3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation.

Small RNA-sequencing (small RNA-seq) has revealed the presence of small RNA-naturally occurring variants such as microRNA (miRNA) isoforms or isomiRs. Due to their small size and the sequence similarity among miRNA isoforms, their validation by RT-qPCR is challenging. We previously identified two miR-31-5p isomiRs-the canonical and a 3'isomiR variant (3' G addition)-which were differentially expressed between individuals with azoospermia of different origin. Here, we sought to determine the discriminatory capacity between these two closely-related miRNA isoforms of three alternative poly(A) based-RT-qPCR strategies in both synthetic and real biological context. We found that these poly(A) RT-qPCR strategies exhibit a significant cross-reactivity between these miR-31-5p isomiRs which differ by a single nucleotide, compromising the reliable quantification of individual miRNA isoforms. Fortunately, in the biological context, given that the two miRNA variants show changes in the same direction, RT-qPCR results were consistent with the findings of small RNA-seq study. We suggest that miRNA selection for RT-qPCR validation should be performed with care, prioritizing those canonical miRNAs that, in small RNA-seq, show parallel/homogeneous expression behavior with their most prevalent isomiRs, to avoid confounding RT-qPCR-based results. This is suggested as the current best strategy for robust biomarker selection to develop clinically useful tests.

Immune and spermatogenesis-related loci are involved in the development of extreme patterns of male infertility.

We conducted a genome-wide association study in a large population of infertile men due to unexplained spermatogenic failure (SPGF). More than seven million genetic variants were analysed in 1,274 SPGF cases and 1,951 unaffected controls from two independent European cohorts. Two genomic regions were associated with the most severe histological pattern of SPGF, defined by Sertoli cell-only (SCO) phenotype, namely the MHC class II gene HLA-DRB1 (rs1136759, P = 1.32E-08, OR = 1.80) and an upstream locus of VRK1 (rs115054029, P = 4.24E-08, OR = 3.14), which encodes a protein kinase involved in the regulation of spermatogenesis. The SCO-associated rs1136759 allele (G) determines a serine in the position 13 of the HLA-DRß1 molecule located in the antigen-binding pocket. Overall, our data support the notion of unexplained SPGF as a complex trait influenced by common variation in the genome, with the SCO phenotype likely representing an immune-mediated condition.

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.

Common Variation in the PIN1 Locus Increases the Genetic Risk to Suffer from Sertoli Cell-Only Syndrome.

We aimed to analyze the role of the common genetic variants located in the PIN1 locus, a relevant prolyl isomerase required to control the proliferation of spermatogonial stem cells and the integrity of the blood-testis barrier, in the genetic risk of developing male infertility due to a severe spermatogenic failure (SPGF). Genotyping was performed using TaqMan genotyping assays for three PIN1 taggers (rs2287839, rs2233678 and rs62105751). The study cohort included 715 males diagnosed with SPGF and classified as suffering from non-obstructive azoospermia (NOA, n = 505) or severe oligospermia (SO, n = 210), and 1058 controls from the Iberian Peninsula. The allelic frequency differences between cases and controls were analyzed by the means of logistic regression models. A subtype specific genetic association with the subset of NOA patients classified as suffering from the Sertoli cell-only (SCO) syndrome was observed with the minor alleles showing strong risk effects for this subset (ORaddrs2287839 = 1.85 (1.17-2.93), ORaddrs2233678 = 1.62 (1.11-2.36), ORaddrs62105751 = 1.43 (1.06-1.93)). The causal variants were predicted to affect the binding of key transcription factors and to produce an altered PIN1 gene expression and isoform balance. In conclusion, common non-coding single-nucleotide polymorphisms located in PIN1 increase the genetic risk to develop SCO.

Common genetic variation in KATNAL1 non-coding regions is involved in the susceptibility to severe phenotypes of male infertility.

