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Background: High-throughput single-cell RNA sequencing (scRNA-seq) is widely used in spermatogenesis. However, it only reveals short reads in germ and somatic cells, limiting the discovery of novel transcripts and genes., Aim: This study shows the long-read transcriptional landscape of spermatogenesis in obstructive azoospermia (OA) and Sertoli cell-only patients., Design: Single cells were isolated from testicular biopsies of OA and non-obstructive azoospermia (NOA) patients. Cell culture was identified by comparing PacBio long-read single-cell sequencing (OA n = 3, NOA n = 3) with short-read scRNA-seq (OA n = 6, NOA n = 6). Ten germ cell types and eight somatic cell types were classified based on known markers., Methods: PacBio long-read single-cell sequencing, short-read scRNA-seq, polymerase chain reaction., Results: A total of 130 426 long-read transcripts (100 517 novel transcripts and 29 909 known transcripts) and 49 508 long-read transcripts (26 002 novel transcripts and 23 506 known transcripts) have been detected in OA and NOA patients, respectively. Moreover, 36 373 and 1642 new genes are identified in OA and NOA patients, respectively. Importantly, specific expressions of long-read transcripts were detected in germ and stomatic cells during normal spermatogenesis., Conclusion: We have identified total full-length transcripts in OA and NOA, and new genes were found. Furthermore, specific expressed full-length transcripts were detected, and the genomic structure of transcripts was mapped in different cell types. These findings may provide valuable information on human spermatogenesis and the treatment of male infertility., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Azoospermia is a form of male infertility characterized by a complete lack of spermatozoa in the ejaculate. Sertoli cell-only syndrome (SCOS) is the most severe form of azoospermia, where no germ cells are found in the tubules. Recently, FANCM gene variants were reported as novel genetic causes of spermatogenic failure. At the same time, FANCM variants are known to be associated with cancer predisposition. We performed whole-exome sequencing on a male patient diagnosed with SCOS and a healthy father. Two compound heterozygous missense mutations in the FANCM gene were found in the patient, both being inherited from his parents. After the infertility assessment, the patient was diagnosed with diffuse astrocytoma. Immunohistochemical analyses in the testicular and tumor tissues of the patient and adequate controls showed, for the first time, not only the existence of a cytoplasmic and not nuclear pattern of FANCM in astrocytoma but also in non-mitotic neurons. In the testicular tissue of the SCOS patient, cytoplasmic anti-FANCM staining intensity appeared lower than in the control. Our case report raises a novel possibility that the infertile carriers of FANCM gene missense variants could also be prone to cancer development.

Testing for AZoospermia Factor (AZF) deletions of the Y chromosome is a key component of the diagnostic workup of azoospermic and severely oligozoospermic men. This revision of the 2013 European Academy of Andrology (EAA) and EMQN CIC (previously known as the European Molecular Genetics Quality Network) laboratory guidelines summarizes recent clinically relevant advances and provides an update on the results of the external quality assessment program jointly offered by both organizations. A basic multiplex PCR reaction followed by a deletion extension analysis remains the gold-standard methodology to detect and correctly interpret AZF deletions. Recent data have led to an update of the sY84 reverse primer sequence, as well as to a refinement of what were previously considered as interchangeable border markers for AZFa and AZFb deletion breakpoints. More specifically, sY83 and sY143 are no longer recommended for the deletion extension analysis, leaving sY1064 and sY1192, respectively, as first-choice markers. Despite the transition, currently underway in several countries, toward a diagnosis based on certified kits, it should be noted that many of these commercial products are not recommended due to an unnecessarily high number of tested markers, and none of those currently available are, to the best of our knowledge, in accordance with the new first-choice markers for the deletion extension analysis. The gr/gr partial AZFc deletion remains a population-specific risk factor for impaired sperm production and a predisposing factor for testicular germ cell tumors. Testing for this deletion type is, as before, left at the discretion of the diagnostic labs and referring clinicians. Annual participation in an external quality control program is strongly encouraged, as the 22-year experience of the EMQN/EAA scheme clearly demonstrates a steep decline in diagnostic errors and an improvement in reporting practice., (© 2023 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Objective: To investigate whether Azoospermia Factor c (AZFc) microdeletions affect Assisted Reproductive Technology (ART) outcomes., Design: Systematic review and meta-analysis., Setting: Not applicable., Patients: Infertile men with and without AZFc microdeletions., Intervention(s): Electronic databases were searched for case-control studies reporting sperm retrieval rates and outcomes of ART in infertile men with and without AZFc microdeletions from inception to April 2023. Study quality was assessed using the Newcastle-Ottawa Scale. Summary effect sizes (odds ratio [OR] with 95% confidence interval [CI]) were calculated for both categories of infertile men., Main Outcome Measures: The primary outcome was successful sperm retrieval and the secondary outcomes were outcomes of ART., Results: Case-control studies reporting sperm retrieval rates and ART outcomes in men with AZFa and AZFb deletions were unavailable. On the basis of the data from 3,807 men, sperm retrieval rates were found to be higher in men with AZFc microdeletions compared to their non-deleted counterparts [OR = 1.82, 95% CI 0.97, 3.41], but the difference was not statistically significant. A significantly lower fertilization rate (OR = 0.61, 95% CI [0.50, 0.74]), clinical pregnancy rate (OR = 0.61, 95% CI [0.42, 0.89]), and live birth rate (OR = 0.54, 95% CI [0.40, 0.72]) were observed in men with AZFc deletions compared with men without deletions. There was no statistically significant difference in rates of embryo cleavage, blastocyst formation, good-quality embryos, implantation, and miscarriage between the two groups. On correcting for female factors, the fertilization rate (OR = 0.76, 95% CI [0.71, 0.82]), cleavage rate (OR = 0.54, 95% CI [0.41, 0.72]), clinical pregnancy rate (OR = 0.39, 95% CI [0.30, 0.52]), and live birth rate (OR = 0.48, 95% CI [0.35, 0.65]) were significantly lower in men with AZFc deletions compared with controls., Conclusions: Presence of AZFc microdeletions adversely affects outcomes of ART in infertile men. Further in-depth studies delineating the role of the AZF genes in embryonic development are necessary to understand the full-impact of this finding., Clinical Trial Registration Number: CRD42022311738., Competing Interests: Declaration of interests S.C. reports funding from Department of Health Research-Young Scientist Scheme Fellowship. D.M. reports funding from Indian Council of Medical Research (ICMR) for the submitted work., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Sertoli cell-only syndrome (SCOS), a severe testicular spermatogenic failure, is characterized by total absence of male germ cells. To better expand the understanding of the potential molecular mechanisms of SCOS, we used microarray datasets from the Gene Expression Omnibus (GEO) and ArrayExpress databases to determine the differentially expressed genes (DEGs). In addition, functional enrichment analysis including the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed. Protein-protein interaction (PPI) networks, modules, and miRNA-mRNA regulatory networks were constructed and analyzed and the validation of hub genes was performed. A total of 601 shared DEGs were identified, including 416 down-regulated and 185 up-regulated genes. The findings of the enrichment analysis indicated that the shared DEGs were mostly enriched in sexual reproduction, reproductive process, male gamete generation, immune response, and immunity-related pathways. In addition, six hub genes (CCNA2, CCNB2, TOP2A, CDC20, BUB1, and BUB1B) were selected from the PPI network by using the cytoHubba and MCODE plug-ins. The expression levels of the hub genes were significantly decreased in patients with SCOS compared to that in normal spermatogenesis controls as indicated by the microarray data, single-cell transcriptomic data, and clinical sample levels. Furthermore, the potential miRNAs were predicted via the miRNA-mRNA network construction. These hub genes and miRNAs can be used as potential biomarkers that may be related to SCOS. However, it has not been proven that the differential expression of these biomarkers is the molecular pathogenesis mechanisms of SCOS. Our findings suggest that these biomarkers can be serve as clinical tool for diagnosis targets and may have some impact on the spermatogenesis of SCOS from a testicular germ cell perspective., (© 2023. The Author(s).)

