors (PDE-5) are the first-line treatment. ED diagnostic and evaluation methods are clinical: erection self-perception and evaluation questionnaires. It is necessary to identify predictive markers to choose the best drug and the best dose for each individual patient. This study seeks to evaluate the effect of tadalafil 5mg daily treatment on penile haemodynamics (penis in flaccidity, using doppler ultrasound, without using alprostadil) and to study if these changes are clinically associated. MATERIAL AND METHODS: 40 patients who started treatment with tadalafil 5mg daily were consecutively selected in a prospective study. Each patient underwent a penile doppler ultrasound and completed questionnaires (IIEF-6, EHS, SEP-2, SEP-3) at three different times: before starting the treatment, at 3 months and at 6 months (within the last 30 days without treatment). RESULTS AND CONCLUSIONS: Tadalafil 5mg daily treatment for 3 months did not result in significant modifications to VPS and AS. There was clinical improvement according to the questionnaires after three months of treatment, which was not maintained at 6 months. Neither a clinical-radiological correlation nor a haemodynamic value predicting response to treatment could be established.

Correction: gr/gr deletion predisposes to testicular germ cell tumour independently from altered spermatogenesis: results from the largest European study.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic dissection of spermatogenic arrest through exome analysis: clinical implications for the management of azoospermic men.

PURPOSE: Azoospermia affects 1% of men and it can be the consequence of spermatogenic maturation arrest (MA). Although the etiology of MA is likely to be of genetic origin, only 13 genes have been reported as recurrent potential causes of MA. METHODS: Exome sequencing in 147 selected MA patients (discovery cohort and two validation cohorts). RESULTS: We found strong evidence for five novel genes likely responsible for MA (ADAD2, TERB1, SHOC1, MSH4, and RAD21L1), for which mouse knockout (KO) models are concordant with the human phenotype. Four of them were validated in the two independent MA cohorts. In addition, nine patients carried pathogenic variants in seven previously reported genes-TEX14, DMRT1, TEX11, SYCE1, MEIOB, MEI1, and STAG3-allowing to upgrade the clinical significance of these genes for diagnostic purposes. Our meiotic studies provide novel insight into the functional consequences of the variants, supporting their pathogenic role. CONCLUSION: Our findings contribute substantially to the development of a pre-testicular sperm extraction (TESE) prognostic gene panel. If properly validated, the genetic diagnosis of complete MA prior to surgical interventions is clinically relevant. Wider implications include the understanding of potential genetic links between nonobstructive azoospermia (NOA) and cancer predisposition, and between NOA and premature ovarian failure.

Short anogenital distance is associated with testicular germ cell tumour development.

BACKGROUND: Testicular germ cell tumour is a multifactorial disease in which various genetic and environmental factors play a role. Testicular germ cell tumour is part of the testicular dysgenesis syndrome which includes also cryptorchidism, hypospadias, oligo/azoospermia and short anogenital distance. OBJECTIVES: The primary objective was to examine anogenital distance in testicular germ cell tumour cases and healthy fertile controls. The secondary objective was to assess the (CAG)n polymorphism of the Androgen Receptor gene in relationship with anogenital distances and testicular germ cell tumour development. MATERIAL AND METHODS: 156 testicular germ cell tumour patients and 110 tumour-free normozoospermic controls of Spanish origin. All subjects underwent full andrological workup (including semen and hormone analysis) and genetic analysis (Androgen Receptor (CAG)n). The main outcome measures were the anopenile distance (AGDap), the anoscrotal distance (AGDas) and AR(CAG)n. RESULT: We observed significantly shorter anogenital distances in the group of testicular germ cell tumour patients in respect to controls (P < .001) independently from sperm count and testis histology. Threshold values, applicable only to our cohort, were calculated for anogenital distances with the best sensitivity and specificity. Subjects with AGDap and AGDas below threshold showed a significantly increased risk for testicular germ cell tumour (OR = 4.97, 95% CI = 2.01-12.33, P = .001 and OR = 4.11, 95% CI = 1.89-8.92, P ≤ .001, respectively). No significant correlation was observed between AR(CAG)n polymorphism and anogenital distances. The median values of the AR(CAG)n were similar between cases and controls, excluding a major role for this polymorphism in the etiopathogenesis of these testicular dysgenesis syndrome components. CONCLUSIONS: Ours is the first study focusing on anogenital distances in testicular germ cell tumour patients. We identified short anogenital distances (which is a surrogate biomarker of androgen action during foetal life) as a significant risk factor for this disease. After further validation of our preliminary data, anogenital distance measurement could become part of testicular germ cell tumour screening in order to better define those individuals who would benefit from long-term active follow-up.

Clinical factors affecting semen improvement after microsurgical subinguinal varicocelectomy: which subfertile patients benefit from surgery?