BACKGROUND: Previous studies in animal models evidenced that genetic mutations of KATNAL1, resulting in dysfunction of its encoded protein, lead to male infertility through disruption of microtubule remodelling and premature germ cell exfoliation. Subsequent studies in humans also suggested a possible role of KATNAL1 single-nucleotide polymorphisms in the development of male infertility as a consequence of severe spermatogenic failure. OBJECTIVES: The main objective of the present study is to evaluate the effect of the common genetic variation of KATNAL1 in a large and phenotypically well-characterised cohort of infertile men because of severe spermatogenic failure. MATERIALS AND METHODS: A total of 715 infertile men because of severe spermatogenic failure, including 210 severe oligospermia and 505 non-obstructive azoospermia patients, as well as 1058 unaffected controls were genotyped for three KATNAL1 single-nucleotide polymorphism taggers (rs2077011, rs7338931 and rs2149971). Case-control association analyses by logistic regression assuming different models and in silico functional characterisation of risk variants were conducted. RESULTS: Genetic associations were observed between the three analysed taggers and different severe spermatogenic failure groups. However, in all cases, the haplotype model (rs2077011*C | rs7338931*T | rs2149971*A) better explained the observed associations than the three risk alleles independently. This haplotype was associated with non-obstructive azoospermia (adjusted p = 4.96E-02, odds ratio = 2.97), Sertoli-cell only syndrome (adjusted p = 2.83E-02, odds ratio = 5.16) and testicular sperm extraction unsuccessful outcomes (adjusted p = 8.99E-04, odds ratio = 6.13). The in silico analyses indicated that the effect on severe spermatogenic failure predisposition could be because of an alteration of the KATNAL1 splicing pattern. CONCLUSIONS: Specific allelic combinations of KATNAL1 genetic polymorphisms may confer a risk of developing severe male infertility phenotypes by favouring the overrepresentation of a short non-functional transcript isoform in the testis.

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)

Contribution of TEX15 genetic variants to the risk of developing severe non-obstructive oligozoospermia.

Background: Severe spermatogenic failure (SPGF) represents one of the most relevant causes of male infertility. This pathological condition can lead to extreme abnormalities in the seminal sperm count, such as severe oligozoospermia (SO) or non-obstructive azoospermia (NOA). Most cases of SPGF have an unknown aetiology, and it is known that this idiopathic form of male infertility represents a complex condition. In this study, we aimed to evaluate whether common genetic variation in TEX15, which encodes a key player in spermatogenesis, is involved in the susceptibility to idiopathic SPGF. Materials and Methods: We designed a genetic association study comprising a total of 727 SPGF cases (including 527 NOA and 200 SO) and 1,058 unaffected men from the Iberian Peninsula. Following a tagging strategy, three tag single-nucleotide polymorphisms (SNPs) of TEX15 (rs1362912, rs323342, and rs323346) were selected for genotyping using TaqMan probes. Case-control association tests were then performed by logistic regression models. In silico analyses were also carried out to shed light into the putative functional implications of the studied variants. Results: A significant increase in TEX15-rs1362912 minor allele frequency (MAF) was observed in the group of SO patients (MAF = 0.0842) compared to either the control cohort (MAF = 0.0468, OR = 1.90, p = 7.47E-03) or the NOA group (MAF = 0.0472, OR = 1.83, p = 1.23E-02). The genotype distribution of the SO population was also different from those of both control (p = 1.14E-02) and NOA groups (p = 4.33-02). The analysis of functional annotations of the human genome suggested that the effect of the SO-associated TEX15 variants is likely exerted by alteration of the binding affinity of crucial transcription factors for spermatogenesis. Conclusion: Our results suggest that common variation in TEX15 is involved in the genetic predisposition to SO, thus supporting the notion of idiopathic SPGF as a complex trait.

Ten-year experience with penile prosthetic surgery for the treatment of erectile dysfunction: outcomes of a tertiary referral center and predictors of early prosthetic infection.

We aimed to evaluate ten-year outcomes of penile prosthesis (PP) implantation for the treatment of erectile dysfunction and to assess predictors of early prosthetic infection (EPI). We identified 549 men who underwent 576 PP placements between 2008 and 2018. Univariate and multivariate analyses were used to identify potential predictors of EPI. An EPI predictive nomogram was developed. Thirty-five (6.1%) cases of EPI were recorded with an explant rate of 3.1%. In terms of satisfaction, 82.0% of the patients defined themselves as "satisfied," while partner's satisfaction was 88.3%. Diabetes (P = 0.012), longer operative time (P = 0.032), and reinterventions (P = 0.048) were associated with EPI risk, while postoperative ciprofloxacin was inversely associated with EPI (P = 0.014). Rifampin/gentamicin-coated 3-piece inflatable PP (r/g-c 3IPP) showed a higher EPI risk (P = 0.019). Multivariate analyses showed a two-fold higher risk of EPI in diabetic patients, redo surgeries, or when a r/g-c 3IPP was used (all P < 0.03). We showed that diabetes, longer operative time, and secondary surgeries were the risk factors for EPI. Postoperative ciprofloxacin was associated with a reduced risk of EPI, while r/g-c 3IPP had higher EPI rates without an increased risk of PP explant. After further validation, the proposed nomogram could be a useful tool for the preoperative counseling of PP implantation.