Background: Sertoli cell-only syndrome (SCOS) is the most serious pathological type of non-obstructive azoospermia. Recently, several genes related to SCOS have been identified, including FANCM, TEX14, NR5A1, NANOS2, PLK4, WNK3, and FANCA, but they cannot fully explain the pathogenesis of SCOS. This study attempted to explain spermatogenesis dysfunction in SCOS through testicular tissue RNA sequencing and to provide new targets for SCOS diagnosis and therapy., Methods: We analyzed differentially expressed genes (DEGs) based on RNA sequencing of nine patients with SCOS and three patients with obstructive azoospermia and normal spermatogenesis. We further explored the identified genes using ELISA and immunohistochemistry., Results: In total, 9406 DEGs were expressed (Log2|FC|≥ 1; adjusted P value < 0.05) in SCOS samples, and 21 hub genes were identified. Three upregulated core genes were found, including CASP4, CASP1, and PLA2G4A. Thus, we hypothesized that testis cell pyroptosis mediated by CASP1 and CASP4 might be involved in SCOS occurrence and development. ELISA verified that CASP1 and CASP4 activities in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenesis. Immunohistochemical results showed that CASP1 and CASP4 in the normal spermatogenesis group were mainly expressed in the nuclei of spermatogenic, Sertoli, and interstitial cells. CASP1 and CASP4 in the SCOS group were mainly expressed in the nuclei of Sertoli and interstitial cells because of the loss of spermatogonia and spermatocytes. CASP1 and CASP4 expression levels in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenisis. Furthermore, the pyroptosis-related proteins GSDMD and GSDME in the testes of patients with SCOS were also significantly higher than those in control patients. ELISA also showed that inflammatory factors (IL-1 β, IL-18, LDH, and ROS) were significantly increased in the SCOS group., Conclusions: For the first time, we found that cell pyroptosis-related genes and key markers were significantly increased in the testes of patients with SCOS. We also observed many inflammatory and oxidative stress reactions in SCOS. Thus, we propose that testis cell pyroptosis mediated by CASP1 and CASP4 could participate in SCOS occurrence and development., (© 2023. The Author(s).)

Background: The precise contribution of each chromosome gene or gene family in achieving male fertility is still the subject of debate. Most studies have examined male populations with heterogeneous causes of infertility, and have therefore reached controversial or uncertain conclusions. This study uses Y-chromosome array-based comparative genomic hybridization (aCGH) to examine a population of males with a uniform sertoli cell-only syndrome (SCOS) infertility phenotype., Methods: Initial analysis of gene copy number variations in 8 SCOS patients, with determination of the log-ratio of probe signal intensity against a DNA reference, was performed using the Y-chromosome NimbleGen aCGH. To confirm the role of candidate genes, real-time quantitative RT-PCR was used to compare 19 patients who had SCOS non-obstructive azoospermia with 15 patients who had obstructive azoospermia but normal spermatogenesis., Results: Our initial aCGH experiments identified CDY1a and CDY1b double deletions in all 8 patients who had total germ cell depletion. However, 5 patients had DAZ1/2 and DAZ3/4 deletions, 1 patient had a DAZ2 and DAZ3/4 deletion, and 2 patients had no DAZ1/2 or DAZ3/4 deletions. Examination of testicular mRNA expression in another 19 patients with SCOS indicated all patients had no detectable levels of CDY1., Conclusions: Our findings indicate that CDY1 deletion in SCOS patients, and analysis of the expression of DAZ and CDY1 genes using aCGH and quantitative RT-PCR, may be useful to predict the presence of mature spermatozoa., Competing Interests: Conflicts of interest The authors declare that they have no competing interests., (Copyright © 2022 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Sertoli cell -only syndrome (SCOS) is a type of testicular pathological failure that causes male infertility and no effective treatment strategy, is available for this condition. Moreover, the molecular mechanism underlying its development remains unknown. We identified DExD/H-Box helicase 58 ( DDX58 ) as a key gene in SCOS based on four datasets of testicular tissue samples obtained from the Gene Expression Synthesis database. DDX58 was significantly upregulated in SCOS testicular Sertoli cells. Moreover, high expression of DDX58 was positively correlated with the expression of several testicular inflammatory factors, such as IL -1β, IL-18, and IL-6. Interestingly, DDX58 could be induced in the D-galactose (D-gal)-stimulated TM4 cell injury model. Whereas silencing of DDX58 inhibited D-gal -mediated p65 expression, inflammatory cytokine release, and growth arrest. Mechanistically, we found that DDX58 acts as an RNA-binding protein, which enhances p65 expression by promoting mRNA stability. Furthermore, p65 gene silencing decreased the expression of inflammatory cytokines and inhibition of cell growth in D-gal-induced cells. In conclusion, our findings demonstrate that DDX58 promotes inflammatory responses and growth arrest in SCOS Sertoli cells by stabilizing p65 mRNA. Accordingly, the DDX58/p65 regulatory axis might be a therapeutic target for SCOS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sun, Yang, Teng, Zhang, Han, Xu, Wang, Wang, Wen, Chen, Qu and Wang.)

Male infertility can be caused by quantitative and/or qualitative abnormalities in spermatogenesis, which affects men's physical and mental health. Sertoli cell-only syndrome (SCOS) is the most severe histological phenotype of male infertility characterized by the depletion of germ cells with only Sertoli cells remaining in the seminiferous tubules. Most SCOS cases cannot be explained by the already known genetic causes including karyotype abnormalities and microdeletions of the Y chromosome. With the development of sequencing technology, studies on screening new genetic causes for SCOS are growing in recent years. Directly sequencing of target genes in sporadic cases and whole-exome sequencing applied in familial cases have identified several genes associated with SCOS. Analyses of the testicular transcriptome, proteome, and epigenetics in SCOS patients provide explanations regarding the molecular mechanisms of SCOS. In this review, we discuss the possible relationship between defective germline development and SCOS based on mouse models with SCO phenotype. We also summarize the advances and challenges in the exploration of genetic causes and mechanisms of SCOS. Knowing the genetic factors of SCOS offers a better understanding of SCO and human spermatogenesis, and it also has practical significance for improving diagnosis, making appropriate medical decisions, and genetic counseling. For therapeutic implications, SCOS research, along with the achievements in stem cell technologies and gene therapy, build the foundation to develop novel therapies for SCOS patients to produce functional spermatozoa, giving them hope to father children., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Spermatogenesis is regulated by several Y chromosome-specific genes located in a specific region of the long arm of the Y chromosome, the azoospermia factor region (AZF). AZF microdeletions are the main structural chromosomal abnormalities that cause male infertility. Assisted reproductive technology (ART) has been used to overcome natural fertilization barriers, allowing infertile couples to have children. However, these techniques increase the risk of vertical transmission of genetic defects. Despite widespread awareness of AZF microdeletions, the occurrence of de novo deletions and overexpression, as well as the expansion of AZF microdeletion vertical transmission, remains unknown. This review summarizes the mechanism of AZF microdeletion and the function of the candidate genes in the AZF region and their corresponding clinical phenotypes. Moreover, vertical transmission cases of AZF microdeletions, the impact of vertical inheritance on male fertility, and the prospective direction of research in this field are also outlined., Competing Interests: None

