Nonobstructive azoospermia (NOA) represents the most severe expression of male infertility, involving around 1% of the male population and 10% of infertile men. This condition is characterised by the inability of the testis to produce sperm cells, and it is considered to have an important genetic component. During the last two decades, different genetic anomalies, including microdeletions of the Y chromosome, karyotype defects, and missense mutations in genes involved in the reproductive function, have been described as the primary cause of NOA in many infertile men. However, these alterations only explain around 25% of azoospermic cases, with the remaining patients showing an idiopathic origin. Recent studies clearly suggest that the so-called idiopathic NOA has a complex aetiology with a polygenic inheritance, which may alter the spermatogenic process. Although we are far from a complete understanding of the molecular mechanisms underlying NOA, the use of the new technologies for genetic analysis has enabled a considerable increase in knowledge during the last years. In this review, we will provide a comprehensive and updated overview of the genetic basis of NOA, with a special focus on the possible application of the recent insights in clinical practice.

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.

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.

STUDY QUESTION: Can genome-wide genotyping data be analysed using a hypothesis-driven approach to enhance the understanding of the genetic basis of severe spermatogenic failure (SPGF) in male infertility? SUMMARY ANSWER: Our findings revealed a significant association between SPGF and the SHOC1 gene and identified three novel genes (PCSK4, AP3B1, and DLK1) along with 32 potentially pathogenic rare variants in 30 genes that contribute to this condition. WHAT IS KNOWN ALREADY: SPGF is a major cause of male infertility, often with an unknown aetiology. SPGF can be due to either multifactorial causes, including both common genetic variants in multiple genes and environmental factors, or highly damaging rare variants. Next-generation sequencing methods are useful for identifying rare mutations that explain monogenic forms of SPGF. Genome-wide association studies (GWASs) have become essential approaches for deciphering the intricate genetic landscape of complex diseases, offering a cost-effective and rapid means to genotype millions of genetic variants. Novel methods have demonstrated that GWAS datasets can be used to infer rare coding variants that are causal for male infertility phenotypes. However, this approach has not been previously applied to characterize the genetic component of a whole case–control cohort. STUDY DESIGN, SIZE, DURATION: We employed a hypothesis-driven approach focusing on all genetic variation identified, using a GWAS platform and subsequent genotype imputation, encompassing over 20 million polymorphisms and a total of 1571 SPGF patients and 2431 controls. Both common (minor allele frequency, MAF > 0.01) and rare (MAF < 0.01) variants were investigated within a total of 1797 loci with a reported role in spermatogenesis. This gene panel was meticulously assembled through comprehensive searches in the literature and various databases focused on male infertility genetics. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study involved a European cohort using previously and newly generated data. Our analysis consisted of three independent methods: (i) variant-wise association analyses using logistic regression models, (ii) gene-wise association analyses using combined multivariate and collapsing burden tests, and (iii) identification and characterisation of highly damaging rare coding variants showing homozygosity only in SPGF patients. MAIN RESULTS AND THE ROLE OF CHANCE: The variant-wise analyses revealed an association between SPGF and SHOC1-rs12347237 (P = 4.15E−06, odds ratio = 2.66), which was likely explained by an altered binding affinity of key transcription factors in regulatory regions and the disruptive effect of coding variants within the gene. Three additional genes (PCSK4, AP3B1, and DLK1) were identified as novel relevant players in human male infertility using the gene-wise burden test approach (P < 5.56E−04). Furthermore, we linked a total of 32 potentially pathogenic and recessive coding variants of the selected genes to 35 different cases. LARGE SCALE DATA: Publicly available via GWAS catalog (accession number: GCST90239721). LIMITATIONS, REASONS FOR CAUTION: The analysis of low-frequency variants presents challenges in achieving sufficient statistical power to detect genetic associations. Consequently, independent studies with larger sample sizes are essential to replicate our results. Additionally, the specific roles of the identified variants in the pathogenic mechanisms of SPGF should be assessed through functional experiments. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the benefit of using GWAS genotyping to screen for both common and rare variants potentially implicated in idiopathic cases of SPGF, whether due to complex or monogenic causes. The discovery of novel genetic risk factors for SPGF and the elucidation of the underlying genetic causes provide new perspectives for personalized medicine and reproductive counselling. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Spanish Ministry of Science and Innovation through the Spanish National Plan for Scientific and Technical Research and Innovation (PID2020-120157RB-I00) and the Andalusian Government through the research projects of ‘Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020)’ (ref. PY20_00212) and ‘Proyectos de Investigación aplicada FEDER-UGR 2023’ (ref. C-CTS-273-UGR23). S.G.-M. was funded by the previously mentioned projects (ref. PY20_00212 and PID2020-120157RB-I00). A.G.-J. was funded by MCIN/AEI/10.13039/501100011033 and FSE ‘El FSE invierte en tu futuro’ (grant ref. FPU20/02926). IPATIMUP integrates the i3S Research Unit, which is partially supported by the Portuguese Foundation for Science and Technology (FCT), financed by the European Social Funds (COMPETE-FEDER) and National Funds (projects PEstC/SAU/LA0003/2013 and POCI-01-0145-FEDER-007274). S.S. is supported by FCT funds (10.54499/DL57/2016/CP1363/CT0019), ToxOmics-Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, and is also partially supported by the Portuguese Foundation for Science and Technology (UIDP/00009/2020 and UIDB/00009/2020). S. Larriba received support from Instituto de Salud Carlos III (grant: DTS18/00101), co-funded by FEDER funds/European Regional Development Fund (ERDF)—a way to build Europe) and from ‘Generalitat de Catalunya’ (grant 2021SGR052). S. Larriba is also sponsored by the ‘Researchers Consolidation Program’ from the SNS-Dpt. Salut Generalitat de Catalunya (Exp. CES09/020). All authors declare no conflict of interest related to this study.

