TYPE Original Research PUBLISHED 15 December 2022 DOI 10.3389/fcell.2022.1089782 Contribution of TEX15 genetic OPEN ACCESS variants to the risk of developing severe non-obstructive EDITED BY Mingrong Lv, First Affiliated Hospital of Anhui Medical University, China REVIEWED BY Rong Hua, oligozoospermia Anhui Medical University, China Aminata Toure, Institute for Advanced Biosciences, France Andrea Guzmán-Jiménez 1,2‡, Sara González-Muñoz 1,2‡, Chunyu Liu, Fudan University, China Miriam Cerván-Martín 1,2, Rocío Rivera-Egea 3,4, *CORRESPONDENCE Nicolás Garrido 4,5, Saturnino Luján 5, Samuel Santos-Ribeiro 6,7, F. David Carmona, José A. Castilla 2,8,9, M. Carmen Gonzalvo 2,8, Ana Clavero 2,8, dcarmona@ugr.es Lara Bossini-Castillo, F. Javier Vicente 2,10, Vicente Maldonado 11, lbossinicastillo@ugr.es Javier Villegas-Salmerón 1, Miguel Burgos 1, Rafael Jiménez 1, # IVIRMA Group, and Lisbon Clinical Group authors are listed at the end of the article Maria Graça Pinto 12, Isabel Pereira 13, Joaquim Nunes 13, ‡ These authors share first authorship Josvany Sánchez-Curbelo 14, Olga López-Rodrigo 14, §These authors share last authorship Iris Pereira-Caetano 15, Patricia Isabel Marques 16,17, SPECIALTY SECTION Filipa Carvalho 16,18, Alberto Barros 16,18, Lluís Bassas 14, This article was submitted to Molecular and Cellular Reproduction, Susana Seixas 16,17, João Gonçalves 16,19, Alexandra M. Lopes 16,20, a section of the journal Frontiers in Cell and Developmental Biology Sara Larriba 21, Rogelio J. Palomino-Morales 2,22, RECEIVED 04 November 2022 F. David Carmona  1,2*§, Lara Bossini-Castillo 1,2*§, ACCEPTED 28 November 2022 PUBLISHED 15 December 2022 IVIRMA Group# and Lisbon Clinical Group# CITATION 1 Departamento de Genética e Instituto de Biotecnología, Centro de Investigación Biomédica (CIBM), Guzmán-Jiménez A, González-Muñoz S, Cerván-Martín M, Rivera-Egea R, Garrido N, Luján S, Universidad de Granada, Granada, Spain, 2Instituto de Investigación Biosanitaria ibs.GRANADA, Santos-Ribeiro S, Castilla JA, Gonzalvo MC, Granada, Spain, 3Andrology Laboratory and Sperm Bank, IVIRMA Valencia, Valencia, Spain, 4IVI Clavero A, Vicente FJ, Maldonado V, Foundation, Health Research Institute La Fe, Valencia, Spain, 5Servicio de Urología. Hospital Universitari Villegas-Salmerón J, Burgos M, Jiménez R, Pinto MG, i Politecnic La Fe e Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain, 6IVI-RMA Lisbon, Pereira I, Nunes J, Sánchez-Curbelo J, Lisbon, Portugal, 7Department of Obstetrics and Gynecology, Faculty of Medicine, University of Lisbon, López-Rodrigo O, Pereira-Caetano I, Marques PI, Carvalho F, Barros A, Bassas L, Seixas S, Gonçalves J, Lisbon, Portugal, 8Unidad de Reproducción, UGC Obstetricia y Ginecología, HU Virgen de Las Nieves, Lopes AM, Larriba S, Palomino-Morales RJ, Granada, Spain, 9CEIFER Biobanco—GAMETIA, Granada, Spain, 10UGC de Urología, HU Virgen de las Carmona FD, Bossini-Castillo L, IVIRMA Group and Nieves, Granada, Spain, 11UGC de Obstetricia y Ginecología, Complejo Hospitalario de Jaén, Jaén, Lisbon Clinical Group (2022), Contribution of TEX15 Spain, 12Centro de Medicina Reprodutiva, Maternidade Alfredo da Costa, Centro Hospitalar genetic variants to the risk of developing severe non- Universitário de Lisboa Central, Lisboa, Portugal, 13Departamento de Obstetrícia, Ginecologia e obstructive oligozoospermia. Front. Cell Dev. Biol. 10:1089782. Medicina da Reprodução, Hospital de Santa Maria, Centro Hospitalar Universitário de Lisboa Norte, doi: 10.3389/fcell.2022.1089782 Lisboa, Portugal, 14Laboratory of Seminology and Embryology, Andrology Service-Fundació Puigvert, Barcelona, Spain, 15Departamento de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo COPYRIGHT Jorge, Lisbon, Portugal, 16i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Copyright © 2022 Guzmán-Jiménez, González- Porto, Portugal, 17Institute of Molecular Pathology and Immunology of the University of Porto Muñoz, Cerván-Martín, Rivera-Egea, Garrido, Luján, Santos-Ribeiro, Castilla, Gonzalvo, Clavero, Vicente, (IPATIMUP), Porto, Portugal, 18Serviço de Genética, Departamento de Patologia, Faculdade de Maldonado, Villegas-Salmerón, Burgos, Jiménez, Medicina da Universidade do Porto, Porto, Portugal, 19ToxOmics—Centro de Toxicogenómica e Saúde Pinto, Pereira, Nunes, Sánchez-Curbelo, López- Humana, Nova Medical School, Lisbon, Portugal, 20CGPP-IBMC—Centro de Genética Preditiva e Rodrigo, Pereira-Caetano, Marques, Carvalho, Preventiva, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal, 21Human Barros, Bassas, Seixas, Gonçalves, Lopes, Larriba, Molecular Genetics Group, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Palomino-Morales, Carmona, Bossini-Castillo, Llobregat, Barcelona, Spain, 22Departamento de Bioquímica y Biología Molecular I, Universidad de IVIRMA Group and Lisbon Clinical Group. This is an Granada, Granada, Spain open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the Background: Severe spermatogenic failure (SPGF) represents one of the most copyright owner(s) are credited and that the original relevant causes of male infertility. This pathological condition can lead to publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or extreme abnormalities in the seminal sperm count, such as severe reproduction is permitted which does not comply oligozoospermia (SO) or non-obstructive azoospermia (NOA). Most cases of with these terms. SPGF have an unknown aetiology, and it is known that this idiopathic form of Frontiers in Cell and Developmental Biology 01 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 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. KEYWORDS oligozoospermia, spermatogenesis, TEX15, polymorphisms, association study Introduction double-strand breaks (DSBs) for homologous chromosome pairing and crossing over (CO) (Cannarella et al., 2020). Infertility is a growing health concern involving over Consequently, DNA repair mechanisms are essential to 50–70 million childbearing age couples worldwide, with the produce fit mature sperm cells. In fact, the assessment of male factor contributing in approximately 50% of cases DNA integrity is a relevant marker to determine the sperm (Fainberg and Kashanian, 2019). Non-obstructive azoospermia quality in assisted reproductive techniques (ART) (Leduc (NOA) and severe oligozoospermia (SO) due to spermatogenic et al., 2008; Gonzalez-Marin et al., 2012). Thus, while the failure (SPGF) represent the most severe phenotypes of male generation of genetic variability through changes in DNA is factor infertility. However, the causes of such conditions are the basis for evolution, it is strictly controlled at the individual poorly understood and the aetiology of most affected men is level (Capilla et al., 2016; Alves et al., 2017). usually defined as being idiopathic (Cannarella et al., 2019; Consistent with the above, the spermatogenic process shows Agarwal et al., 2021). Recent advances clearly point to a very complex molecular and cellular control, involving over idiopathic male infertility as a complex trait, in which the 2,000 genes, among which 900 are solely expressed in the male combined effect of polymorphic risk variants (causing subtle germline (Chalmel et al., 2012; Cervan-Martin et al., 2020b). The changes in gene expression) may increase the susceptibility of an so-called Testis expressed (TEX) gene family plays a crucial role in individual to suffer from this disorder (Singh and Jaiswal, 2011; spermatogenesis and some of its members have been strongly Cervan-Martin et al., 2020b; Salas-Huetos and Aston, 2021). associated with male infertility (Cannarella et al., 2019; Bellil In this regard, common variants in the human genome may et al., 2021). Specifically, TEX15, which is predominantly increase the