Sertoli cell-only syndrome (SCOS) is the most severe and common pathological type of non-obstructive azoospermia. The etiology of SCOS remains largely unknown to date despite a handful of studies reported in this area. According to the gene expression of testicular tissue samples in six datasets from the Gene Expression Omnibus, we detected 1441 differentially expressed genes (DEGs) between SCOS and obstructive azoospermia (OA) testicular tissue samples. Enriched GO terms and KEGG pathways for the downregulated genes included various terms and pathways related to cell cycle and reproduction, while the enrichment for the upregulated genes yielded many inflammation-related terms and pathways. In accordance with the protein-protein interaction (PPI) network, all genes in the most critical module belonged to the downregulated DEGs, and we obtained nine hub genes, including CCNB1, AURKA, CCNA2, BIRC5, TYMS, UBE2C, CDC20, TOP2A, and OIP5. Among these hub genes, six were also found in the most significant SCOS-specific module obtained from consensus module analysis. In addition, most of SCOS-specific modules did not have a consensus counterpart. Based on the downregulated genes, transcription factors (TFs) and kinases within the upstream regulatory network were predicted. Then, we compared the difference in infiltrating levels of immune cells between OA and SCOS samples and found a significantly higher degree of infiltration for most immune cells in SCOS than OA samples. Moreover, CD56 bright natural killer cell was significantly associated with six hub genes. Enriched hallmark pathways in SCOS had remarkably more upregulated pathways than the downregulated ones. Collectively, we detected DEGs, significant modules, hub genes, upstream TFs and kinases, enriched downstream pathways, and infiltrated immune cells that might be specifically implicated in the pathogenesis of SCOS. These findings provide new insights into the pathogenesis of SCOS and fuel future advances in its theranostics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Wang, Tian, Guo, Xia, Wang and Song.)

Spermatogenesis is a multistep biological process. In addition to somatic cells, it involves the orderly differentiation of dozens of spermatogenic cells. In this process, the regulatory networks between different spermatogenic cell populations are significantly different. RNA m6A regulators and miRNAs have been found to be closely related to spermatogenesis in recent years, and they are an important part of the above regulatory networks. Understanding gene expression and its rules in different spermatogenic cell populations will help in the in-depth exploration of their detailed roles in spermatogenesis. This study collected a public dataset of nonobstructive azoospermia (NOA). Based on the Johnson score, the testicular samples of NOA were divided into three types, Sertoli-cell only syndrome, meiotic arrest and postmeiotic arrest, which represented the loss of three germ cell populations, including whole spermatogenic cells, postmeiotic spermatogenic cells, and a mixture of late spermatids and spermatozoa, respectively. The aforementioned three types of testis data were compared with normal testis data, and the molecular expression characteristics of the abovementioned three germ cell populations were obtained. Our study showed that different germ cell populations have different active molecules and their pathways. In addition, RNA m6A regulators, including METTL3, IGF2BP2 and PRRC2A, and miRNAs, including hsa-let-7a-2, hsa-let-7f-1, hsa-let-7g, hsa-miR-15a, hsa-miR-197, hsa-miR-21, hsa-miR-30e, hsa-miR-32, hsa-miR-503 and hsa-miR-99a, also presented regulatory roles in almost all germ cells., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Sertoli cell only syndrome (SCOS) is characterized by complete absence of germ cells in seminiferous tubules of testis. SCOS is multifactorial but genetic factors play a major role in pathogenesis of the disorder with idiopathic origin. Genetic factors majorly include sex chromosomal aneuploidy and Yq Microdeletion. But a large number of cases are still idiopathic. The study aimed to evaluate the genomic imbalances (CNVs and LOH) in idiopathic SCOS patients. The study is based on 28 apparent idiopathic SCOS cases and 10 controls. Molecular cytogenetic techniques viz., FISH, STS-Multiplex PCR and Affymetrix cytoscan microarray (750 K) were used. The microarray screened whole genomic imbalances in DNA from peripheral blood of 25 cases (excluded Klinefelter syndrome patients) and testicular FNAC sample of 2 cases. High FSH and low Inhibin B were observed in cases as compared to control controls groups. Four cases of sex chromosomal abnormality (i.e., three non-mosaic 47, XXY males and one non-mosaic 46, XX male) as well as four cases of Yq microdeletion (i.e., three cases with AZFc deletion and one case with complete AZFa, b and c deletion) were identified. Microarray detected unbalanced translocation of two segments of Y-chromosome i.e., Yp11.31-p11.2 (~4.o mb region, involving SRY) and Yp11.2 (~2.5 mb region) on X-chromosome in XX male. Also, loss of segment on same X-chromosome involving PAR1 region was identified. We have identified both autosomal and sex chromosomal CNVs (recurrent as well as private) involving candidate genes like SYCE1, ZFPM2, SRPK1, DAZ1, BPY2, HSFY1, VCY1 etc. All these CNVs are possibly associated with SCOS pathogenesis. CNVs identified in cases were already reported as pathogenic variant in clinical database DECIPHER. Microarray also detected many LOH (all autosomal, >3.0 mb size) that covered genes with spermatogenesis related function. The mechanism of action of LOH in pathogenesis of SCOS still remains unravelled. CNVs and LOH related to spermatogenesis identified from two different sample types (blood vs. testicular tissue) were discordant. This study should be extended for larger cohort of patients., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Male infertility is a major health concern in couples of childbearing ages. Nonobstructive azoospermia (NOA) is an extreme form of male infertility that affects ∼1% of adult men, and the etiology remains unknown in most cases. Sertoli cell-only syndrome (SCOS) is the most severe type of NOA. Aims: To explore novel human candidate variants that cause SCOS. Methods: (1) Whole exome sequencing (WES) of 20 men with SCOS, (2) Sanger sequencing of the HELQ gene in an additional 163 men with SCOS, (3) in vitro functional assays, and (4) in vivo studies. Results: WES of 20 patients with SCOS led to the identification of two heterozygous missense mutations (M1 and M2) in two unrelated Chinese patients with infertility. Using subsequent Sanger sequencing covering all the coding regions of the HELQ gene for 163 additional SCOS cases, we identified four additional heterozygous mutations (M3-M6) in unrelated patients. In vitro functional analyses revealed that two of these mutations (M5, c.2538T > G and M6, c.2945G > T) might affect the function of the HELQ protein. Two heterozygous mutant mouse models with mutations similar to those of two patients (M5 and M6) did not show any considerable spermatogenic defects. Conclusion: Assuming that the mouse models accurately reflect the impact of the mutations, heterozygous HELQ variants alone did not lead to the development of the SCOS phenotype in mice. However, we cannot rule out the risk variants in Chinese or other human populations, and a larger dataset is needed to confirm the association between HELQ mutations with SCOS.

