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IVIRMA Group, Lisbon Clinical Group: Alberto Pacheco, Cristina González, Susana Gómez, David Amorós, Jesus Aguilar, Fernando Quintana, Carlos Calhaz-Jorge, Ana Aguiar, Joaquim Nunes, Sandra Sousa, Maria Graça Pinto, Sónia Correia 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

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., Spanish Ministry of Economy and Competitiveness through the Spanish State Plan for Scientific and Technical Research and Innovation SAF201678722-R, Spanish Government RYC-2014-16458, "Juan de la Cierva Incorporacion" program IJC2018-038026-I, European Union (EU), Spanish Government FIS-ISCIII DTS18/00101, Generalitat de Catalunya 2017SGR191, "Plan Propio" program of the University of Granada ("Becas de Iniciacion a la Investigacion para estudiantes de Grado"), "Researchers Consolidation Program" from the SNS-Dpt. Salut Generalitat de Catalunya CES09/020, FCT/MCTES, through national funds attributed to Center for Toxicogenomics and Human Health-ToxOmics UIDB/00009/2020, Portuguese Foundation for Science and Technology SFRH/BPD/120777/2016, Portuguese State Budget of the Ministry for Science, Technology and High Education, European Social Fund through the Programa Operacional do Capital Humano, Portuguese Foundation for Science and Technology IF/01262/2014, FCT in the framework of the project "Institute for Research and Innovation in Health Sciences" POCI-01-0145-FEDER-007274

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. info:eu-repo/semantics/publishedVersion

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&times, g and the miRCURY Cell/Urine/CSF 1500&times, 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.