Changes in environmental exposures over decades may influence the genetic architecture of severe spermatogenic failure.

STUDY QUESTION: Do the genetic determinants of idiopathic severe spermatogenic failure (SPGF) differ between generations? SUMMARY ANSWER: Our data support that the genetic component of idiopathic SPGF is impacted by dynamic changes in environmental exposures over decades. WHAT IS KNOWN ALREADY: The idiopathic form of SPGF has a multifactorial etiology wherein an interaction between genetic, epigenetic, and environmental factors leads to the disease onset and progression. At the genetic level, genome-wide association studies (GWASs) allow the analysis of millions of genetic variants across the genome in a hypothesis-free manner, as a valuable tool for identifying susceptibility risk loci. However, little is known about the specific role of non-genetic factors and their influence on the genetic determinants in this type of conditions. STUDY DESIGN, SIZE, DURATION: Case-control genetic association analyses were performed including a total of 912 SPGF cases and 1360 unaffected controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants had European ancestry (Iberian and German). SPGF cases were diagnosed during the last decade either with idiopathic non-obstructive azoospermia (n = 547) or with idiopathic non-obstructive oligozoospermia (n = 365). Case-control genetic association analyses were performed by logistic regression models considering the generation as a covariate and by in silico functional characterization of the susceptibility genomic regions. MAIN RESULTS AND THE ROLE OF CHANCE: This analysis revealed 13 novel genetic association signals with SPGF, with eight of them being independent. The observed associations were mostly explained by the interaction between each lead variant and the age-group. Additionally, we established links between these loci and diverse non-genetic factors, such as toxic or dietary habits, respiratory disorders, and autoimmune diseases, which might potentially influence the genetic architecture of idiopathic SPGF. LARGE SCALE DATA: GWAS data are available from the authors upon reasonable request. LIMITATIONS, REASONS FOR CAUTION: Additional independent studies involving large cohorts in ethnically diverse populations are warranted to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Overall, this study proposes an innovative strategy to achieve a more precise understanding of conditions such as SPGF by considering the interactions between a variable exposome through different generations and genetic predisposition to complex diseases. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the "Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020)" (ref. PY20_00212, P20_00583), the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (ref. PID2020-120157RB-I00 funded by MCIN/ AEI/10.13039/501100011033), and the 'Proyectos I+D+i del Programa Operativo FEDER 2020' (ref. B-CTS-584-UGR20). ToxOmics-Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, is also partially supported by the Portuguese Foundation for Science and Technology (Projects: UIDB/00009/2020; UIDP/00009/2020). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Using Small Non-Coding RNAs in Extracellular Vesicles of Semen as Biomarkers of Male Reproductive System Health: Opportunities and Challenges.

Reproductive dysfunction and urogenital malignancies represent a serious health concern in men. This is in part as a result of the absence of reliable non-invasive tests of diagnosis/prognosis. Optimizing diagnosis and predicting the patient's prognosis will affect the choice of the most appropriate treatment and therefore increase the chances of success and the result of therapy, that is, it will lead to a more personalized treatment of the patient. This review aims firstly to critically summarize the current knowledge of the reproductive roles played by extracellular vesicle small RNA components, which are typically altered in diseases affecting the male reproductive tract. Secondly, it aims to describe the use of semen extracellular vesicles as a non-invasive source of sncRNA-based biomarkers for urogenital diseases.

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.

The PxIxIT motif of the RCAN3 inhibits angiogenesis and tumor progression in Triple Negative breast cancer in immunocompetent mice.

RCAN proteins are endogenous regulators of the calcineurin-cytosolic nuclear factor of activated T cells (CN-NFATc) pathway that bind CN through similar conserved motifs PxIxIT and LxVP of the NFATc family. RCAN1 and RCAN3 protein levels were reported to correlate with overall survival of breast cancer patients. We additionally provided supporting results about RCAN3 role on cancer showing that overexpression of the native PxIxIT sequence of RCAN3-derived R3 peptide (PSVVVH, EGFP-R3178-210) dramatically inhibits tumor growth and tumor angiogenesis in an orthotopic mouse model of Triple Negative Breast Cancer (TNBC) in nude mice. On the other hand, RCAN3 protein and its derived peptide EGFP-R3178-210 bind to CN and inhibit NFAT-mediated cytokine gene expression without affecting CN phosphatase activity suggesting that RCAN3 and EGFP-R3178-210 peptide have tumor suppressor and immunosuppressant activity. Due to the known relationship between tumor development and immune system, as well as the relevance of CN-NFATc in the regulation of the immune system, in the present study we decided to assess the effect of EGFP-R3178-210 peptide in an orthotopic syngeneic TNBC mouse model, in order to ensure that the role of RCAN3 as immunosuppressant do not override its tumor suppressor activity. Our results evidence that EGFP-R3178-210 peptide displays an inhibitory potential on tumor growth and tumor angiogenesis similar to those obtained in the previous orthotopic TNBC model. These results highlight the importance of the RCAN3 peptide as a tumor suppressor protein and totally complement our previous results, indicating that this antitumor activity role is maintained in the presence of a complete functional immune system.