Studies increasingly show the involvement of circular RNAs (circRNAs) in several diseases. This study aims to explore the circRNA expression pattern in the testicular tissues of patients with Sertoli only cell syndrome (SCOS) and their potential functions. High throughput circRNA microarray analysis indicated that 399 circRNAs were upregulated and 1195 were down-regulated (fold change >2, P < 0.05) in SCOS relative to obstructive azoospermia (OA). The hsa&#95;circRNA&#95;101222, hsa&#95;circRNA&#95;001387, hsa&#95;circRNA&#95;001153, hsa&#95;circRNA&#95;101373 and hsa&#95;circRNA&#95;103864 were validated by qRT-PCR. Furthermore, the hosting genes of the differentially expressed circRNAs (DEcircRNAs) were enriched in biological processes related to cell cycle and intercellular communication. Also, the overlapping genes between the hosting genes of SCOS-related DEcircRNAs and those highly expressed in Sertoli cells of non-obstructive azoospermia (NOA) were enriched in immune cell development and cell communication. Taken together, aberrantly expressed circRNAs likely mediate SCOS development by regulating the function of Sertoli cells and the spermatogenic microenvironment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: To investigate microRNA (miRNA) expression profiles in the seminal plasma of nonobstructive azoospermia (NOA) patients with different histopathologic patterns and evaluate potential noninvasive diagnostic biomarkers of NOA., Design: Sequencing and validation using quantitative reverse transcription polymerase chain reaction (qRT-PCR)., Setting: Reproductive center and research institute., Patient(s): Thirteen patients with NOA (7 Sertoli cell-only syndrome [SCOS] and 6 hypospermatogenesis to spermatogenesis arrest [SA]) and 7 normal fertile controls for sequencing, six samples per group for validation; 54 patients with NOA (27 SCOS and 27 SA) and 19 normal fertile controls for large-sample qRT-PCR analysis., Intervention(s): None., Main Outcome Measure(s): MicroRNA expression profiles in the seminal plasma of patients with NOA with different histopathologic patterns were assessed using high-throughput sequencing and validated using qRT-PCR., Result(s): There were 78 overexpressed and 132 underexpressed miRNAs in patients with SCOS and 32 up-regulated and 90 down-regulated miRNAs in patients with SA compared with fertile men with normozoospermia. Two down-regulated and one up-regulated miRNA were validated using qRT-PCR, which indicated that the qRT-PCR and sequencing results were basically consistent. Hsa-miR-34c-5p expression was significantly lower in the seminal plasma of patients with NOA than normal fertile controls. The area under the receiver operating characteristic curve(AUC) for hsa-miR-34c-5p was 0.979 and 0.987 in the seminal plasma of patients with SA and patients with SCOS, respectively, compared with normal fertile controls. The AUC was 0.799 for hsa-miR-34c-5p in the seminal plasma between patients with SA and patients with SCOS. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially expressed miRNA target genes revealed that the Notch signaling pathway was one of the most abundant signaling pathways. The expression of Hes5, an effector of the Notch signaling pathway, was significantly higher in the seminal plasma of patients with NOA than normal fertile controls., Conclusion(s): MicroRNA expression profiles in seminal plasma were altered in patients with NOA compared with normal fertile controls. The profiles differed in patients with NOA with different pathologic patterns. We speculate that miR-34c-5p in seminal plasma could be a potential noninvasive biomarker to diagnose patients with NOA and distinguish different pathologic types of NOA. The Notch signaling pathway may be involved in the pathogenesis of NOA., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Decreased testosterone (T) to LH ratio and increased 17β-estradiol (E2) serum concentrations represent a common finding among patients with severe spermatogenic failure, suggesting a concurrent Leydig cell steroidogenic dysfunction. Aromatase overexpression has been associated with increased serum and intratesticular E2 in these patients. However, it is unknown whether the sulfatase pathway contributes to the increased availability of active estrogens in patients with primary spermatogenic failure., Objectives: To assess estrogen sulfotransferase (SULT1E1) and steroid sulfatase (STS) mRNA abundance in testicular tissue of patients with Sertoli cell-only syndrome (SCOS) and normal tissues, its association with serum and intratesticular hormone levels, and to explore the mRNA and protein testicular localization of both enzymes., Materials and Methods: Testicular tissues of 23 subjects with SCOS (cases) and 22 patients with obstructive azoospermia and normal spermatogenesis (controls) were obtained after biopsy. SULT1E1 and STS transcripts accumulation was quantified by RT-qPCR. For mRNA and protein localization, we performed RT-qPCR in Leydig cell clusters and seminiferous tubules isolated by laser-capture microdissection and immunofluorescence in testicular tissues. Serum and intratesticular hormones were measured by immunoradiometric assays., Results: SULT1E1 mRNA accumulation was similar in both groups. The amount of STS mRNA was higher in cases (p = 0.007) and inversely correlated with T/LH ratio (r = -0.402; p = 0.02). Also, a near significant correlation was observed with intratesticular E2 (r = 0.329, p = 0.057), in agreement with higher intratesticular E2 in cases (p < 0.001). Strong STS immunoreaction was localized in the wall of small blood vessels but not in Leydig cells. Both SULT1E1 and STS mRNA abundance was similar in Leydig cell clusters and the tubular compartment, except for lower SUTL1E1 mRNA in the seminiferous tubules of SCOS patients (p = 0.001)., Conclusions: Our results suggest that an unbalance of the STS/SULT1E1 pathway contributes to the testicular hyperestrogenic microenvironment in patients with primary spermatogenic failure and Leydig cell dysfunction., (© 2020 American Society of Andrology and European Academy of Andrology.)

Background: Non-obstructive azoospermia (NOA), identified in approximately 10% of infertile males, is a multifactorial disease whose molecular mechanisms remain unknown., Objectives: The aim of this study was to identify the role of hsa&#95;circ&#95;0000116 in NOA and illustrate its predictive value in testicular sperm retrieval., Materials and Methods: The study included 78 individuals, 58 with NOA and 20 with obstructive azoospermia (OA). Serum hormones including testosterone (T), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and estradiol II (E2) were measured. Testicular histopathology was analyzed by at least two pathologists. The expression of hsa&#95;circ&#95;0000116 in testicular tissue samples was detected using real-time PCR, and the circRNA-miRNA-mRNA networks were predicted using bioinformatics analysis., Results: Our study illustrated that the expression of hsa&#95;circ&#95;0000116 was significantly higher in testicular tissue samples of NOA patients than in that of OA patients. Moreover, hsa&#95;circ&#95;0000116 was aberrantly expressed in three different pathological types of NOA: It was significantly up-regulated in patients with Sertoli cell-only syndrome (SCOS) when compared to patients with hypospermatogenesis (HS). In addition, the expression of hsa&#95;circ&#95;0000116 was negatively correlated with Johnsen score, while it was positively correlated with serum FSH level. A multivariate logistic regression model demonstrated that a high level of hsa&#95;circ&#95;0000116 was associated with a low rate of successful testicular sperm retrieval. Bioinformatics analysis and verification experiments showed that one of the most probable potential target miRNA for hsa&#95;circ&#95;0000116 was hsa-miR-449a. Further analysis indicated that hsa&#95;circ&#95;0000116 may be affecting the fertility function through a hsa&#95;circ&#95;0000116-miR-449-autophagy-related competing endogenous RNA (ceRNA) network., Discussion and Conclusion: We report for the first time that hsa&#95;circ&#95;0000116 may play pivotal roles in regulating spermatogenesis and may also be a potential biomarker for the diagnosis and treatment of NOA, while acting as a predictive tool for the rate of successful testicular sperm retrieval in NOA patients., (© 2020 American Society of Andrology and European Academy of Andrology.)