[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.

Effect and in silico characterization of genetic variants associated with severe spermatogenic disorders in a large Iberian cohort.

BACKGROUND: Severe spermatogenic failure (SpF) represents the most extreme manifestation of male infertility, as it decreases drastically the semen quality leading to either severe oligospermia (SO, <5 million spermatozoa/mL semen) or non-obstructive azoospermia (NOA, complete lack of spermatozoa in the ejaculate without obstructive causes). OBJECTIVES: The main objective of the present study is to analyze in the Iberian population the effect of 6 single-nucleotide polymorphisms (SNPs) previously associated with NOA in Han Chinese through genome-wide association studies (GWAS) and to establish their possible functional relevance in the development of specific SpF patterns. MATERIALS AND METHODS: We genotyped 674 Iberian infertile men (including 480 NOA and 194 SO patients) and 1058 matched unaffected controls for the GWAS-associated variants PRMT6-rs12097821, PEX10-rs2477686, CDC42BPA-rs3000811, IL17A-rs13206743, ABLIM1-rs7099208, and SOX5-rs10842262. Their association with SpF, SO, NOA, and different NOA phenotypes was evaluated by logistic regression models, and their functional relevance was defined by comprehensive interrogation of public resources. RESULTS: ABLIM1-rs7099208 was associated with SpF under both additive (OR = 0.86, p = 0.036) and dominant models (OR = 0.78, p = 0.026). The CDC42BPA-rs3000811 minor allele frequency was significantly increased in the subgroup of NOA patients showing maturation arrest (MA) of germ cells compared to the remaining NOA cases under the recessive model (OR = 4.45, p = 0.044). The PEX10-rs2477686 SNP was associated with a negative testicular sperm extraction (TESE) outcome under the additive model (OR = 1.32, p = 0.034). The analysis of functional annotations suggested that these variants affect the testis-specific expression of nearby genes and that lincRNA may play a role in SpF. CONCLUSIONS: Our data support the association of three previously reported NOA risk variants in Asians (ABLIM1-rs7099208, CDC42BPA-rs3000811, and PEX10-rs2477686) with different manifestations of SpF in Iberians of European descent, likely by influencing gene expression and lincRNA deregulation.

Penile low intensity shock wave treatment for PDE5I refractory erectile dysfunction: a randomized double-blind sham-controlled clinical trial.

PURPOSE: Over the last decade, penile low-intensity extracorporeal shockwave therapy (LI-ESWT) has emerged as a promising alternative for the treatment of erectile dysfunction (ED). The aim of this trial is to assess the effect of electromagnetic LI-ESWT on the erectile function of vascular phosphodiesterase type 5 inhibitor (PDE5I) refractory ED patients. METHODS: Randomized, double-blind, sham-controlled study. 76 patients with vascular PDE5I-refractory ED completed the study. 40 men were treated with LI-ESWT (1 session/week for 4 weeks, 5000 shocks/session, 0.09 mJ/mm2 energy density) and 36 were treated with a sham probe. Baseline and post-treatment (1, 3 and 6 months) evaluations were performed using validated erectile function questionnaires (IIEF-EF, EHS, SEP2, SEP3 and GAQ1). The groups were compared using Mann-Whitney-Wilcoxon and chi-squared tests, with results considered statistically significant at p < 0.05. RESULTS: At the 3-month follow-up, median change in IIEF-EF score for active and sham groups was 3.5 (IQR 0-10) and - 0.5 (IQR - 11 to 1), respectively (p < 0.05). Six months after treatment, 52.5% of patients (21/40) in the active group and 27.8% of patients (10/36) in the sham group presented an EHS > 2 (p < 0.05). At the same evaluation, 40.0% (16/40) and 13.9% (5/36) of patients had positive answers to GAQ-1, in the treated and sham groups, respectively (p < 0.05). No adverse events were observed during the study. CONCLUSION: This study showed that penile electromagnetic shockwave therapy may improve erectile function, to a modest extent, on certain patients that do not respond to PDE5I; making it an alternative for vascular ED patients that reject more invasive therapies.

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.

Intronic variation of the SOHLH2 gene confers risk to male reproductive impairment.