susceptibility to develop male infertility by altering expressed in spermatogonial and primary spermatocytes, key events during spermatogenesis, in which DNA integrity is encodes a 2,789 amino acid protein necessary for meiotic crucial (Neto et al., 2016). Having such in mind, it is important to recombination and DSB repair in the germ line (Yang et al., stress that alterations in the DNA structure frequently occur in 2008). Additionally, TEX15 also has a significant epigenetic meiosis prophase I during the transition from primary to function in spermatogenesis by interacting with several PIWI secondary spermatocytes, due to the generation of like RNA-mediated gene silencing proteins (namely PIWIL2, Frontiers in Cell and Developmental Biology 02 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 PIWIL4), in order to maintain spermatogonial stem cell integrity general population (most of them with self-reported fatherhood), through the silencing of transposable elements (Schopp et al., and 358 men with normal sperm counts, as previously described 2020; Yang et al., 2020). Indeed, inactivation of Tex15 expression (Cervan-Martin et al., 2020a; Cervan-Martin et al., 2020c). Cases in mutant male mice causes sterility by meiotic arrest and and controls were matched by age, geographical origin, and impaired activity of RAD51 and DMC1 recombinases (Yang ethnicity, and signed an informed written consent in accordance et al., 2008; Chen et al., 2016). with the Declaration of Helsinki. Each participating centre received In this context, high-penetrance pathogenic variants in ethical approval and complied with the requirements of their local TEX15 have been widely linked to human male infertility due regulatory authorities prior to the study. to spermatogenic impairment in different studies. For instance, SPGF patients were diagnosed after two high-speed non-sense mutations producing truncated TEX15 proteins have centrifugations in two different semen samples in different been reported to lead to cryptozoospermia (where spermatozoa fertility clinics managed in public and private health Hospitals are apparently absent in fresh semen samples but are recovered in and centres from Portugal and Spain, based on the guidelines for centrifuged pellets) and SPGF in unrelated families (Okutman the management of infertile men by the American Urological et al., 2015; Colombo et al., 2017; Wang et al., 2018; Cannarella Association (AUA)/American Society for Reproductive Medicine et al., 2021; Tian et al., 2021). Moreover, mutations in this gene (ASRM), the Canadian Urological Association (CUA), and the have been associated with NOA due to Sertoli cell-only (SCO) World Health Organization (WHO) (Cooper et al., 2010; Jarvi phenotype (characterised by a complete lack of germ cells in the et al., 2010; Bjorndahl and Kirkman Brown, 2022). Moreover, the seminiferous tubules) (Araujo et al., 2020). NOA diagnosis was further confirmed by histological On the other hand, case-control genetic association studies examination of testicular biopsies of the NOA patients that have attempted to assess the involvement of TEX15 polymorphic decided to undergo testicular sperm extraction (TESE) for use variation, such as single nucleotide polymorphisms (SNPs), in in ART (who represented around half of the NOA cohort). We the development of SPGF resulting in NOA or SO. The first established stringent selection criteria to include only infertile approach was conducted in a cohort of European ancestry and men due to idiopathic SPGF, which involved an exhaustive identified two TEX15 SNPs putatively associated with SPGF medical evaluation, screening for possible karyotype (Aston et al., 2010). However, such associations were not abnormalities and Y-chromosome microdeletions, as well as a replicated in a subsequent study carried out in an thorough revision of the medical records to discard physical independent South-Eastern European population (Plaseski testicular disorders (e.g., orchitis and obstruction of vas deferens) et al., 2012). Similarly, two novel TEX15 variants have been and other known causes of male infertility. However, no recently proposed to be involved in the genetic predisposition to screening for high-penetrance point mutations was conducted SPGF in the Iranian population (Ghadirkhomi et al., 2022). because this procedure is not part of the routine diagnostic Finally, although TEX15 common variation was initially workup of infertile men due to SPGF (Krausz et al., 2018). associated with SPGF risk in Han Chinese (Ruan et al., 2012), another report could not confirm this observation in this ethnicity (Zhang et al., 2015). Therefore, no conclusive results Study design and single nucleotide are currently available on the possible role of the common TEX15 polymorphism selection genetic variation in SPGF development. In view of the above, we aimed to clarify the involvement of A candidate gene study was conducted to shed light on the TEX15 common variation in SPGF predisposition in a European possible association of common genetic variation in the genetic background, by analysing a large and phenotypically TEX15 locus with idiopathic SPGF risk. During the selection well-characterised study cohort. process, we confirmed that, according to publicly available single- cell RNA-seq data in the puberty and adult (Guo et al., 2018; Guo et al., 2020), the highest TEX15 expression was found in Materials and methods spermatogonia and spermatocytes (Supplementary Figure S1). TEX15 is located in human chromosome 8, spanning an Patient cohort characterisation 81.5 kb region that constitutes a single linkage disequilibrium (LD) block in the 1,000 Genomes phase III (1KGPh3) European This study was carried out in a large SPGF cohort with population (Auton et al., 2015), according to LDlink (Machiela European descent. In total, the infertile men group comprised and Chanock, 2015) (Figure 1A). Therefore, we followed a SNP 727 SPGF cases from Spain and Portugal, diagnosed as NOA tagging strategy using the European data of the 1KGPh3 and the (n = 527), if a total absence of spermatozoa was observed in the Haploview V.4.2 software (Barrett, 2009; Auton et al., 2015) to ejaculate, or SO (n = 200), when showing less than 5 million cover most of the genetic variability of the region, prioritising spermatozoa/mL semen. The control set included 1,058 Iberian those taggers that tagged, at least, more than five variants. Three men, of whom 700 were healthy individuals representative of the taggers representative of three different minor allele frequency Frontiers in Cell and Developmental Biology 03 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 FIGURE 1 Genomic context and predicted functional relevance of the proxy variants tagged by TEX15-rs1362912. Both the CADD scores of each variant (left x-axis) and the recombination rate of the region (right x axis, blue line) are displayed. Variants with functional annotations in testis are represented as diamonds. (MAF) ranges were selected with this method: rs1362912 Association Studies (Skol et al., 2006) (Supplementary (MAF <0.1), and rs323346 (0.1 < MAF <0.2), and rs323342 Table S2). Possible deviances from Hardy- (MAF >0.2) (Supplementary Table S1). Weinberg equilibrium (HWE) were evaluated at the 5% significance level in both case and control groups using a χ 2 test. Sample preparation and genotyping The statistical analyses were conducted with the software Plink v1.9 (Chang et al., 2015). Case-control comparisons of the For all the recruited individuals, genomic DNA was extracted allele and genotype frequencies were performed by logistic from peripheral blood mononuclear cells with the QIAamp® regression on the genotypes using geographical origin (Spain DNA Blood Midi/Maxi kit (Qiagen, Hilden, Germany), the or Portugal) as a covariate and assuming different association MagNA Pure LC-DNA LV Isolation kit I (Roche, Basel, models for genetic risk (including additive, dominant, recessive, Switzerland), or the Wizard® Genomic DNA Purification Kit and genotypic). p-values, odds ratios (ORs) and their 95% Protocol (Promega, Madison, WI, United States), following the confidence intervals (CIs) were then calculated setting the manufacturers’ protocols. The genotyping was performed using significance threshold at p-value < 0.05 after correction of ™ the TaqMan SNP genotyping technology (Applied Biosystems, possible multiple testing effects by the Benjamini and Foster City, CA, United States). Real-time quantitative Hochberg False Discovery Rate (FDR-BH) method (Benjamini polymerase chain reactions (qPCR) were performed in a et al., 2001). 