Retinoic acid (RA), the active metabolite of vitamin A, is one of the most important factors regulating spermatogenesis. RA activates downstream pathways through its receptors (retinoic acid receptor alpha [RARA], retinoic acid receptor beta, and retinoic acid receptor gamma [RARG]) and retinoid X receptors (retinoid X receptor alpha [RXRA], retinoid X receptor beta [RXRB], and retinoid X receptor gamma [RXRG]). These receptors may serve as therapeutic targets for infertile men. However, the localization and expression of retinoid receptors in normal and infertile men were unknown. In this study, we found RARA and RARG were mostly localized in spermatocytes and round spermatids, RXRB was mainly expressed in Sertoli cells, and RXRG was expressed in most cell types in the fertile human testis. The localization of RARA, RARG, RXRB, and RXRG in men with hypospermatogenesis (HYPO) was similar to that of men with normal fertility. In addition, the messenger RNA expression levels of RARA, RARG, RXRA, RXRB, and RXRG were significantly decreased in men with Sertoli cell-only syndrome (SCOS) and maturational arrest (MA), but not in men with HYPO. These results suggest that reduced levels of RARA, RARG, RXRB, RXRA, and RXRG are more closely associated with SCOS and MA spermatogenetic failure. These results could contribute to the development of new molecular indicators of spermatogenic dysfunction and might provide novel therapeutic targets for treating male infertility., (© 2020 Wiley Periodicals LLC.)

Purpose: To assess testicular mRNA and protein expression levels of MRE11 and RAD50 in human azoospermia patients., Methods: Patients diagnosed with maturation arrest at the spermatocyte stage (MA) and Sertoli cell-only syndrome (SCOS) were recruited through diagnostic testicular biopsy. Patients with normal spermatogenesis were studied as controls. In addition, knockdown of MRE11 and RAD50 was performed in GC-2spd(ts) cells to investigate their roles in cellular proliferation and apoptosis., Results: mRNA and protein expression levels of MRE11 and RAD50 were measured using quantitative polymerase chain reaction, western blotting, and immunohistochemistry, respectively. Knockdown of both MRE11 and RAD50 utilized transfection with small interfering RNAs., Conclusion: Our findings demonstrated altered expression levels of MRE11 and RAD50 in human testes with MA and SCOS, and showed that these alterations might be associated with impaired spermatogenesis. These results offer valuable new perspectives into the molecular mechanisms of male infertility.

Sertoli cell-only syndrome (SCOS) is a severe phenotype of male infertility; autosomal gene defects are thought to be the causes for this disease. The iPSC line generated from a SCOS patient carrying a mutation in PIWIL2 gene expresses pluripotent markers, has a normal karyotype and the mutation c.731&#95;732delAT in PIWIL2 gene and is able to differentiate into three germ layers. This cell line will help to study the pathogenesis of SCOS, and the roles of PIWIL2 in human germ cells development and spermatogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Sertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). Here, we report the in vitro reprogramming of fibroblasts to human induced Sertoli-like cells (hiSCs). Initially, five transcriptional factors and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles and cellular properties that are similar to those of primary human Sertoli cells. Moreover, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. hiSCs suppress the proliferation of human T lymphocytes and protect xenotransplanted human cells in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli cell only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells., Competing Interests: JL, NW, JH, JD, YG, LL, WW, CY, ZL, KK No competing interests declared, (© 2019, Liang et al.)

Androgens play a key role in spermatogenesis, and their functions are mediated by the androgen receptor (AR). Some mutations in the AR gene have the potential to alter the primary structure and function of the protein. The aim of this study was to investigate the AR gene mutations in a cohort of males with idiopathic azoospermia referred to Royan Institute. Fifty-one biopsy samples were obtained for routine clinical purposes from 15 men with hypospermatogenesis (HS), 17 patients with maturation arrest (MA) and 19 patients with Sertoli cell-only syndrome (SCOS). The AR cDNAs were prepared from tissue mRNAs and were sequenced. One synonymous variant and three nonsynonymous protein coding single nucleotide polymorphisms (nsSNPs) were detected. Protein structure prediction demonstrated that the S815I and M746T nonsynonymous variants would affect protein structure and its normal function. Our study suggests that mutations in the AR gene would change or disturb the receptor's normal activity. Although these variations may influence spermatogenesis, it is difficult to say that they lead to a lack of spermatogenesis., (© 2019 Blackwell Verlag GmbH.)

Purpose: To identify genomic imbalances and candidate loci in idiopathic male infertility., Methods: Affymetrix CytoScan 750K Array was used to analyze genomic imbalances and candidate loci in 34 idiopathic infertile cases of different phenotypes (hypo-spermatogenesis, n = 8; maturation arrest, n = 7; and Sertoli cell-only syndrome, n = 13, severe oligozoospermia, n = 6, and 10 normozoospermic fertile men). Ten ethnically matched controls were screened for comparison., Results: The cytogenetic array analysis detected a genomic gain at the 19p13.3 region in 9 (26.47%) cases, with the highest frequency in patients with Sertoli cell-only syndrome (SCOS) (38%). Its complete absence in the control group suggests its likely pathogenic nature. In addition to Y-classical, micro, and partial deletions, the duplication in 19p13.3 could serve as a unique biomarker for evaluation of infertility risk. The common region across the individuals harboring the duplication identified STK11, ATP5D, MIDN, CIRBP, and EFNA2 genes which make them strong candidates for further investigations. The largest duplicated region identified in this study displayed a major network of 7 genes, viz., CIRBP, FSTL3, GPX4, GAMT, KISS1R, STK11, and PCSK4, associated with reproductive system development and function. The role of chance was ruled out by screening of ethnically matched controls., Conclusion: The result clearly indicates the significance of 19p13.3 duplication in infertile men with severe testicular phenotypes. The present study underlines the utility and significance of whole genomic analysis in the cases of male infertility which goes undiagnosed due to limitations in the conventional cytogenetic techniques and for identifying genes that are essential for spermatogenesis.

