González-Muñoz, SaraCerván-Martín, MiriamGuzmán-Jiménez, AndreaRodríguez-Martín, Ana IsabelGarrido, NicolásCastilla, José A.Gonzalvo, M. CarmenClavero, AnaMolina, MartaVilches, Miguel ÁngelEspuch-Oliver, AndreaMaldonado, VicenteGarcía-Peña, María LuisaGaliano-Gutiérrez, NoeliaSantamaría, EstherGonzález, CristinaQuintana-Ferraz, FernandoGómez, SusanaAmorós, DavidMartínez-Granados, LuisOrtega-González, YaniraBurgos, MiguelPereira-Caetano, IrisPinto, Graça S.Aguiar, AnaPereira, Isabel S.López-Rodrigo, OlgaBassas, LluísSeixas, SusanaGonçalves, JoãoLopes, Alexandra M.Larriba, SaraBossini-Castillo, LaraCarmona, F. DavidPalomino-Morales, Rogelio J.2026-01-272026-01-272025-06-11Hum Reprod . 2025 Sep 1;40(9):1762-1772. doi: 10.1093/humrep/deaf1070268-1161http://hdl.handle.net/10400.18/10765Erratum in: Hum Reprod. 2025 Sep 1;40(9):1797. doi: 10.1093/humrep/deaf142Study question: What is the functional impact of the rs508485 genetic polymorphism, located in the 3'-untranslated region (UTR) region of the PIWIL4 gene, on non-obstructive azoospermia (NOA)? Summary answer: The rs508485 genetic variant contributes to the pathogenesis of extreme patterns of NOA by modulating PIWIL4 expression through microRNA (miRNA) interactions. What is known already: Male infertility represents a significant global health challenge with profound societal and economic consequences. One of the most severe forms of male infertility is NOA, which is characterized by severe spermatogenic failure (SPGF) of idiopathic origin in most cases. Cumulating knowledge increasingly suggests that this idiopathic form of NOA may represent a multifactorial condition involving complex interactions between genetic and environmental factors. The PIWI protein subfamily, particularly PIWIL4, plays a pivotal role in spermatogenesis by processing PIWI-interacting RNAs, which silence retrotransposons to protect genomic integrity. Genetic variations in this gene have been found to be associated with susceptibility to NOA. Study design, size, duration: A case-control study was conducted in a European cohort including 1516 infertile men with SPGF and 2451 fertile controls. Logistic regression and functional assays were employed to investigate the functional role of the rs508485 polymorphism in PIWIL4. Participants/materials, setting, methods: Participants were genotyped for the rs508485 polymorphism. Associations between the polymorphism and NOA phenotypes, including Sertoli cell-only (SCO) syndrome and testicular sperm extraction (TESE) outcomes, were assessed. In silico tools predicted miRNA binding effects, which were subsequently validated using luciferase reporter assays. Main results and the role of chance: The T allele of rs508485 was significantly associated with the SCO phenotype (P = 2.69E-03, OR = 1.34) and unfavourable TESE outcomes (P = 1.09E-03, OR = 1.54). In silico analyses predicted that the rs508485 variant might alter binding sites in the 3'-UTR region of PIWIL4 for different miRNAs, such as hsa-miR-215-3p and hsa-miR-136-3p. Functional validation using luciferase assays confirmed that these miRNAs differentially bind to the T and C alleles of this polymorphism, influencing PIWIL4 regulation. Large scale data: N/A. Limitations, reasons for caution: The study is limited to a single genetic polymorphism and functional assays were performed in vitro. Additional studies are required to validate these findings across diverse populations and explore additional genetic interactions. Wider implications of the findings: These findings highlight the critical role of miRNA regulation in extreme forms of male infertility by influencing the expression of essential spermatogenesis genes, such as PIWIL4. Our study sheds light on the genetic mechanisms underlying spermatogenesis and suggests potential therapeutic targets for NOA.engNOASCOTESEPIWIL4piRNAmiRNASNPMale InfertilityDoenças Genéticasjournal article10.1093/humrep/deaf1071460-235040499151