Erdheim–Chester Disease (ECD) is a rare histiocytosis characterized by a wide spectrum of clinical manifestations. Although somatic mutations have been involved in ECD, its etiology remains poorly understood. This study aimed to identify novel molecular mechanisms involved in ECD through the first integrated methylome and transcriptome analysis. Peripheral blood samples were collected from 137 ECD patients and 410 controls. Genome-wide DNA methylation and transcriptome analyses were performed, followed by functional in silico studies using different online bioinformatics tools. Subsequently, methylome and transcriptome data were integrated, and a drug repurposing approach was undertaken. Our results revealed 2511 differentially methylated positions and 1484 differentially expressed genes associated with ECD. The integrative analysis identified 46 alterations in DNA methylation patterns that regulate the expression levels of 29 altered genes in ECD patients, highlighting key genes involved in immune response and tumorigenesis. Remarkably, our results identified B cells and NF-kB signaling pathway as novel contributors of ECD pathogenesis. Finally, the drug repurposing analysis identified potential therapeutic options for ECD patients. In conclusion, this study represents an important advance in understanding the molecular basis of ECD, proposing novel cell types and pathways involved in ECD pathogenesis and suggesting new avenues for clinical management.

STUDY 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. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (grant: PID2020-120157RB-I00 funded by MICIU/AEI/10.13039/501100011033; and grant: PID2023-152215OB-I00 funded by MICIU/AEI/10.13039/501100011033 and ERDF, EU). M.C.-M is beneficiary of a Juan de la Cierva 2022 postdoctoral fellowship (reference number JDC2022-048561-I) funded by MCIN/AEI/10.13039/501100011033 and European Union NextGenerationEU/PRTR. S.L. received support from “Generalitat de Catalunya” (grant 2021SGR052). S.L. is sponsored by the “Researchers Consolidation Program” from the SNS-Dpt. Salut Generalitat de Catalunya (Exp. CES09/020). S.G.-M. was funded by grant ref. FPU23/02674. S.S. was supported by funds from the Portuguese Foundation for Science and Technology (FCT; 10.54499/DL57/2016/CP1363/CT0019). The authors declare no competing interests.