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, California, United States), using specific ™ predesigned TaqMan probes (assay IDs: C___8867446_10; In silico functional characterisation of risk C____622151_10; C____622153_10) and the SDS 2.3 software variants for allele discrimination (both from Applied Biosystems, Foster City, California, United States). In an attempt to provide a plausible functional effect of the observed associations and identify possible molecular or cellular mechanisms underlying the pathogenic phenotypes, Case-control statistical association we implemented a bioinformatic workflow to extract and analysis explore the functional annotation data available in different public databases. In a first step, we extended our functional An estimation of the statistical power of this study was characterisation to all proxies (genetic variants showing a LD calculated with the CaTS Power Calculator for Genetic r2 ≥ 0.8) of the selected taggers in the reference European Frontiers in Cell and Developmental Biology 04 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 TABLE 1 Analysis of the genotype and allele frequencies of the TEX15 tagger variants comparing groups of male infertility against the unaffected control group. Allelic model Genotypic model SNP Change Group Genotypes (11/ MAF p Adjusted p OR [CI 95%] p Adjusted (1/2) 12/22) p rs1362912 G/A Controls (n = 1/96/949 0.0468 NA NA NA NA NA 1,046) SPGF (n = 715) 4/74/637 0.0573 0.2571 NS 1.20 [0.88–1.65] 0.2367 NS SO (n = 196) 2/29/165 0.0842 7.47E- 2.24E-02 1.90 [1.19–3.03] 1.14E- 3.41E-02 03 02 NOA (n = 519) 2/45/472 0.0472 0.9884 NS 1.00 [0.70–1.43] 0.4932 NS rs323342 A/T Controls (n = 67/420/562 0.2641 NA NA NA NA NA 1,049) SPGF (n = 714) 60/277/377 0.2780 0.6212 NS 1.04 [0.89–1.22] 0.5412 NS SO (n = 195) 18/67/110 0.2641 0.6740 NS 0.94 [0.72–1.23] 0.3335 NS NOA (n = 519) 42/210/267 0.2832 0.4275 NS 1.07 [0.90–1.27] 0.6689 NS rs323346 C/T Controls (n = 32/318/699 0.1821 NA NA NA NA NA 1,049) SPGF (n = 718) 29/225/464 0.1971 0.5822 NS 1.05 [0.88–1.26] 0.7808 NS SO (n = 197) 7/64/126 0.1980 0.7353 NS 1.05 [0.78–1.43] 0.9444 NS NOA (n = 521) 22/161/338 0.1967 0.5371 NS 1.06 [0.88–1.29] 0.6949 NS CI, confidence interval; MAF, minor allele frequency; NA, not applicable; NOA, non-obstructive azoospermia; NS: not significant; OR, odds ratio (for the minor allele); SNP, single- nucleotide polymorphism; SO, severe oligozoospermia; SPGF, spermatogenic failure. Significant p-values are highlighted in bold. 1: reference (minor) allele. 2: alternative (major) allele. population using the tools for that purpose implemented in Results LDLink (Machiela and Chanock, 2015). All proxies were equally considered candidates for explaining the observed The genotyping success rate of the three analysed variants associations, as described elsewhere (Cervan-Martin et al., was above 98% and none of them showed a significant deviation 2020a; Cervan-Martin et al., 2021). Briefly, the prioritisation from HWE either in cases or controls. Additionally, the MAFs in processes were performed with the following resources: the control group were concordant with those described for both GTExPortal (Carithers and Moore, 2015), SNPnexus the Iberian subpopulation and the European super population of (Oscanoa et al., 2020), ENCODE (Luo et al., 2020), the 1KGPh3 project (Auton et al., 2015), with no statistically Haploreg v.4.1. (Ward and Kellis, 2016), RegulomeDB significant differences observed between the population- (Dong and Boyle, 2019), SNP2TFBS (Kumar et al., 2017), representative group and the normozoospermic group in amongst others. Several scores of deleteriousness were also either the allele or the genotype frequencies. Furthermore, our used, such as CADD, DeepSEA, EIGEN, FATHMM, fitCons, study population had an appropriate overall statistical power to FunSeq2 GWAVA, REMM, and RegulomeDB identify genetic associations with moderate to high effects, as (Supplementary Tables S3,S4). In addition, we carried out detailed in Supplementary Table S2. an enrichment analysis of both gene ontology (GO) terms and protein-protein interactions (PPIs), including all transcription factors whose reported binding site (TFBS) Common TEX15 gene variation confers sequences overlapped with the SNPs included in the susceptibility to severe oligozoospermia prioritisation analysis, as implemented in STRINGv11.5 (Szklarczyk et al., 2019). Finally, to determine the TEX15 First, we compared the allele and genotype frequencies of expression at the single cell level, we used the data the selected taggers between the overall SPFG group and the available at both the Human Testis Atlas Browser (Guo unaffected control population. These analyses revealed no et al., 2018) and the Single Cell Expression Atlas significant differences under any of the tested models (Papatheodorou et al., 2020). (Table 1). Frontiers in Cell and Developmental Biology 05 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 TABLE 2 Analysis of the genotype and allele frequencies of the TEX15 tagger variants comparing the severe oligozoospermia (SO) group against the non- obstructive azoospermia (NOA) group. Allelic model Genotypic model SNP MAF (SO/NOA) p Adjusted p OR [CI 95%]^ p Adjusted p rs1362912 0.0842/0.0472 1.23E-02 3.69E-02 1.83 [1.14–2.94] 4.33E-02 0.13 rs323342 0.2641/0.2832 0.3614 NS 0.88 [0.67–1.15] 0.2672 NS rs323346 0.1980/0.1967 0.6516 NS 0.93 [0.69–1.26] 0.7451 NS CI, confidence interval; MAF, minor allele frequency; NS: not significant; OR, odds ratio (for the minor allele); SNP, single nucleotide polymorphism; Significant p-values are highlighted in bold. Subsequently, we compared the groups of cases showing different MAFs rather than on their possible functional features. specific phenotypes of male infertility stablished by semen Therefore, to investigate the biological meaning of the observed analysis (that is, NOA and SO) against the control cohort. association between rs1362912 and SO further, we first identified all Regarding NOA, no statistically significant differences were proxies (r2 ≥ 0.8) of this tagger and, subsequently, we carried out a observed for any of the three tested taggers. In fact, the MAFs functional prioritisation to elucidate the putative causal variant/s. for the NOA group were very similar to those observed for the The tagger rs1362912 represents a synonymous SNP in exon control group (Table 1). eight of the TEX15 gene (Figure 1), whereas most of its proxies are Conversely, the comparison between the SO group and the non-coding variants located in both intronic and intergenic control cohort revealed statistically significant differences in the regions (Figure 1; Supplementary Table S5). We decided to allele/genotype frequencies of rs1362912 under the additive base our prioritisation mostly on the overlaps with possible (pADD = 7.47E-03, OR = 1.90, CI 95% = 1.19–3.03) and regulatory elements