OBJECTIVE: To evaluate whether SOHLH2 intronic variation contributes to the genetic predisposition to male infertility traits, including severe oligospermia (SO) and different nonobstructive azoospermia (NOA) clinical phenotypes. DESIGN: Genetic association study. SETTING: Not applicable. PATIENT(S): Five hundred five cases (455 infertile patients diagnosed with NOA and 50 with SO) and 1,050 healthy controls from Spain and Portugal. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genomic DNA extraction from peripheral blood mononuclear cells, genotyping of the SOHLH2 polymorphisms rs1328626 and rs6563386 using the TaqMan allelic discrimination technology, case-control association analyses using logistic regression models, and exploration of functional annotations in publicly available databases. RESULT(S): Evidence of association was observed for both rs6563386 with SO and rs1328626 with unsuccessful sperm retrieval after testicular sperm extraction (TESE-) in the context of NOA. A dominant effect of the minor alleles was suggested in both associations, either when the subset of patients with the manifestation were compared against the control group (rs6563386/SO: P=.021, odds ratio [OR] = 0.51; rs1328626/TESE-: P=.066, OR = 1.46) or against the group of patients without the manifestation (rs6563386/SO: P=.014, OR = 0.46; rs1328626/TESE-: P=.012, OR = 2.43). The haplotype tests suggested a combined effect of both polymorphisms. In silico analyses evidenced that this effect could be due to alteration of the isoform population. CONCLUSION(S): Our data suggest that intronic variation of SOHLH2 is associated with spermatogenic failure. The genetic effect is likely caused by different haplotypes of rs6563386 and rs1328626, which may predispose to SO or TESE- depending on the specific allelic combination.

Rates of hypogonadism forms in Klinefelter patients undergoing testicular sperm extraction: A multicenter cross-sectional study.

INTRODUCTION AND OBJECTIVES: Adult patients with Klinefelter syndrome (KS) may present with testicular volume loss and a decrease in circulating testosterone (T) levels. However, the actual rate of hypogonadism in adult KS men is unknown. We aimed to (a) assess the prevalence of different forms of hypogonadism in a cohort of KS patients with non-obstructive azoospermia (NOA); and (b) investigate potential preoperative predictor of positive sperm retrieval (SR) at surgery in the same cohort of men. METHODS: Complete data from 103 KS men with NOA who underwent testicular sperm extraction (TESE) between 2008 and 2019 at five centers were analyzed. Comorbidities were scored with the Charlson Comorbidity Index (CCI). Patients were categorized into four groups of hypogonadism as follows: eugonadism [normal total T (tT) (≥3.03 ng/mL) and normal luteinizing hormone (LH) (≤9.4 mUI/mL)], secondary hypogonadism [low tT (≤3.03 ng/mL) and low/normal LH (≤9.4 mUI/mL)], primary hypogonadism [low tT (≤3.03 ng/mL) and elevated LH (≥9.4 mUI/mL)], and compensated hypogonadism [normal tT (≥3.03 ng/mL) and elevated LH (≥9.4 mUI/mL)]. Descriptive statistics tested the association between clinical characteristics and laboratory values among the four groups. RESULTS: Median (IQR) patients age was 32 (24, 37) years. Baseline follicle-stimulating hormone and tT levels were 29.5 (19.9, 40.9) mUI/mL and 3.8 (2.5, 11.0) ng/mL, respectively. Eugonadism, primary hypogonadism, and compensated hypogonadism were found in 16 (15.6%), 34 (33.0%), and 53 (51.4%) men, respectively. No patients had secondary hypogonadism. Positive SR rate at TESE was 21.4% (22 patients); of 22, 15 (68.2%) patients underwent assisted reproductive technology and five (22.7%) ended in live birth children. Patients' age, BMI, CCI, FSH levels, and positive SR rates were comparable among hypogonadism groups. No preoperative parameters were associated with positive SR at logistic regressions analysis. CONCLUSIONS: Findings from this cross-sectional study showed that 15.6% of adult KS men have normal tT values at presentation in the real-life setting. Most KS patients presented with either compensated or primary hypogonadism. Sperm retrieval rates were not associated with different forms of hypogonadism.

Sperm retrieval rates in non-mosaic Klinefelter patients undergoing testicular sperm extraction: What expectations do we have in the real-life setting?