Genome-wide compound heterozygote analysis highlights DPY19L2 alleles in a non-consanguineous Spanish family with total globozoospermia.

RESEARCH QUESTION: Would the use of genome-wide genotyping be an advantageous strategy to identify the molecular aetiology of two brothers from a non-consanguineous family, clinically diagnosed with total globozoospermia? DESIGN: Two related Spanish globozoospermic patients were studied. Eight first- and second-degree family members were also included in the study. The clinical procedure included anamnesis, physical examination and semen analyses. Acrosome visualization was performed by fluorescein isothiocyanate-Pisum sativum agglutinin labelling and ultrastructural electron microscope sperm analysis. Sperm DNA fragmentation was determined by TUNEL and SCD. Molecular analysis included: the detection of deletion of the DPY19L2 gene by a BPa (break point "a") gap-polymerase chain reaction, and genotyping by using a high-throughput genome-wide genotyping platform and a genotype imputation strategy. RESULTS: The biological characteristics of the two globozoospermic siblings included round-headed spermatozoa without an acrosome; ultrastructural defects in spermatozoa; increased sperm fragmentation and aneuploidies, inability of spermatozoa to activate oocytes (correctable with artificial activation) and good developmental potential of embryos generated by IVF/intracytoplasmic sperm injection. This genetic study focused on a genome-wide compound heterozygote analysis that identified two deleterious rare coding variants in the DPY19L2 gene [rs771726551 (c.431T>A exon 3) and rs147579680 (c.869G>A exon 8)]. CONCLUSION: A genome-wide compound heterozygote analysis strategy should be considered for molecular screening in globozoospermia and other rare congenital diseases, particularly in cases from non-consanguineous families.

Impact of Extracellular Vesicle Isolation Methods on Downstream Mirna Analysis in Semen: A Comparative Study.

Seminal plasma (SP) contains a unique concentration of miRNA, mostly contained in small extracellular vesicles (sEVs) such as exosomes, some of which could be clinically useful for diagnosis and/or prognosis of urogenital diseases such as prostate cancer (PCa). We optimized several exosome-EV isolation technologies for their use in semen, evaluating EV purifying effectiveness and impact on the downstream analysis of miRNAs against results from the standard ultracentrifugation (UC) method to implement the use of SP sEV_miRNAs as noninvasive biomarkers for PCa. Our results evidenced that commercial kits designed to isolate exosomes/EVs from blood or urine are mostly applicable to SP, but showed quantitative and qualitative variability between them. ExoGAG 3500× g and the miRCURY Cell/Urine/CSF 1500× g methods resulted as equivalent alternative procedures to UC for isolating exosomes/sEVs from semen for nanoparticle characteristics and quality of RNA contained in vesicles. Additionally, the expression profile of the altered semen sEV-miRNAs in PCa varies depending on the EV isolation method applied. This is possibly due to different extraction techniques yielding different proportions of sEV subtypes. This is evidence that the exosome-EV isolation method has a significant impact on the analysis of the miRNAs contained within, with important consequences for their use as clinical biomarkers. Therefore, miRNA analysis results for EVs cannot be directly extrapolated between different EV isolation methods until clear markers for delineation between microvesicles and exosomes are established. However, EV extraction methodology affects combined models (semen exosome miRNA signatures plus blood Prostate specific antigen (PSA) concentration for PCa diagnosis) less; specifically our previously described (miR-142-3p + miR-142-5p + miR-223-3p + PSA) model functions as molecular marker from EVs from any of the three isolation methods, potentially improving the efficiency of PSA PCa diagnosis.

Exosomal microRNAs in seminal plasma are markers of the origin of azoospermia and can predict the presence of sperm in testicular tissue.