in the testis related with changes in gene genotypic (pGENO = 1.14E-02) models (Table 1). Such expression that could affect the spermatogenic process. Amongst associations remained significant after multiple testing the 44 SNPs that were evaluated, the variants rs114435820, correction (pADD-FDR = 2.24E-02; pGENO-FDR = 3.41E-02) rs4733201, and rs1381559038 stood out from the rest using (Table 1). such criterion (Figure 2; Supplementary Table S5). Specifically, To assess the consistency of the rs1362912 association with the SNP rs114435820 was located in a CpG island, which may SO, we decided to eliminate the possible confounding effect of indicate a possible effect on nearby gene expression. having SPFG in the analysis by using the NOA group as the Interestingly, this proxy overlaps with a CTCF binding site, as reference cohort. The comparison between SO and NOA showed well as with histone marks to active enhancers (H3K27ac) and a similar risk effect on SO for the rs1362912*G minor allele in the promoters (H3K4me3) in the adult testis, according to ChIP- seq additive model (pADD = 1.23E-02, 1.83, CI 95% = 1.14–2.94). The data from ENCODE (Davis et al., 2018) (Figure 2; observed differences in the genotypic test for this SNP were also Supplementary Table S5). Additionally, the position weight at the same range (pGENO = 4.33E-02) (Table 2). Interestingly, matrix (PWM) data extracted from HaploReg (Ward and despite the lower statistical power of this case-case comparison, Kellis, 2016) showed that the SNP rs114435820 could alter the the association assuming an additive effect of rs1362912*G binding affinity of the transcription factor YY1, which plays an rameined after multiple testing correction (pADD-FDR = 3.69E- important role in DNA repair during spermatogenesis (Wu et al., 02) (Table 2). 2009), amongst others (Figure 2; Supplementary Tables S5, S6). No evidence of additional association with the SO group was The data of the testis from ENCODE also showed an overlap of evident for the remaining selected variants (i.e., rs323346 and the variant rs4733201 with binding sites of CTCF and POLR2A, rs323342) (Tables 1, 2). with the same previously described histone marks, and with a DNAse hypersensitive sites (Figure 2; Supplementary Table S5). Furthermore, this variant was predicted to alter the TFBS of The severe oligozoospermia-associated E2F1, which has been previously associated with male infertility variants in TEX15 might affect chromatin due to SPGF (Jorgez et al., 2015; Rocca et al., 2019). Moreover, activity in the testis 31 additional proteins were observed to be bound to this site through ChIP-Seq experiments, being some of them (such as The statistical analyses suggested an involvement of low CTCF, YY1, EGR1, TBP, or CCNT2) involved in frequency variants, tagged by rs1362912, in the susceptibility to spermatogenesis (Tourtellotte et al., 2000; Wu et al., 2009; SO. However, our study design relied on a tagging strategy, and the Teng et al., 2011; Hernandez-Hernandez et al., 2016; studied SNPs were selected based on their representativeness of Ponomarenko et al., 2020) (Figure 2; Supplementary Tables Frontiers in Cell and Developmental Biology 06 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 FIGURE 2 Enrichment in functional annotations of the human genome for TEX15-rs1362912 and its proxies. Functional prediction scores, according to different algorithms, are marked in pink (CADD), violet (Funseq2), and yellow (RegulomeDB); blue-coloured cells represent overlap with functional annotations in testis based on the ENCODE and Roadmap Epigenomics projects; variants located within transcription factor binding sites (TFBS) related to spermatogenesis (based on either position weight matrix data or Chip-seq experiments) are shown in green; and variants in CpG sites are highlighted in orange. Colour intensity is correlated with the probability or relevance (dark colours indicate higher probability). Bp, base pairs; GRCh38, Genome Reference Consortium Human Build 38; DHS, DNase I hypersensitive site. S5, S6). With regards to the scores indicative of functionality, pathways considering the 112 TFs whose binding affinity to although some proxies (such as rs114433201) showed relevant this genetic region was predicted to be altered by the analysed values, the strongest evidence of deleteriousness was observed for SNPs (Supplementary Tables S5, S6). The PPI network showed the TEX15 variant rs4733201, which is located in the 5’ significantly more interactions than expected (p = 1.00E-16) untranslated region of the nearby gene PPP2CB and had a (Figure 3). In relation to the functional enrichment of this considerably high CADD score (score = 21.9) (Figures 1, 2; protein set, “regulation of transcription” and “gene Supplementary Table S5). expression” were the GO terms showing the most significant In view of the possible influence on TFBSs of the most likely enrichment p-values (p = 3.94E-61 and p = 2.60E-45, causal variants of the TEX15 association with SO, we decided to respectively). Interestingly, other significantly enriched perform an enrichment analysis of both PPIs and biological biological pathways included “reproductive process” (GO: Frontiers in Cell and Developmental Biology 07 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 FIGURE 3 Protein-protein interaction network of the 122 transcription factors with predicted binding sites overlapping with TEX15-rs1362912 and its proxies based on ChIP-seq experiments or protein weight matrix data. Blue lines indicate that the interaction is established from curated databases; pink lines indicate experimentally determined connections; green lines link neighbour genes; black lines represent co-expression; and grey lines correspond to proteins with homology. Bubble colour meaning: light blue, formation of primary germ layer; orange, developmental process involved in reproduction; yellow, reproductive process; pink, reproductive structure development; dark green, pituitary gland development; light green, urogenital system development; purple, ovarian infertility and red, male infertility. 0022414; p = 2.98E-07), “developmental process involved in (WP34; p = 4.48E-06) were also highlighted amongst the reproduction” (GO:0003006; p = 1.31E-07), “reproductive enriched pathways (Figure 3; Supplementary Table S8). structure development” (GO:0048608; p = 3.51E-07), “urogenital system development” (GO:0001655; p = 2.8E-04), “formation of primary germ layer” (GO:0001704; p = 1.70E-03), Discussion and “pituitary gland development” (GO:0021983; p = 4.26E-02) (Figure 3; Supplementary Table S7). Furthermore, “male Understanding the genetic basis of SPGF is a necessary step infertility” (WP4673; p = 9.00E-03) and “ovarian infertility” to improve the clinical management and genetic counselling of Frontiers in Cell and Developmental Biology 08 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 infertile patients (Fainberg and Kashanian, 2019; Sharma et al., Remarkably, animal models clearly support the role of the 2021). In the present study, we conducted a comprehensive TEX15 locus in SO development (Yang et al., 2008; Schopp et al., evaluation of the possible involvement of TEX15 genetic 2020; Yang et al., 2020). In fact, TEX15-defficient murine models variation in the predisposition to SPGF, using a large show reproductive system abnormalities, including male European cohort that ensured a high statistical power. infertility, altered spermatogenesis, arrest of male meiosis, Our results clearly suggest that TEX15-rs1362912, or any of decreased male germ cell number, DSB repair anomalies, and its tagged SNPs, is directly involved in the pathological reduced testis weight (Yang et al., 2008; Carvalho-Silva et al., mechanisms underlying