Sertoli cell-only (SCO) syndrome is a severe form of human male infertility seemingly characterized by the lack all spermatogenic cells. However, tubules of some SCO testes contain small patches of active spermatogenesis and thus spermatogonial stem cells. We hypothesized that these stem cells cannot replicate and seed spermatogenesis in barren areas of tubule because as-of-yet unrecognized deficits in Sertoli cell gene expression disable most stem cell niches. Performing the first thorough comparison of the transcriptomes of human testes exhibiting complete spermatogenesis with the transcriptomes of testes with SCO syndrome, we defined transcripts that are both predominantly expressed by Sertoli cells and expressed at aberrant levels in SCO testes. Some of these transcripts encode proteins required for the proper assembly of adherent and gap junctions at sites of contact with other cells, including spermatogonial stem cells (SSCs). Other transcripts encode GDNF, FGF8 and BMP4, known regulators of mouse SSCs. Thus, most SCO Sertoli cells can neither organize junctions at normal sites of cell-cell contact nor stimulate SSCs with adequate levels of growth factors. We propose that the critical deficits in Sertoli cell gene expression we have identified contribute to the inability of spermatogonial stem cells within small patches of spermatogenesis in some SCO testes to seed spermatogenesis to adjacent areas of tubule that are barren of spermatogenesis. Furthermore, we predict that one or more of these deficits in gene expression are primary causes of human SCO syndrome., Competing Interests: DAP is affiliated with 'Consulting Research Services, Inc.' This service was founded and funded by DAP. Funds came from his lecture fees. DAP does not receive any financial benefit from this service and the service has no employees. Thus, this service does not present a conflict of interest with the research presented in this manuscript. The authors declare that there are no other conflicts of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Approximately 15% of couples are infertile, with half of these cases being due to a male factor. Testis-specific cytoplasmic poly(A) polymerase beta (PAPOLB) is known to be critical for spermatogenesis. In mice, the loss of function of the Papolb gene results in the arrest of spermiogenesis and in male infertility. To analyse the role of the PAPOLB gene in human male infertility, this study investigated the relevance of this gene to human Sertoli-cell-only syndrome (SCOS) with azoospermia. Mutation analysis of the PAPOLB coding region was performed on 139 Japanese patients by PCR and direct sequence analysis. No critical mutations directly causing SCOS were detected, but three single-nucleotide polymorphisms (SNPs; SNP1 (c1101C > T), SNP2 (c1347T > C) and SNP3 (c1903C > A)) were found in the coding region. However, there were no significant associations in the allelic and genotypic distributions of these three SNPs between the SCOS and control groups (p>.05). This study suggests a lack of association of PAPOLB with azoospermia due to SCOS in humans.

Objectives: To detect autosomal genetic defects and to determine candidate genes in Sertoli cell-only syndrome infertile men., Methods: Single-nucleotide polymorphism + comparative genomic hybridization microarray technology was carried out on 39 Sertoli cell-only syndrome infertile patients in the present study. Array comparative genomic hybridization compares the patient's genome against a reference genome, and identifies uncover deletions, amplifications and loss of heterozygosity., Results: A link between defective spermatogenesis genes and infertility was examined, and amplifications and deletions in several genes were detected, including homeobox gene; synaptonemal complex element protein 1; collagen, type I, alpha 1; imprinted maternally expressed transcript; and potassium voltage-gated channel subfamily Q member 1., Conclusions: The present data suggest that several genes can play an important role in spermatogenesis and progression of Sertoli cell-only syndrome., (© 2018 The Japanese Urological Association.)

Background: Sertoli cells are the most important somatic cells contributing to the microenvironment (named niche) for spermatogonial stem cells (SSCs). They produce amounts of crucial growth factors and structure proteins that play essential roles in the complex processes of male SSCs survival, proliferation, and differentiation. It has been suggested that Sertoli cell abnormalities could result in spermatogenesis failure, eventually causing azoospermia in humans. However, to the end, the gene expression characteristics and protein functions of human Sertoli cells remained unknown. In this study, we aimed to evaluate the effect of fibroblast growth factor-5 (FGF5), a novel growth factor downregulated in Sertoli cells from Sertoli cell-only syndrome (SCOS) patients compared to Sertoli cells from obstructive azoospermia (OA) patients, on SSCs., Methods: We compared the transcriptome between Sertoli cell from SCOS and OA patients. Then, we evaluated the expression of FGF5, a growth factor which is downregulated in SCOS Sertoli cells, in human primary cultured Sertoli cells and testicular tissue. Also, the proliferation effect of FGF5 in mice SSCs was detected using EDU assay and CCK-8 assay. To investigate the mechanism of FGF5, Phospho Explorer Array was performed. And the results were verified using Western blot assay., Results: Using RNA-Seq, we found 308 differentially expressed genes (DEGs) between Sertoli cells from SCOS and OA patients. We noted and verified that the expression of fibroblast growth factor-5 (FGF5) was higher in Sertoli cells of OA patients than that of SCOS patients at both transcriptional and translational levels. Proliferation assays showed that rFGF5 enhanced the proliferation of mouse SSCs line C18-4 in a time- and dose-dependent manner. Moreover, we demonstrated that ERK and AKT were activated and the expression of Cyclin A2 and Cyclin E1 was enhanced by rFGF5., Conclusion: The distinct RNA profiles between Sertoli cells from SCOS and OA patients were identified using RNA-Seq. Also, FGF5, a growth factor that downregulated in SCOS Sertoli cells, could promote SSCs proliferation via ERK and AKT activation.

Purpose: In about 10% of patients affected by Fanconi anemia (FA) the diagnosis is delayed until adulthood, and the presenting symptom in these "occult" FA cases is often a solid cancer and cancer treatment-related toxicity. Highly predictive clinical parameter(s) for diagnosing such an adult-onset cases are missing., Methods: (1) Exome sequencing (ES), (2) Sanger sequencing of FANCA, (3) diepoxybutane (DEB)-induced chromosome breakage test., Results: ES identified a pathogenic homozygous FANCA variant in a patient affected by Sertoli cell-only syndrome (SCOS) and in his azoospermic brother. Although they had no overt anemia, chromosomal breakage test revealed a reverse somatic mosaicism in the former and a typical FA picture in the latter. In 27 selected SCOS cases, 1 additional patient showing compound heterozygous pathogenic FANCA variants was identified with positive chromosomal breakage test., Conclusion: We report an extraordinarily high frequency of FA in a specific subgroup of azoospermic patients (7.1%). The screening for FANCA pathogenic variants in such patients has the potential to identify undiagnosed FA before the appearance of other severe clinical manifestations of the disease. The definition of this high-risk group for "occult" FA, based on specific testis phenotype with mild/borderline hematological alterations, is of unforeseen clinical relevance.

Androgen receptor (AR) mediates androgen activities such as the growth of accessory sex organs, and initiation and promotion of spermatogenesis. There are two trinucleotide polymorphisms (CAG and GGN repeats) in the first exon of AR gene that their association with infertility is still controversial. The variants of both polymorphic repeats were investigated by PCR-Sequencing in 220 infertile men (80 azoospermic, 60 oligospermic and 80 asthenospermic) and 80 healthy fertile controls. AR Expression level was quantified by RT-qPCR on 30 patients (20 patients with nonobstructive azoospermia (NOA) and 10 obstructive azoospermia patients as controls). Our results demonstrated that the medians of CAG and GGN repeats length in infertile group were significantly higher than fertile men (p < 0.05). AR expression results showed a significant increase in SCOS group compared to control (p < 0.05). Long stretches of tandem repeats of AR gene may negatively affect the function of the gene and consequently lead to male infertility. In patients with SCOS, AR expression increases because of the lack of germ cells. Therefore, with increasing AR expression, the probability of SCOS occurrence is also increased. It can be concluded that increasing AR expression in testes tissue decreases the probability of sperm presence., (© 2018 Blackwell Verlag GmbH.)