STUDY QUESTION: Are exosomal microRNAs (miRNAs) in seminal plasma (SP) useful as markers of the origin of azoospermia and the presence of sperm in the testis? SUMMARY ANSWER: Our study demonstrated the potential of several miRNAs contained in small extracellular vesicles (sEVs) of seminal fluid as sensitive and specific biomarkers for selecting those azoospermic individuals with real chances of obtaining spermatozoa from the testicular biopsy. WHAT IS KNOWN ALREADY: There are no precise non-invasive diagnostic methods for classifying the origin of the sperm defects in semen and the spermatogenic reserve of the testis in those infertile men with a total absence of sperm in the ejaculate (azoospermia). The diagnosis of such individuals is often based on the practice of biopsies. In this context it is reasonable to study the presence of organ-specific markers in human semen that contains fluid from the testis and the male reproductive glands, which could help in the diagnosis and prognosis of male infertility. Additionally, seminal fluid contains high concentrations of sEVs that are morphologically and molecularly consistent with exosomes, which originate from multiple cellular sources in the male reproductive tract. STUDY DESIGN, SIZE, DURATION: A case and control prospective study was performed. This study compares the miRNA content of exosomes in semen samples obtained from nine normozoospermic fertile individuals (control group), 14 infertile men diagnosed with azoospermia due to spermatogenic failure, and 13 individuals with obstructive azoospermia and conserved spermatogenesis. Additionally, three severe oligozoospermic individuals (<5 × 106 sperm/ml) were included in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: A differential high-throughput miRNA profiling analysis using miRNA quantitative PCR panels was performed in SP exosomes from azoospermic patients and fertile individuals. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 623 miRNAs were included in the miRNA profiling stage of the study. A total of 397 miRNAs (63.7%) were consistently detected in samples from all groups and statistically analysed, which revealed altered patterns of miRNA expression in infertile patients. We focused on the miRNAs that were differentially expressed between azoospermia as a result of an obstruction in the genital tract (i.e. having conserved spermatogenesis) and azoospermia caused by spermatogenic failure, and described, in a miRNA validation stage of the study, the expression values of one miRNA (miR-31-5p) in exosomes from semen as a predictive biomarker test for the origin of azoospermia with high sensitivity and specificity (>90%). The efficacy of the predictive test was even better when the blood FSH values were included in the analysis. Furthermore a model that included miR-539-5p and miR-941 expression values is also described as being useful for predicting the presence of residual spermatogenesis in individuals with severe spermatogenic disorders with diagnostic accuracy. LIMITATIONS, REASONS FOR CAUTION: Further studies, with an independent second population involving a larger number of samples, are needed to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Our findings contribute to the search for the most valuable genetic markers that are potentially useful as tools for predicting the presence of testicular sperm in azoospermic individuals. STUDY FUNDING/COMPETING INTEREST(S): This work was financially supported by grants from the Fondo de Investigaciones Sanitarias/Fondo Europeo de Desarrollo Regional "Una manera de hacer Europa" (FIS/FEDER) [Grant number PI15/00153], the Generalitat de Catalunya [Grant number 2014SGR5412]. S.L. is sponsored by the Researchers Stabilization Program (ISCIII/Generalitat de Catalunya) from the Spanish National Health System [CES09/020].

Aberrant DNA methylation patterns of spermatozoa in men with unexplained infertility.

STUDY QUESTION: Are there DNA methylation alterations in sperm that could explain the reduced biological fertility of male partners from couples with unexplained infertility? SUMMARY ANSWER: DNA methylation patterns, not only at specific loci but also at Alu Yb8 repetitive sequences, are altered in infertile individuals compared with fertile controls. WHAT IS KNOWN ALREADY: Aberrant DNA methylation of sperm has been associated with human male infertility in patients demonstrating either deficiencies in the process of spermatogenesis or low semen quality. STUDY DESIGN, SIZE, DURATION: Case and control prospective study. This study compares 46 sperm samples obtained from 17 normospermic fertile men and 29 normospermic infertile patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Illumina Infinium HD Human Methylation 450K arrays were used to identify genomic regions showing differences in sperm DNA methylation patterns between five fertile and seven infertile individuals. Additionally, global DNA methylation of sperm was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek) in 14 samples, and DNA methylation at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4) measured by bisulfite pyrosequencing in 44 sperm samples. A sperm-specific DNA methylation pattern was obtained by comparing the sperm methylomes with the DNA methylomes of differentiated somatic cells using data obtained from methylation arrays (Illumina 450 K) of blood, neural and glial cells deposited in public databases. MAIN RESULTS AND THE ROLE OF CHANCE: In this study we conduct, for the first time, a genome-wide study to identify alterations of sperm DNA methylation in individuals with unexplained infertility that may account for the differences in their biological fertility compared with fertile individuals. We have identified 2752 CpGs showing aberrant DNA methylation patterns, and more importantly, these differentially methylated CpGs were significantly associated with CpG sites which are specifically methylated in sperm when compared with somatic cells. We also found statistically significant (P < 0.001) associations between DNA hypomethylation and regions corresponding to those which, in somatic cells, are enriched in the repressive histone mark H3K9me3, and between DNA hypermethylation and regions enriched in H3K4me1 and CTCF, suggesting that the relationship between chromatin context and aberrant DNA methylation of sperm in infertile men could be locus-dependent. Finally, we also show that DNA methylation patterns, not only at specific loci but also at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4), were lower in sperm than in somatic cells. Interestingly, sperm samples at Alu Yb8 repetitive sequences of infertile patients showed significantly lower DNA methylation levels than controls. LIMITATIONS, REASONS FOR CAUTION: Our results are descriptive and further studies would be needed to elucidate the functional effects of aberrant DNA methylation on male fertility. WIDER IMPLICATIONS OF THE FINDINGS: Overall, our data suggest that aberrant sperm DNA methylation might contribute to fertility impairment in couples with unexplained infertility and they provide a promising basis for future research. STUDY FUNDING/COMPETING INTERESTS: This work has been financially supported by Fundación Cientifica de la AECC (to R.G.U.); IUOPA (to G.F.B.); FICYT (to E.G.T.); the Spanish National Research Council (CSIC; 200820I172 to M.F.F.); Fundación Ramón Areces (to M.F.F); the Plan Nacional de I+D+I 2008-2011/2013-2016/FEDER (PI11/01728 to AF.F., PI12/01080 to M.F.F. and PI12/00361 to S.L.); the PN de I+D+I 2008-20011 and the Generalitat de Catalunya (2009SGR01490). A.F.F. is sponsored by ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (CP11/00131). S.L. is sponsored by the Researchers Stabilization Program from the Spanish National Health System (CES09/020). The IUOPA is supported by the Obra Social Cajastur, Spain.