BACKGROUND: A recent meta-analysis (Human Reproduction Update 23, 2017 and 265) reported positive sperm retrieval rates (SRR) in 50% of patients with Klinefelter syndrome (KS) undergoing testicular sperm extraction (TESE). However, these results do not reflect the rates of SR that we observe in clinical practice. We assessed the rate and potential predictors of SR in Klinefelter patients in the real-life setting. MATERIALS AND METHODS: We reviewed clinical data of 103 KS men who underwent TESE between 08/2008 and 03/2019 at five tertiary referral Andrology centers. Patients underwent testis ultrasound, hormonal evaluation, and genetic testing. All patients were azoospermic based on the 2010 WHO reference criteria. Conventional TESE (cTESE) or microsurgical TESE (mTESE) was performed based on the surgeon's preference. We used descriptive statistics and logistic regression models to describe the whole cohort. RESULTS: Median (IQR) patient's age was 32 (24-37) years. Baseline serum FSH and total testosterone levels were 29.5 (19.9-40.9) mUI/mL and 3.8 (2.5-11.0) ng/mL, respectively. Conventional TESE and mTESE were performed in 38 (36.5%) and 65 (63.5%) men, respectively. The sperm retrieval rate was 21.4% (22/103 men). Fifteen patients used spermatozoa for ICSI and five ended in live birth children. Patients with positive SR were similar to those with a negative TESE in terms of clinical, hormonal, and procedural parameters (all P > .05). Logistic regression analyses confirmed the lack of association between clinical, hormonal, and procedural parameters with SR outcome. DISCUSSION: Given the conflicting results in the literature regarding SRR in KS, patients should be carefully counseled regarding TESE outcomes based on data from published literature and local results. CONCLUSIONS: In the real-life setting, we observed a lower SRR (21.4%) than that reported in meta-analyses in our cohort of KS patients. No associations between clinical, hormonal, and procedural variables with TESE success were found.

Evaluation of Male Fertility-Associated Loci in a European Population of Patients with Severe Spermatogenic Impairment.

Infertility is a growing concern in developed societies. Two extreme phenotypes of male infertility are non-obstructive azoospermia (NOA) and severe oligospermia (SO), which are characterized by severe spermatogenic failure (SpF). We designed a genetic association study comprising 725 Iberian infertile men as a consequence of SpF and 1058 unaffected controls to evaluate whether five single-nucleotide polymorphisms (SNPs), previously associated with reduced fertility in Hutterites, are also involved in the genetic susceptibility to idiopathic SpF and specific clinical entities. A significant difference in the allele frequencies of USP8-rs7174015 was observed under the recessive model between the NOA group and both the control group (p = 0.0226, OR = 1.33) and the SO group (p = 0.0048, OR = 1.78). Other genetic associations for EPSTI1-rs12870438 and PSAT1-rs7867029 with SO and between TUSC1-rs10966811 and testicular sperm extraction (TESE) success in the context of NOA were observed. In silico analysis of functional annotations demonstrated cis-eQTL effects of such SNPs likely due to the modification of binding motif sites for relevant transcription factors of the spermatogenic process. The findings reported here shed light on the molecular mechanisms leading to severe phenotypes of idiopathic male infertility, and may help to better understand the contribution of the common genetic variation to the development of these conditions.

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.

Clinical relevance of Y-linked CNV screening in male infertility: new insights based on the 8-year experience of a diagnostic genetic laboratory.

AZF microdeletion screening is routinely performed in the diagnostic work-up for male infertility; however, some issues remain debated. In this study, we provide insights into the sperm concentration cutoff value for routine testing, the predictive value of AZFc deletion for testicular sperm retrieval and the Y-background contribution to the interpopulation variability of deletion frequencies. In the Spanish population, partial AZFc rearrangements have been poorly explored and no data exist on partial duplications. In our study, 27/806 (3.3%) patients carried complete AZF deletions. All were azoo/cryptozoospermic, except for one whose sperm concentration was 2 × 10(6)/ml. In AZFc-deleted men, we observed a lower sperm recovery rate upon conventional TESE (9.1%) compared with the literature (60-80% with microTESE). Haplogroup E was the most represented among non-Spanish and hgr P among Spanish AZF deletion carriers. The analysis of AZFc partial rearrangements included 330 idiopathic infertile patients and 385 controls of Spanish origin. Gr/gr deletion, but not AZFc partial duplications, was significantly associated with spermatogenic impairment. Our data integrated with the literature suggest that: (1) routine AZF microdeletion testing could eventually include only men with ≤2 × 10(6)/ml; (2) classical TESE is associated with low sperm recovery rate in azoospermic AZFc-deleted men, and therefore microTESE should be preferred; (3) Y background could partially explain the differences in deletion frequencies among populations. Finally, our data on gr/gr deletion further support the inclusion of this genetic test in the work-up of infertile men, whereas partial AZFc duplications do not represent a risk for spermatogenic failure in the Spanish population.