Context Abnormalities in the hypothalamo-pituitary-gonadal axis have long been reported in Noonan syndrome (NS) males with only few data available in prepubertal children. Objective The aim of this study was to describe the gonadal function of NS males from childhood to adulthood. Design It is a retrospective chart review. Patients and methods A total of 37 males with a genetically confirmed diagnosis of NS were included. Clinical and genetic features, as well as serum hormone levels (LH, FSH, testosterone, anti-Müllerian hormone (AMH), and inhibin B) were analysed. Results Of the 37 patients, 16 (43%) children had entered puberty at a median age of 13.5 years (range: 11.4-15.0 years); age at pubertal onset was negatively correlated with BMI SDS (r = -0.541; P = 0.022). In pubertal boys, testosterone levels were normal suggesting a normal Leydig cell function. In contrast, NS patients had significant lower levels of AMH (mean SDS: -0.6 ± 1.1; P = 0.003) and inhibin B (mean SDS: -1.1 ± 1.2; P < 0.001) compared with the general population, suggesting a Sertoli cell dysfunction. Lower AMH and inhibin B levels were found in NS-PTPN11 patients, whereas these markers did not differ from healthy children in SOS1 patients. No difference was found between cryptorchid and non-cryptorchid patients for AMH and inhibin B levels (P = 0.43 and 0.62 respectively). Four NS-PTPN11 patients had a severe primary hypogonadism with azoospermia/cryptozoospermia. Conclusions NS males display Sertoli cell-specific primary testicular insufficiency, whereas Leydig cell function seems to be unaffected.

Objective: To study the expression of CLAUDIN-11 in the testis tissue of non-obstructive azoospermia (NOA) patients with different severities and investigate its clinical significance., Methods: Sixty-two NOA patients were divided into a hypospermatogenesis (HS) group (n = 30) and a Sertoli cell only syndrome (SCO) group (n =32). The expression of CLAUDIN-11 in the testicular tissue of the patients was detected by immunohistochemistry, that of CLAUDIN-11 mRNA determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the levels of serum reproductive hormones measured by chemiluminescent immunoassay., Results: Immunohistochemistry showed that the expression of CLAUDIN-11 was mainly in the cytoplasm of the Sertoli cells around the seminiferous tubule wall in the HS group, but diffusely distributed in the membrane of the Sertoli cells in the SCO group. RT-qPCR revealed a significantly lower expression of CLAUDIN-11 mRNA in the HS than in the SCO group (0.008 ± 0.001 vs 0.013 ± 0.002, t = 10.616, P<0.01). The level of serum luteotropic hormone (LH) was also markedly lower in the HS than in the SCO group (［3.62 ± 1.34］ vs ［4.96 ± 3.10］ IU/L, P<0.05) and so was that of follicle-stimulating hormone (FSH) (［5.36 ± 2.80］ vs ［10.65 ± 9.18］ IU/L, P<0.05)., Conclusions: The up-regulated expression of CLAUDIN-11 in Sertoli cells may play an important role in the development and progression of spermatogenic dysfunction in NOA patients.

In recent decades, infertility has been considered a major widespread public health issue of very high concern. Currently, almost 50% of infertility cases are due to male factors, including semen disorders, obstructions, cryptorchidism, varicocele and testicular failures, which can occur due to malfunctions in both somatic and germ cells. In this context, besides other approaches, different miRNAs have been used as biomarkers for the diagnosis of male infertility, with different pathologic conditions such as Sertoli cell-only syndrome, mixed atrophy, and germ cell arrest. However, most studies related to male fertility do not point out the functions and cell targets of the described miRNAs. Initial investigations using experimental assays in murine and porcine models were performed, providing the first evidence of the influence of miRNAs on Sertoli cell function including, for instance, proliferation, maturation and hormone responses of these cells. The aim of this mini-review is therefore to summarize our present knowledge of this relevant subject and to highlight the importance of future investigations concerning the miRNA influence in the control of Sertoli cells, spermatogenesis and male fertility.

This study aimed to define the expression patterns of HENMT1 and PIWI proteins in human testis and investigate their association with transposon expression, infertility sub-type or development of testicular germ cell tumours (TGCTs). Testis biopsies showing normal spermatogenesis were used to identify normal localisation patterns of HENMT1 and PIWIL1 by immunolocalisation and RT-PCR after laser microdissection. 222 testis biopsies representing normal spermatogenesis, hypospermatogenesis, spermatogenic arrests, Sertoli cell-only (SCO) tumours and TGCTs were analysed by RT-qPCR for expression of HENMT1/PIWIL1/PIWIL2/PIWIL3/PIWIL4 and LINE-1 Additionally, HENMT1 -overexpressing TCam2 seminoma cell lines were analysed for the same parameters by RT-qPCR. We found that HENMT1 and PIWIL1 are coexpressed in pachytene spermatocytes and spermatids. Expression of HENMT1 , PIWIL1 and PIWIL2 was mainly dependent on germ cell content but low levels of expression were also detected in some SCO samples. Levels of HENMT1 , PIWIL1 and PIWIL2 expression were low in TGCT. Samples with HENMT1, PIWIL2 and PIWIL4 expression showed significantly ( P  < 0.05) lower transposon expression compared to samples without expression in the same histological group. HENMT1-overexpressing TCam2 cells showed lower LINE-1 expression than empty vector-transfected control lines. Our findings support that the transposon-regulating function of the piRNA pathway found in the mouse is conserved in adult human testis. HENMT1 and PIWI proteins are expressed in a germ-cell-specific manner and required for transposon control., (© 2017 Society for Reproduction and Fertility.)

Genetic mechanisms are implicated in some cases of male infertility. Recently, it was demonstrated that male mice lacking the gene for RAD21L exhibited azoospermia caused by meiotic arrest. Mouse RAD21L is a functionally relevant meiotic α-kleisin that is essential for male fertility. Therefore, we hypothesized that RAD21L mutations or polymorphisms may be associated with male infertility, especially azoospermia secondary to meiotic arrest. To determine if RAD21L defects are associated with azoospermia in groups of patients with meiotic arrest, we performed direct sequencing of the RAD21L coding regions in 38 Japanese patients with meiotic arrest and in 200 normal controls. Three coding single-nucleotide polymorphisms (SNP1-SNP3) were detected in the meiotic arrest patient group. Sertoli cell-only syndrome is considered a common cause of non-obstructive azoospermia. For comparison, the RAD21L coding regions in which SNP1-SNP3 were detected were sequenced in 140 patients with Sertoli cell-only syndrome. Statistical analyses were used to compare the two groups of patients with the control group. Genotype and allele frequencies of SNP2 and SNP3 were notably higher in the two patient groups compared with the control group (Bonferroni adjusted p value <0.016). These results suggest a critical role for RAD21L in human spermatogenesis.

Several observational studies have showed a combination of lower testosterone (T) to LH ratio and higher estradiol (E 2 ) to T ratio in secretory infertile men compared to men with normal spermatogenesis, suggesting a steroidogenic dysfunction of Leydig cells (Lc) that may involve increased aromatase activity. Low T/LH ratio is associated with Lc hyperplasia, which together with LH hyperstimulation may represent compensation for impaired T production. Aromatase expression and oestrogen production are mainly detected in Lc of the testis, although Sertoli and germ cells also contribute to testicular aromatase activity. The aim of this study was to assess the transcriptional expression of CYP19A1 (aromatase) in isolated Lc of subjects with Sertoli cell-only syndrome (SCOS) and signs of Lc impairment. Nineteen patients with SCOS and 10 controls with normal spermatogenesis who had medical indication of testicular biopsy for sperm retrieval were studied. Leydig cells were isolated by Laser Capture Microdissection (LCM) and CYP19A1 mRNA expression was quantified by SYBR ® Green-based qPCR. In addition, testicular T and E 2 and serum hormonal levels were measured. Relative to control group, CYP19A1 was overexpressed more than twofold in 10/19 cases (2.3-12.2-fold increase), showing a significant increment in cases with low T/LH ratio (T/LH < 2) compared to cases with T/LH ≥ 2 (p = 0.038, REST ® ). Moreover, Rq data for CYP19A1 had a direct correlation with testicular levels of E 2 and the E 2 /T ratio (r = 0.869; p < 0.001 and r = 0.633; p = 0.005). In summary, Lc from infertile patients with signs of Lc dysfunction overexpressed aromatase and showed an increment of testicular E 2 . Our results suggest that increased expression of aromatase in Lc leads to higher E 2 production and may account for the functional impairment of the Lc in patients with SCOS., (© 2016 American Society of Andrology and European Academy of Andrology.)