Seminal extracellular vesicle sncRNA sequencing reveals altered miRNA/isomiR profiles as sperm retrieval biomarkers for azoospermia.

BACKGROUND: Non-invasive molecular biomarkers for classifying azoospermia by origin into either obstructive or non-obstructive/secretory azoospermia, as well as for inferring the spermatogenic reserve of the testis of non-obstructive/secretory azoospermia patients, are of great interest for testicular sperm retrieval outcome prediction for assisted reproduction. Prior analyses of semen small non-coding RNA expression in azoospermia have focused on microRNAs, but there has been a lack of attention on other regulatory small RNA species. In this regard, studying more in-depth expression changes of small non-coding RNA subtypes in small extracellular vesicles from semen of azoospermic individuals could be useful to select additional non-invasive biomarkers with diagnostic/prognostic purposes. MATERIAL AND METHODS: A high-throughput small RNA profiling analysis to determine the expression pattern of seminal small extracellular vesicle microRNAs (analyzed at the isomiR level), PIWI-interacting RNAs, and transfer RNA-derived small RNAs in normozoospermic (n = 4) and azoospermic (obstructive azoospermia because of pathological occurring obstruction in the genital tract, n = 4; secretory azoospermic individuals with positive testicular sperm extraction value, n = 5; secretory azoospermic individuals with negative testicular sperm extraction value, n = 4) individuals was carried out. Reverse transcriptase-quantitative real-time polymerase chain reaction validation analysis of selected microRNAs was additionally performed in a larger number of individuals. RESULTS AND DISCUSSION: Clinically relevant quantitative changes in the small non-coding RNA levels contained in semen small extracellular vesicles can be used as biomarkers for the origin of azoospermia and for predicting the presence of residual spermatogenesis. In this regard, canonical isoform microRNAs (n = 185) but also other isomiR variants (n = 238) stand out in terms of numbers and fold-change differences in expression, underlining the need to consider isomiRs when investigating microRNA-based regulation. Conversely, although transfer RNA-derived small RNAs are shown in our study to represent a high proportion of small non-coding RNA sequences in seminal small extracellular vesicle samples, they are not able to discriminate the origin of azoospermia. PIWI-interacting RNA cluster profiles and individual PIWI-interacting RNAs with significant differential expression were also not able to discriminate. Our study demonstrated that expression values of individual and/or combined canonical isoform microRNAs (miR-10a-5p, miR-146a-5p, miR-31-5p, miR-181b-5p; area under the receiver operating characteristic curve >0.8) in small extracellular vesicles provide considerable clinical value in identifying samples with a high likelihood of sperm retrieval while discriminating azoospermia by origin. Although no individual microRNA showed sufficient discriminating power on its own to identify severe spermatogenic disorders with focal spermatogenesis, multivariate microRNA models in semen small extracellular vesicles have the potential to identify those individuals with residual spermatogenesis. Availability and adoption of such non-invasive molecular biomarkers would represent a great improvement in reproductive treatment decision protocols for azoospermia in clinical practice.