BACKGROUND: The exact mechanism of varicocele-related infertility is still elusive, therefore, the current challenges for its management lie in determining which patients stand to benefit most from surgical correction. The authors aimed to assess the clinical factors affecting semen improvement after left microsurgical subinguinal varicocelectomy (MSV) in relation to patient age, ultrasound varicocele grading (USVG), and presence of a right subclinical varicocele (RSV). METHODS: From 2010 to 2017 a total of 228 infertile patients underwent left MSV for clinical varicocele. Descriptive statistics were used to describe the cohort and verify the surgical benefit in terms of semen improvement, in addition, subsets of patients were selected according to clinical covariates. Logistic regression modeling was applied to evaluate the presence of RSV, operative time, age, and USVG as explanatory variables. RESULTS: Sperm concentration (SC), progressive sperm motility (PSM), and normal sperm morphology (NSM) increased significantly after surgery (p = 0.002; p = 0.011; p = 0.024; respectively). Mean SC improved after MSV in ⩾35 year-old patients and the grade 3 USVG group (p = 0.01; p = 0.02; respectively). Logistic regression modeling showed a that the probability of SC improvement was 76% lower in subjects presenting RSV (p = 0.011). In addition, patients with a grade 3 USVG presented a three-times greater probability of SC improvement compared with patients with a lower USVG (p = 0.035). In addition, older patients showed a greater probability of SC improvement after MSV (p = 0.041). CONCLUSIONS: MSV is an effective varicocele-related infertility treatment that should also be offered to older patients. In addition, patients with a higher USVG benefit from surgery. In infertile men with an RSV in association with a left clinical disease, a bilateral varicocele repair should be considered.

gr/gr deletion predisposes to testicular germ cell tumour independently from altered spermatogenesis: results from the largest European study.

The association between impaired spermatogenesis and TGCT has stimulated research on shared genetic factors. Y chromosome-linked partial AZFc deletions predispose to oligozoospermia and were also studied in TGCT patients with controversial results. In the largest study reporting the association between gr/gr deletion and TGCT, sperm parameters were unknown. Hence, it remains to be established whether this genetic defect truly represents a common genetic link between TGCT and impaired sperm production. Our aim was to explore the role of the following Y chromosome-linked factors in the predisposition to TGCT: (i) gr/gr deletion in subjects with known sperm parameters; (ii) other partial AZFc deletions and, for the first time, the role of partial AZFc duplications; (iii) DAZ gene dosage variation. 497 TGCT patients and 2030 controls from two Mediterranean populations with full semen/andrological characterization were analyzed through a series of molecular genetic techniques. Our most interesting finding concerns the gr/gr deletion and DAZ gene dosage variation (i.e., DAZ copy number is different from the reference sequence), both conferring TGCT susceptibility. In particular, the highest risk was observed when normozoospermic TGCT and normozoospermic controls were compared (OR = 3.7; 95% CI = 1.5-9.1; p = 0.006 for gr/gr deletion and OR = 1.8; 95% CI = 1.1-3.0; p = 0.013 for DAZ gene dosage alteration). We report in the largest European study population the predisposing effect of gr/gr deletion to TGCT as an independent risk factor from impaired spermatogenesis. Our finding implies regular tumour screening/follow-up in male family members of TGCT patients with gr/gr deletion and in infertile gr/gr deletion carriers.

From exome analysis in idiopathic azoospermia to the identification of a high-risk subgroup for occult Fanconi anemia.

PURPOSE: In about 10% of patients affected by Fanconi anemia (FA) the diagnosis is delayed until adulthood, and the presenting symptom in these "occult" FA cases is often a solid cancer and cancer treatment-related toxicity. Highly predictive clinical parameter(s) for diagnosing such an adult-onset cases are missing. METHODS: (1) Exome sequencing (ES), (2) Sanger sequencing of FANCA, (3) diepoxybutane (DEB)-induced chromosome breakage test. RESULTS: ES identified a pathogenic homozygous FANCA variant in a patient affected by Sertoli cell-only syndrome (SCOS) and in his azoospermic brother. Although they had no overt anemia, chromosomal breakage test revealed a reverse somatic mosaicism in the former and a typical FA picture in the latter. In 27 selected SCOS cases, 1 additional patient showing compound heterozygous pathogenic FANCA variants was identified with positive chromosomal breakage test. CONCLUSION: We report an extraordinarily high frequency of FA in a specific subgroup of azoospermic patients (7.1%). The screening for FANCA pathogenic variants in such patients has the potential to identify undiagnosed FA before the appearance of other severe clinical manifestations of the disease. The definition of this high-risk group for "occult" FA, based on specific testis phenotype with mild/borderline hematological alterations, is of unforeseen clinical relevance.