mild forms of SPGF, i.e., SO, with 2019). This gene is mostly expressed in spermatogonia and little or no contribution to extreme patterns, such as NOA. spermatocytes, in which it has a relevant regulatory role in Taking into account the high relevance of TEX15 in the the control of gene expression by interacting with a vast reproductive function and the insight provided by number of transcription factors related to the reproductive previously published genetic studies on this gene (Aston function, as reported in the Open Targets Platform (Carvalho- et al., 2010; Plaseski et al., 2012; Ruan et al., 2012; Okutman Silva et al., 2019). et al., 2015; Zhang et al., 2015; Colombo et al., 2017; Wang According to the results of our in silico functional analyses, et al., 2018; Araujo et al., 2020; Cannarella et al., 2021; amongst the linked polymorphisms of the tagger rs1362912, the Ghadirkhomi et al., 2022), we hypothesise that NOA could non-coding TEX15 variant rs4733201 may be responsible for an be mostly influenced by high-penetrance damaging mutations important contribution to the final phenotype. This SNP showed in this locus rather than by common SNPs, which would be strong evidence of functionality, including high scoring by responsible for increasing SO predisposition instead. different functional impact prediction methods as well as an Consistent with this idea, only 27 SO-associated point overlap with testis-specific active chromatin epigenetic marks. mutations are currently annotated in both the “Male Interestingly, the sequence surrounding this SNP corresponds to Infertility Genomic Consortium (IMIGC) database” and the a TFBS for different key transcription factors related with “Infertility Disease Database (IDDB)”, whereas NOA has spermatogenesis, including EGR1 (Tourtellotte et al., 2000; 138 entries, amongst which TEX15 is included (Houston Man et al., 2014), CTCF (Hernandez-Hernandez et al., 2016; et al., 2021; Wu et al., 2021). Moreover, a higher proportion Rivero-Hinojosa et al., 2021), SMC3 (Eijpe et al., 2000; James of NOA cases present Mendelian causes of their infertility et al., 2002), YY1 (Wu et al., 2009; Kim et al., 2016), CCNT2 when compared to SO (e.g., AZF microdeletions and (Teng et al., 2011), E2F6 (Pohlers et al., 2005; Dahlet et al., 2021), chromosomal abnormalities are three times more prevalent and MAX (Maeda et al., 2013), and it is annotated to be in NOA than in SO) (Krausz and Riera-Escamilla, 2018). transcriptionally active in the testis (Davis et al., 2018). Although our data are consistent with this hypothesis, the Moreover, this SNP was predicted to modify the activity of causality of the TEX15 common variation in SO predisposition E2F1, which has been associated with human SPGF (Jorgez remains controversial. Previous genetic association reports in this et al., 2015; Rocca et al., 2019). locus have not been fully replicated likely due to differences in the Similarly, the results of the biological pathway enrichment genetic architecture of the different study groups or, more analysis of the transcription factors with TFBSs influenced by the probably, to type I or II errors caused by the limitations in analysed variants also support their involvement in SPGF, as the statistical power of the studied cohorts to detect low or some of the enriched pathways included “reproductive process”, modest effects. In this regard, the TEX15 SNP rs323346 was not “pituitary gland development”, “ovarian infertility”, and “male associated with SO neither in our study nor in an independent infertility”. To our knowledge, there are no reported studies in population of European ancestry previously assessed by Aston which the possible effects of TEX15 on female infertility were et al. (2010). Inconsistent results were obtained in two additional evaluated (Yang et al., 2008; Bellil et al., 2021). However, TEX15 studies performed in Chinese, as Ruan et al. (2012) described a is expressed in the female reproductive tract (Supplementary genetic association between this SNP and SO that was not Figure S1), “ovarian infertility” was amongst the most enriched replicated by Zhang et al. (2015) in the same ethnicity. pathways, and there is evidence of the implications in female Moreover, there are striking differences between the reported fertility of some of the transcription factors with binding sites in rs323346*C allele frequencies for the human populations the genomic positions of some of the SNPs considered in our included in the 1KGPh3 project (e.g., EUR = 0.17; EAS = functional analysis. Specifically, VDR is associated with female 0.11; SAS = 0.33; AFR = 0.80, Supplementary Figure S2) infertility (Djurovic et al., 2020), whereas CEBPB has been (Auton et al., 2015), which might indicate population-specific described as relevant factor for female reproduction for its causal variants within this region (Aston et al., 2010; Ruan et al., role in ovarian follicle development (Fan et al., 2009), together 2012; Ghadirkhomi et al., 2022). Therefore, subsequent studies in with SMAD3 (Tomic et al., 2004; Li et al., 2008) and ESR2 independent study cohorts should be performed to clarify further (Khristi et al., 2018; Chakravarthi et al., 2020). Such is consistent the possible existence of different SO functionally relevant SNPs with the assumption that the effect of the common variation in in this gene. the genome on pathological conditions is not limited to altering Frontiers in Cell and Developmental Biology 09 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 the expression or function of a single gene or protein, but rather Data availability to unbalance complex molecular networks (Cui et al., 2015). Regarding the possible limitations of this work, it is The data generated in this study are either contained in the important note that our exclusion criteria based on genetic article file and its Supplementary Material or available upon abnormalities considered only karyotype alterations and Y reasonable request to the corresponding author. chromosome microdeletions. Although both type of alterations account for the majority of known genetic causes of SPGF (Cioppi et al., 2021), screening of high-penetrance point Ethics statement mutations in reported SPGF genes was not performed. As a consequence, some cases of our study cohort could harbour The studies involving human participants were reviewed and single-gene mutations that may explain their infertility. However, approved by Ethics Committee “CEIM/CEI Provincial de considering the low frequency of the reported monogenic Granada” (Andalusia, Spain). The patients/participants mutations in SPGF (Cioppi et al., 2021), this limitation is not provided their written informed consent to participate in this expected to have a relevant impact in the allele frequencies of our study. study cohort. The possible statistical noise added by such cases would likely cause a subtle increase in the probability of obtaining type II errors (false negative results), but it would hardly affect Author contributions the consistency of the observed associations. Overall, the insight gained in this study supports the notion Formal analysis, writing—original draft and review and of idiopathic SPGF as a complex trait, where common genetic editing: AG-J and SG-M. Data acquisition, draft review and variation has a determinant role in disease susceptibility and validation: RR-E, NG, SL, GR, SS-R, and IVIRMA group, development. In the case of TEX15, it is likely that the selective Lisbon Clinical group, JC, MG, AC, FV, VM, MP, IP, JN, pressures have prevented the dissemination of high-penetrance JS-C, OL-R, IP-C, PM, FC, AB, LB, SS, JG, SL, and AL. Data deleterious variants leading to NOA in the human populations, interpretation and draft review: MC-M, JV-S, MB, and considering the crucial role that this gene has in the reproductive RJ. Conceptualization, draft review and validation: RP-M. success of an individual (Yang et al., 2008; Chen et al., 2016). Conceptualization, design, supervision, draft review and Under this assumption, it is reasonable to assume that the validation: LB-C and FDC. All authors approved the final common TEX15 polymorphisms affecting male infertility were version of the manuscript. associated with less extreme manifestations of SPGF, such as SO. Funding Lisbon clinical group co-authors and IVIRMA group co-authors This work was supported by the Spanish Ministry of Science and Innovation through the Spanish National Plan Ana Aguiar, (Unidade de Medicina da Reproducao, for Scientific and Technical Research and Innovation (PID Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, 2020-120157RB-I00) and the Andalusian Government Lisboa, Portugal); Carlos Calhaz-Jorge, (Unidade de Medicina through the research projects of “Plan Andaluz de da Reproducao, Hospital de Santa Maria, Centro Hospitalar Investigacion, Desarrollo e Innovacion (PAIDI 2020)” (ref. Lisboa Norte, Lisboa, Portugal); Joaquim Nunes, (Unidade de PY20_00212) and “Programa Operativo FEDER 2020” (ref. Medicina da Reproducao, Hospital de Santa Maria, Centro B-CTS-584-UGR20). LB-C was supported by the Spanish Hospitalar Lisboa Norte, Lisboa, Portugal); Sandra Sousa Ministry of Science and Innovation through the “Juan de la (Unidade de Medicina da Reproducao, Hospital de Santa Cierva Incorporacion” program (Grant ref. IJC 2018-038026- Maria, Centro Hospitalar Lisboa Norte, Lisboa, Portugal), I, funded by MCIN/AEI/10.13039/501100011033), which and Sónia Correia (Centro de Medicina Reprodutiva, includes FEDER funds. AG-J was funded by MCIN/AEI/ Maternidade Alfredo da Costa, Centro Hospitalar Lisboa 10.13039/501100011033 and FSE “El FSE invierte en tu Central, Lisboa, Portugal); Maria Graça Pinto(Centro de futuro” (grant ref. FPU20/02926). IPATIMUP integrates Medicina Reprodutiva, Maternidade Alfredo da Costa, the i3S Research Unit, which is partially supported by the Centro Hospitalar Lisboa Central, Lisboa, Portugal). Portuguese Foundation for Science and Technology (FCT), Alberto Pacheco, (IVIRMA Madrid, Spain); Cristina financed by the European Social Funds (COMPETE-FEDER) González, (IVIRMA Sevilla, Spain); Susana Gómez, and National Funds (projects PEstC/SAU/LA0003/2013 and (IVIRMA Lisboa, Portugal); David Amorós, (IVIRMA POCI-01-0145-FEDER-007274). PM is supported by the FCT Barcelona, Spain); Jesús Aguilar, (IVIRMA Vigo, Spain); post-doctoral fellowship (SFRH/BPD/120777/2016), financed Fernando Quintana, (IVIRMA Bilbao, Spain). from the Portuguese State Budget of the Ministry for Science, Frontiers in Cell and Developmental Biology 10 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 Technology and High Education and from the European Conflict of interests Social Fund, available through the Programa Operacional do Capital Humano. ToxOmics—Centre for The authors declare that the research was conducted in the Toxicogenomics and Human Health, Genetics, Oncology absence of any commercial or financial relationships that could and Human Toxicology, Nova Medical School, Lisbon, is be construed as a potential conflict of interest. also partially supported by FCT (UID/BIM/00009/2016 and UIDB/00009/2020). SL received support from Instituto de Salud Carlos III (grant: DTS18/00101], co-funded by Publisher’s note FEDER funds/European Regional Development Fund (ERDF)-a way to build Europe-), and from “Generalitat de All claims expressed in this article are solely those of the Catalunya” (grant 2017SGR191). SL is sponsored by the authors and do not necessarily represent those of their affiliated “Researchers Consolidation Program” from the SNS-Dpt. organizations, or those of the publisher, the editors and the Salut Generalitat de Catalunya (Exp. CES09/020). This reviewers. Any product that may be evaluated in this article, or article is related to the Ph.D. Doctoral Thesis of AG-J. claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Acknowledgments Supplementary material We thank the National DNA Bank Carlos III (University of Salamanca, Spain) for supplying part of the control DNA samples The Supplementary Material for this article can be found from Spain and all the participants for their essential online at: https://www.frontiersin.org/articles/10.3389/fcell.2022. collaboration. 1089782/full#supplementary-material References Agarwal, A., Baskaran, S., Parekh, N., Cho, C. L., Henkel, R., Vij, S., et al. (2021). Cannarella, R., Condorelli, R. A., Paolacci, S., Barbagallo, F., Guerri, G., Bertelli, Male infertility. Lancet 397, 319–333. doi:10.1016/S0140-6736(20)32667-2 M., et al. (2021). Next-generation sequencing: Toward an increase in the diagnostic yield in patients with apparently idiopathic spermatogenic failure. Asian J. Androl. Alves, I., Houle, A. A., Hussin, J. G., and Awadalla, P. (2017). The impact of 23, 24–29. doi:10.4103/aja.aja_25_20 recombination on human mutation load and disease. Philos. Trans. R. Soc. Lond. B Biol. Sci. 372, 20160465. doi:10.1098/rstb.2016.0465 Capilla, L., Garcia Caldes, M., and Ruiz-Herrera, A. (2016). Mammalian meiotic recombination: A toolbox for genome evolution. Cytogenet. Genome Res. 150, 1–16. Araujo, T. F., Friedrich, C., Grangeiro, C. H. P., Martelli, L. R., Grzesiuk, J. D., doi:10.1159/000452822 Emich, J., et al. (2020). Sequence analysis of 37 candidate genes for male infertility: Challenges in variant assessment and validating genes. Andrology 8, 434–441. Carithers, L. J., and Moore, H. M. (2015). The genotype-tissue expression (GTEx) doi:10.1111/andr.12704 project. Biopreserv. Biobank. 13, 307–308. doi:10.1089/bio.2015.29031.hmm Aston, K. I., Krausz, C., Laface, I., Ruiz-Castane, E., and Carrell, D. T. (2010). Carvalho-Silva, D., Pierleoni, A., Pignatelli, M., Ong, C., Fumis, L., Karamanis, N., Evaluation of 172 candidate polymorphisms for association with oligozoospermia et al. (2019). Open Targets Platform: New developments and updates two years on. or azoospermia in a large cohort of men of European descent. Hum. Reprod. 25, Nucleic Acids Res. 47, D1056–D1065. doi:10.1093/nar/gky1133 1383–1397. doi:10.1093/humrep/deq081 Cervan-Martin, M., Bossini-Castillo, L., Rivera-Egea, R., Garrido, N., Lujan, S., Auton, A., Brooks, L. D., Durbin, R. M., Garrison, E. P., Kang, H. M., Korbel, J. O., Romeu, G., et al. (2021). Effect and in silico characterization of genetic variants et al. (2015). A global reference for human genetic variation. Nature 526, 68–74. associated with severe spermatogenic disorders in a large Iberian cohort. Andrology doi:10.1038/nature15393 9, 1151–1165. doi:10.1111/andr.13009 Barrett, J. C. (2009). Haploview: Visualization and analysis of SNP genotype data. Cervan-Martin, M., Bossini-Castillo, L., Rivera-Egea, R., Garrido, N., Lujan, S., Cold Spring Harb. Protoc. 2009, pdb.ip71. pdb ip71. doi:10.1101/pdb.ip71 Romeu, G., et al. (2020a). Evaluation of male fertility-associated loci in a European population of patients with severe spermatogenic impairment. J. Pers. Med. 11, 22. Bellil, H., Ghieh, F., Hermel, E., Mandon-Pepin, B., and Vialard, F. (2021). doi:10.3390/jpm11010022 Human testis-expressed (TEX) genes: A review focused on spermatogenesis and male fertility. Basic Clin. Androl. 31, 9. doi:10.1186/s12610-021-00127-7 Cervan-Martin, M., Castilla, J. A., Palomino-Morales, R. J., and Carmona, F. D. (2020b). Genetic landscape of nonobstructive azoospermia and new perspectives for Benjamini, Y., Drai, D., Elmer, G., Kafkafi, N., and Golani, I. (2001). Controlling the clinic. J. Clin. Med. 9, 300. doi:10.3390/jcm9020300 the false discovery rate in behavior genetics research. Behav. Brain Res. 125, 279–284. doi:10.1016/s0166-4328(01)00297-2 Cervan-Martin, M., Suazo-Sanchez, M. I., Rivera-Egea, R., Garrido, N., Lujan, S., Romeu, G., et al. (2020c). Intronic variation of the SOHLH2 gene confers risk to Bjorndahl, L., and Kirkman Brown, J.other Editorial Board Members of the WHO male reproductive impairment. Fertil. Steril. 