Experimental validation of seminal miR-31-5p as biomarker for azoospermia and evaluation of the effect of preanalytical variables.

BACKGROUND AND OBJECTIVES: Predicting the origin of azoospermia with non-invasive biomarkers is clinically relevant for determining the chance of successful sperm retrieval from the testes before attempting assisted reproduction treatment. Here, the semen small extracellular vesicle microRNA miR-31-5p-based biomarker test to distinguish obstructive azoospermia from secretory azoospermia (previously described by our group) is validated for clinical use, and additionally, the sample source (seminal small extracellular vesicles vs. total seminal plasma) as a preanalytical variable is considered to optimize the procedure. RESULTS AND DISCUSSION: Our results provide evidence that altered miR-31-5p expression can be determined both from extracellular vesicles and from the whole seminal plasma to discriminate obstructive from secretory azoospermic samples. Not only have we validated this microRNA-based molecular model as a clinically useful test for predicting the origin of azoospermia in a sample from azoospermic individuals, but additionally, and more interestingly for the clinicians, we have evidenced its usefulness for predicting the presence of spermatogenic failure in azoospermic patients with follicle-stimulating hormone values <10 IU/L as a sensitive and specific biomarker (area under the receiver operating characteristic curve >0.88; p-value < 0.006). The use of total seminal plasma as an analytical sample would facilitate the use of a simplified technical procedure for miR-31-5p quantification and would represent a great improvement in reproductive treatment decision protocols for azoospermia in clinical practice.

Sperm gene expression profile is related to pregnancy rate after insemination and is predictive of low fecundity in normozoospermic men.

BACKGROUND: Assessment of male fertility is traditionally based on microscopic evaluation of semen. However, the classical semen parameters do not adequately reflect sperm function, and their clinical value in predicting fertility is limited. We hypothesize that the sperm expression profile could reflect the fertilizing quality of spermatozoa and could be more informative for predicting the in vivo reproductive fitness of men with normal semen parameters. METHODS: Sperm gene expression patterns of 68 normozoospermic donors (43 Phase I and 25 Phase II), used for therapeutic IUI, were analysed via TaqMan Arrays. RESULTS: Significant differences in the expression of individual genes were observed between groups of donors with the lowest and highest pregnancy rates (PRs) after IUI. Additionally, we have developed a molecular means to classify the fertility status of semen donors for IUI based on the expression signature of four genes. In the Phase I study, this model had 90% sensitivity and 97% specificity for discriminating donors resulting in low PRs (cut-off value: <13.6%), far better than that obtained from the combination of sperm parameters. The translation of the model was validated in Phase II donors resulting in a sensitivity of 71.5% and a specificity of 78%. CONCLUSIONS: Our findings contribute to the search for the most valuable genetic markers which are potentially useful as tools for predicting pregnancy. Our expression model could complement classical semen analysis in order to identify sperm donors with a less favourable IUI reproductive outcome despite having normal semen parameters. It may also be useful for the study of sperm function in couples with unexplained infertility.

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.

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.

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.

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.

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.

Seminal plasma microRNAs improve diagnosis/prognosis of prostate cancer in men with moderately altered prostate-specific antigen.

There is an urgent need for accurate non-invasive biomarkers for prostate cancer (PCa) diagnosis and disease risk stratification. Previous data suggests that total seminal plasma (SP) represents a source of miRNAs for screening. We have evaluated a panel of eight PCa-associated miRNAs for their potential use as PCa biomarkers in SP by analyzing their levels using RT-qPCR. Multivariate logistic regression modelling and clinical risk assessment were performed for those SP miRNAs statistically altered between PCa and non-PCa (HCt and/or BPH) groups. Our results provide evidence that altered miRNA expression in PCa tissue can also be detected in total SP. We obtained a clinically useful SP miRNA-based combined model (PSA+miR-142-3p+miR-223-3p+miR-93-5p), which improves PCa specificity of the PSA test, for, firstly, predicting the presence of malignant tumors in a sample from the total population and secondly, and more interestingly for clinicians, for predicting PCa in samples from the positive PSA screening test (PSA>4 ng/ml). Additionally, [PSA+miR-30d-5p+miR-93-5p] and [PSA+miR-30d-5p] models have been shown to be useful for predicting the disease aggressiveness with diagnostic accuracy. In conclusion, our results provide evidence that miRNAs in total SP represent a useful target for evaluation for PCa, which technically simplifies the future use of semen miRNA-based models as non-invasive biomarkers to increase the efficiency of PCa diagnosis and prognosis.

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.

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.