114, 398–406. doi:10.1016/j.fertnstert. Laboratory Manual for the Examination and Processing of Human Semen (2022). 2020.02.115 The sixth edition of the WHO laboratory manual for the examination and processing of human semen: Ensuring quality and standardization in basic Chakravarthi, V. P., Ghosh, S., Roby, K. F., Wolfe, M. W., and Rumi, M. a. K. examination of human ejaculates. Fertil. Steril. 117, 246–251. doi:10.1016/j. (2020). A gatekeeping role of ESR2 to maintain the primordial follicle reserve. fertnstert.2021.12.012 Endocrinology 161, bqaa037. doi:10.1210/endocr/bqaa037 Cannarella, R., Condorelli, R. A., Duca, Y., La Vignera, S., and Calogero, A. E. Chalmel, F., Lardenois, A., Evrard, B., Mathieu, R., Feig, C., Demougin, P., et al. (2019). New insights into the genetics of spermatogenic failure: A review of the (2012). Global human tissue profiling and protein network analysis reveals distinct literature. Hum. Genet. 138, 125–140. doi:10.1007/s00439-019-01974-1 levels of transcriptional germline-specificity and identifies target genes for male infertility. Hum. Reprod. 27, 3233–3248. doi:10.1093/humrep/des301 Cannarella, R., Condorelli, R. A., Mongioi, L. M., La Vignera, S., and Calogero, A. E. (2020). Molecular Biology of spermatogenesis: Novel Chang, C. C., Chow, C. C., Tellier, L. C., Vattikuti, S., Purcell, S. M., and Lee, J. J. Targets of apparently idiopathic male infertility. Int. J. Mol. Sci. 21, 1728. (2015). Second-generation PLINK: Rising to the challenge of larger and richer doi:10.3390/ijms21051728 datasets. Gigascience 4, 7. doi:10.1186/s13742-015-0047-8 Frontiers in Cell and Developmental Biology 11 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 Chen, S. R., Hao, X. X., Zhang, Y., Deng, S. L., Wang, Z. P., Wang, Y. Q., et al. Kim, J. S., Chae, J. H., Cheon, Y. P., and Kim, C. G. (2016). Reciprocal localization (2016). Androgen receptor in Sertoli cells regulates DNA double-strand break of transcription factors YY1 and CP2c in spermatogonial stem cells and their repair and chromosomal synapsis of spermatocytes partially through intercellular putative roles during spermatogenesis. Acta Histochem. 118, 685–692. doi:10.1016/ EGF-EGFR signaling. Oncotarget 7, 18722–18735. doi:10.18632/oncotarget.7916 j.acthis.2016.08.005 Cioppi, F., Rosta, V., and Krausz, C. (2021). Genetics of Azoospermia. Int. J. Mol. Krausz, C., Cioppi, F., and Riera-Escamilla, A. (2018). Testing for genetic Sci. 22, 3264. contributions to infertility: Potential clinical impact. Expert Rev. Mol. diagn. 18, 331–346. doi:10.1080/14737159.2018.1453358 Colombo, R., Pontoglio, A., and Bini, M. (2017). Two novel TEX15 mutations in a family with nonobstructive azoospermia. Gynecol. Obstet. Invest. 82, 283–286. Krausz, C., and Riera-Escamilla, A. (2018). Genetics of male infertility. Nat. Rev. doi:10.1159/000468934 Urol. 15, 369–384. doi:10.1038/s41585-018-0003-3 Cooper, T. G., Noonan, E., Von Eckardstein, S., Auger, J., Baker, H. W., Behre, H. Kumar, S., Ambrosini, G., and Bucher, P. (2017). SNP2TFBS - a database of M., et al. (2010). World Health Organization reference values for human semen regulatory SNPs affecting predicted transcription factor binding site affinity. Nucleic characteristics. Hum. Reprod. Update 16, 231–245. doi:10.1093/humupd/dmp048 Acids Res. 45, D139–D144. doi:10.1093/nar/gkw1064 Cui, H., Dhroso, A., Johnson, N., and Korkin, D. (2015). The variation game: Leduc, F., Nkoma, G. B., and Boissonneault, G. (2008). Spermiogenesis and DNA Cracking complex genetic disorders with NGS and omics data. Methods 79-80, repair: A possible etiology of human infertility and genetic disorders. Syst. Biol. 18–31. doi:10.1016/j.ymeth.2015.04.018 Reprod. Med. 54, 3–10. doi:10.1080/19396360701876823 Dahlet, T., Truss, M., Frede, U., Al Adhami, H., Bardet, A. F., Dumas, M., et al. Li, Q., Pangas, S. A., Jorgez, C. J., Graff, J. M., Weinstein, M., and Matzuk, M. M. (2021). E2F6 initiates stable epigenetic silencing of germline genes during (2008). Redundant roles of SMAD2 and SMAD3 in ovarian granulosa cells in vivo. embryonic development. Nat. Commun. 12, 3582. doi:10.1038/s41467-021- Mol. Cell. Biol. 28, 7001–7011. doi:10.1128/MCB.00732-08 23596-w Luo, Y., Hitz, B. C., Gabdank, I., Hilton, J. A., Kagda, M. S., Lam, B., et al. (2020). Davis, C. A., Hitz, B. C., Sloan, C. A., Chan, E. T., Davidson, J. M., Gabdank, I., New developments on the Encyclopedia of DNA Elements (ENCODE) data portal. et al. (2018). The encyclopedia of DNA elements (ENCODE): Data portal update. Nucleic Acids Res. 48, D882–D889. doi:10.1093/nar/gkz1062 Nucleic Acids Res. 46, D794–D801. doi:10.1093/nar/gkx1081 Machiela, M. J., and Chanock, S. J. (2015). LDlink: A web-based application for Djurovic, J., Stamenkovic, G., Todorovic, J., Aleksic, N., and Stojkovic, O. (2020). exploring population-specific haplotype structure and linking correlated alleles of Polymorphisms and haplotypes in VDR gene are associated with female idiopathic possible functional variants. Bioinformatics 31, 3555–3557. doi:10.1093/ infertility. Hum. Fertil. 23, 101–110. doi:10.1080/14647273.2018.1515503 bioinformatics/btv402 Dong, S., and Boyle, A. P. (2019). Predicting functional variants in enhancer and Maeda, I., Okamura, D., Tokitake, Y., Ikeda, M., Kawaguchi, H., Mise, N., et al. promoter elements using RegulomeDB. Hum. Mutat. 40, 1292–1298. doi:10.1002/ (2013). Max is a repressor of germ cell-related gene expression in mouse embryonic humu.23791 stem cells. Nat. Commun. 4, 1754. doi:10.1038/ncomms2780 Eijpe, M., Heyting, C., Gross, B., and Jessberger, R. (2000). Association of Man, P. S., Wells, T., and Carter, D. A. (2014). Cellular distribution of mammalian SMC1 and SMC3 proteins with meiotic chromosomes and Egr1 transcription in the male rat pituitary gland. J. Mol. Endocrinol. 53, synaptonemal complexes. J. Cell Sci. 113, 673–682. doi:10.1242/jcs.113.4.673 271–280. doi:10.1530/JME-14-0158 Fainberg, J., and Kashanian, J. A. (2019). Recent advances in understanding Neto, F. T., Bach, P. V., Najari, B. B., Li, P. S., and Goldstein, M. (2016). and managing male infertility. F1000Res 8, 670. doi:10.12688/f1000research. Spermatogenesis in humans and its affecting factors. Semin. Cell Dev. Biol. 59, 17076.1 10–26. doi:10.1016/j.semcdb.2016.04.009 Fan, H. Y., Liu, Z., Shimada, M., Sterneck, E., Johnson, P. F., Hedrick, S. M., et al. Okutman, O., Muller, J., Baert, Y., Serdarogullari, M., Gultomruk, M., Piton, A., (2009). MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female et al. (2015). Exome sequencing reveals a nonsense mutation in TEX15 causing fertility. Science 324, 938–941. doi:10.1126/science.1171396 spermatogenic failure in a Turkish family. Hum. Mol. Genet. 24, 5581–5588. doi:10. 1093/hmg/ddv290 Ghadirkhomi, E., Angaji, S. A., Khosravi, M., and Mashayekh, M. R. (2022). Correlation of novel single nucleotide polymorphisms ofUSP26, TEX15, and Oscanoa, J., Sivapalan, L., Gadaleta, E., Dayem Ullah, A. Z., Lemoine, N. R., and TNP2 genes with male infertility in north west of Iran. Int. J. Fertil. Steril. 16, Chelala, C. (2020). SNPnexus: A web server for functional annotation of human 10–16. doi:10.22074/IJFS.2021.521138.1058 genome sequence variation (2020 update). Nucleic Acids Res. 48, W185–W192. doi:10.1093/nar/gkaa420 Gonzalez-Marin, C., Gosalvez, J., and Roy, R. (2012). Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells. Int. J. Mol. Sci. 13, Papatheodorou, I., Moreno, P., Manning, J., Fuentes, A. M., George, N., Fexova, 14026–14052. doi:10.3390/ijms131114026 S., et al. (2020). Expression atlas update: From tissues to single cells. Nucleic Acids Res. 48, D77–D83. doi:10.1093/nar/gkz947 Guo, J., Grow, E. J., Mlcochova, H., Maher, G. J., Lindskog, C., Nie, X., et al. (2018). The adult human testis transcriptional cell atlas. Cell Res. 28, 1141–1157. Plaseski, T., Noveski, P., Popeska, Z., Efremov, G. D., and Plaseska-Karanfilska, D. doi:10.1038/s41422-018-0099-2 (2012). Association study of single-nucleotide polymorphisms in FASLG, JMJDIA, LOC203413, TEX15, BRDT, OR2W3, INSR, and TAS2R38 genes with male Guo, J., Nie, X., Giebler, M., Mlcochova, H., Wang, Y., Grow, E. J., et al. (2020). infertility. J. Androl. 33, 675–683. doi:10.2164/jandrol.111.013995 The dynamic transcriptional cell atlas of testis development during human puberty. Cell Stem Cell 26, 262–276. doi:10.1016/j.stem.2019.12.005 Pohlers, M., Truss, M., Frede, U., Scholz, A., Strehle, M., Kuban, R. J., et al. (2005). A role for E2F6 in the restriction of male-germ-cell-specific gene expression. Curr. Hernandez-Hernandez, A., Lilienthal, I., Fukuda, N., Galjart, N., and Hoog, C. Biol. 15, 1051–1057. doi:10.1016/j.cub.2005.04.060 (2016). CTCF contributes in a critical way to spermatogenesis and male fertility. Sci. Rep. 6, 28355. doi:10.1038/srep28355 Ponomarenko, M. P., Sharypova, E. B., Drachkova, I. A., Savinkova, L. K., Chadaeva, I. V., Rasskazov, D. A., et al. (2020). Candidate SNP-markers altering Houston, B. J., Riera-Escamilla, A., Wyrwoll, M. J., Salas-Huetos, A., Xavier, TBP binding affinity for promoters of the Y-linked genes CDY2A, SHOX, and ZFY M. J., Nagirnaja, L., et al. (2021). A systematic review of the validated are lowering many indexes of reproductive potential in men. Vavilovskii Zhurnal monogenic causes of human male infertility: 2020 update and a discussion Genet. Sel. 24, 785–793. doi:10.18699/VJ20.674 of emerging gene-disease relationships. Hum. Reprod. Update 28, 15–29. doi:10.1093/humupd/dmab030 Rivero-Hinojosa, S., Pugacheva, E. M., Kang, S., Mendez-Catala, C. F., Kovalchuk, A. L., Strunnikov, A. V., et al. (2021). The combined action of CTCF and its testis- James, R. D., Schmiesing, J. A., Peters, A. H., Yokomori, K., and Disteche, C. M. specific paralog BORIS is essential for spermatogenesis. Nat. Commun. 12, 3846. (2002). Differential association of SMC1alpha and SMC3 proteins with meiotic doi:10.1038/s41467-021-24140-6 chromosomes in wild-type and SPO11-deficient male mice. Chromosome Res. 10, 549–560. doi:10.1023/a:1020910601858 Rocca, M. S., Di Nisio, A., Sabovic, I., Ghezzi, M., Foresta, C., and Ferlin, A. (2019). E2F1 copy number variations contribute to spermatogenic impairment and Jarvi, K., Lo, K., Fischer, A., Grantmyre, J., Zini, A., Chow, V., et al. (2010). CUA cryptorchidism by increasing susceptibility to heat stress. Andrology 7, 251–256. Guideline: The workup of azoospermic males. Can. Urol. Assoc. J. 4, 163–167. doi:10.1111/andr.12583 doi:10.5489/cuaj.10050 Ruan, J., He, X. J., Du, W. D., Chen, G., Zhou, Y., Xu, S., et al. (2012). Genetic Jorgez, C. J., Wilken, N., Addai, J. B., Newberg, J., Vangapandu, H. V., Pastuszak, variants in TEX15 gene conferred susceptibility to spermatogenic failure in the A. W., et al. (2015). Genomic and genetic variation in E2F transcription factor-1 in Chinese Han population. Reprod. Sci. 19, 1190–1196. doi:10.1177/ men with nonobstructive azoospermia. Fertil. Steril. 103, 44–52. doi:10.1016/j. 1933719112446076 fertnstert.2014.09.021 Salas-Huetos, A., and Aston, K. I. (2021). Defining new genetic etiologies of male Khristi, V., Chakravarthi, V. P., Singh, P., Ghosh, S., Pramanik, A., Ratri, A., et al. infertility: Progress and future prospects. Transl. Androl. Urol. 10, 1486–1498. (2018). ESR2 regulates granulosa cell genes essential for follicle maturation and doi:10.21037/tau.2020.03.43 ovulation. Mol. Cell. Endocrinol. 474, 214–226. doi:10.1016/j.mce.2018.03.012 Frontiers in Cell and Developmental Biology 12 frontiersin.org Guzmán-Jiménez et al. 10.3389/fcell.2022.1089782 Schopp, T., Zoch, A., Berrens, R. V., Auchynnikava, T., Kabayama, Y., regulation and Leydig cell steroidogenesis. Mol. Cell. Biol. 20, 5261–5268. doi:10. Vasiliauskaite, L., et al. (2020). TEX15 is an essential executor of MIWI2- 1128/mcb.20.14.5261-5268.2000 directed transposon DNA methylation and silencing. Nat. Commun. 11, 3739. Wang, X., Jin, H. R., Cui, Y. Q., Chen, J., Sha, Y. W., and Gao, Z. L. (2018). Case doi:10.1038/s41467-020-17372-5 study of a patient with cryptozoospermia associated with a recessive Sharma, A., Minhas, S., Dhillo, W. S., and Jayasena, C. N. (2021). Male infertility TEX15 nonsense mutation. Asian J. Androl. 20, 101–102. doi:10.4103/1008- due to testicular disorders. J. Clin. Endocrinol. Metab. 106, e442–e459. doi:10.1210/ 682X.194998 clinem/dgaa781 Ward, L. D., and Kellis, M. (2016). HaploReg v4: Systematic mining of putative Singh, K., and Jaiswal, D. (2011). Human male infertility: A complex causal variants, cell types, regulators and target genes for human complex traits and multifactorial phenotype. Reprod. Sci. 18, 418–425. doi:10.1177/1933719111398148 disease. Nucleic Acids Res. 44, D877–D881. doi:10.1093/nar/gkv1340 Skol, A. D., Scott, L. J., Abecasis, G. R., and Boehnke, M. (2006). Joint analysis is Wu, S., Hu, Y. C., Liu, H., and Shi, Y. (2009). Loss of YY1 impacts the more efficient than replication-based analysis for two-stage genome-wide heterochromatic state and meiotic double-strand breaks during association studies. Nat. Genet. 38, 209–213. doi:10.1038/ng1706 mouse spermatogenesis. Mol. Cell. Biol. 29, 6245–6256. doi:10.1128/MCB. 00679-09 Szklarczyk, D., Gable, A. L., Lyon, D., Junge, A., Wyder, S., Huerta-Cepas, J., et al. (2019). STRING v11: Protein-protein association networks with increased coverage, Wu, J., Li, D., Liu, X., Li, Q., He, X., Wei, J., et al. (2021). Iddb: A comprehensive supporting functional discovery in genome-wide experimental datasets. Nucleic resource featuring genes, variants and characteristics associated with infertility. Acids Res. 47, D607–D613. doi:10.1093/nar/gky1131 Nucleic Acids Res. 49, D1218–D1224. doi:10.1093/nar/gkaa753 Teng, Y., Wang, Y., Fu, J., Cheng, X., Miao, S., and Wang, L. (2011). Cyclin T2: A Yang, F., Eckardt, S., Leu, N. A., Mclaughlin, K. J., and Wang, P. J. (2008). novel miR-15a target gene involved in early spermatogenesis. FEBS Lett. 585, Mouse TEX15 is essential for DNA double-strand break repair and 2493–2500. doi:10.1016/j.febslet.2011.06.031 chromosomal synapsis during male meiosis. J. Cell Biol. 180, 673–679. doi:10.1083/jcb.200709057 Tian, R. H., Huang, Y. H., Chen, H. X., Li, P., Zhi, E. L., Yao, C. C., et al. (2021). Successful microsurgical vasoepididymostomy for a case of cryptozoospermia. Yang, F., Lan, Y., Pandey, R. R., Homolka, D., Berger, S. L., Pillai, R. S., et al. Asian J. Androl. 24, 436–437. doi:10.4103/aja202178 (2020). TEX15 associates with MILI and silences transposable elements in male germ cells. Genes. Dev. 34, 745–750. doi:10.1101/gad.335489.119 Tomic, D., Miller, K. P., Kenny, H. A., Woodruff, T. K., Hoyer, P., and Flaws, J. A. (2004). Ovarian follicle development requires Smad3. Mol. Endocrinol. 18, Zhang, X., Ding, M., Ding, X., Li, T., and Chen, H. (2015). Six polymorphisms in 2224–2240. doi:10.1210/me.2003-0414 genes involved in DNA double-strand break repair and chromosome synapsis: Association with male infertility. Syst. Biol. Reprod. Med. 61, 187–193. doi:10.3109/ Tourtellotte, W. G., Nagarajan, R., Bartke, A., and Milbrandt, J. (2000). 19396368.2015.1027014 Functional compensation by Egr4 in Egr1-dependent luteinizing hormone Frontiers in Cell and Developmental Biology 13 frontiersin.org