Objective: To define the stage-by-stage expression of KATNB1 during human spermatogenesis., Design: Gene expression analysis, histologic and immunohistochemical evaluation., Setting: University research laboratories and andrological clinic., Patient(s): Eighty human testicular biopsy samples: 43 showing normal spermatogenesis, 9 with maturation arrest at level of spermatocytes, 8 with maturation arrest at level of spermatogonia, and 20 with a Sertoli cell only syndrome., Intervention(s): None., Main Outcome Measure(s): Evaluation of katanin p80 expression in normal as well as impaired spermatogenesis on mRNA (RT-PCR, RT-qPCR, and in situ hybridization) and protein level (immunohistochemistry/immunofluorescence)., Result(s): KATNB1 messenger RNA is exclusively expressed in germ cells, and quantitatively reduced in maturation arrests at the level of spermatogonia. The KATNB1 protein was detected in type B spermatogonia entering meiosis and in the Golgi complex of pachytene spermatocytes. Immediately before the first meiotic division, it is colocalized with the cleaving centriole. It was also detected in early round spermatids in the dictyosome., Conclusion(s): The expression and localization of KATNB1 support a role in spindle formation. The localization of KATNB1 in early round spermatids suggests an involvement in the formation of microtubule-based structures during spermiogenesis (manchette and flagellum). These data are consistent with the demonstrated role of KATNB1 in mouse meiosis, nuclear shaping, and flagellum formation of sperm and suggest the strong conservation of function even between distantly related species., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Spermatogenesis is a complex process that involves thousands of genes whose expression during different stages is strictly regulated. Small non-coding microRNAs play an important role in the posttranscriptional regulation of mRNA processing during spermatogenesis. Using Agilent SurePrint v16 microRNA 8 × 60 K microarray kit, we investigated the microRNA expression profiles of 24 formalin-fixed paraffin-embedded testicular biopsies from patients with hypospermatogenesis (n = 10), hypospermatogenesis and azoospermia factor c region on the Y chromosome (AZFc) deletion (n = 3), Sertoli cell-only syndrome (n = 3) and maturation arrest (n = 2), in comparison with subjects with normal spermatogenesis (n = 6). After adjusting for multiple testing, six deregulated miRNAs were detected in the patients with AZFc deletion, 30 in maturation arrest group, 52 in Sertoli cell-only syndrome group of patients, and none in the group of patients with hypospermatogenesis. Some of the deregulated microRNAs were shared between groups, resulting in 58 unique differentially expressed microRNAs. The expression of five microRNAs (hsa-miR-34b, hsa-miR-449b, hsa-miR-517c, hsa-miR-181c, and hsa-miR-605) was validated by qRT-PCR in a total of 74 samples. Using mRNA expression profiles of subjects with matching histopathological patterns of impaired spermatogenesis from publically available Gene Expression Omnibus data sets, we have performed integrated mRNA-microRNA regulatory network analysis. Pathway analysis revealed significantly enriched set of genes for tumor necrosis factor-related apoptosis-inducing ligand signaling pathway, previously shown to be involved in regulation of apoptosis in normal functioning testis. Our results should be considered as preliminary as we have analyzed only a small number of patients in each studied group. Further studies with larger number of patients with impaired spermatogenesis as well as more targeted approaches with parallel microRNA and mRNA expression profiling in isolated subpopulations of somatic or germ cells from different stages of spermatogenesis are needed to clarify the role of the microRNAs in the process of spermatogenesis., (© 2016 American Society of Andrology and European Academy of Andrology.)

Cell-free seminal mRNA (cfs-mRNA) contains testis-specific transcripts from bilateral testes. This study determined the presence of DEAD box polypeptide 4 (DDX4) in cfs-mRNA to identify and characterize the incidence of Sertoli cell-only (SCO) syndrome in men with non-obstructive azoospermia (NOA). DDX4 cfs-mRNA was determined in 315 men with NOA, and compared with testicular samples obtained by microdissection from 19 NOA patients. Karyotype and azoospermia factor microdeletion analysis were performed, and clinical features were evaluated. Negative DDX4 cfs-mRNA suggestive of SCO was found in 13.7% of NOA patients, with a similar incidence in NOA men with known genetic causes and those without known genetic causes. DDX4 cfs-mRNA was absent in 44% of SCO cases diagnosed by testicular histopathology, but present in all patients presenting with maturation arrest or hypospermatogenesis. Furthermore, 84.2% of NOA men with DDX4 cfs-positive mRNA had a DDX4-positive testicular sample. In NOA men without genetic causes, SCO patients discriminated by negative DDX4 cfs-mRNA showed different clinical features when compared with non-SCO cases. These results suggest that the evaluation of DDX4 cfs-mRNA is more accurate than testicular histopathology in discriminating SCO, and also permits the identification of a specific group of NOA men with distinct clinical features., (Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: To investigate the 5'-flanking regulatory sequence methylation status of the Boule gene in the testis tissue of infertile men with Sertoli cell-only syndrome (SCOS)., Methods: We collected biopsy samples of the testis tissue from 12 men with obstructive azoospermia (the control group) and 15 cases of SCOS, all without varicocele, cryptorchidism, or infectious disease. We extracted genomic DNA from the testis tissue of the SCOS patients, analyzed the characteristics of the 5'-flanking regulatory sequence of the Boule gene using the bioinformatics method, and detected the methylation status of the Boule gene by sodium bisulfite sequencing., Results: A CpG island was observed in the 5'-flanking regulation region of the Boule gene. The methylation level of the Boule gene was remarkably higher in the SCOS group than in the obstructive azoospermia controls (61.4% vs 21.7%, P<0.01), with significant differences in the methylation levels of 14 CpG sites, namely, －58 bp, －50 bp, －48 bp, －38 bp, －28 bp, －24 bp, －20 bp, －15 bp, －1 bp, +5 bp, +8 bp, +15 bp, +29 bp, and +58 bp., Conclusions: The methylation level of the Boule gene is significantly higher in the SCOS patients than in the obstructive azoospermia males, which suggests that the changes in Boule methylation may be associated with spermatogenic dysfunction.

Purpose: Azoospermia is one of the major causes of male infertility and is basically classified into obstructive (OA) and non-obstructive azoospermia (NOA). The molecular background of NOA still largely remains elusive. It has been shown that the poly(A)-binding proteins (PABPs) essentially play critical roles in stabilization and translational control of the mRNAs during spermatogenesis., Methods: In the present study, we aim to evaluate expression levels of the PABP genes, EPAB, PABPC1, and PABPC3, in the testicular biopsy samples and in the isolated spermatocyte (SC) and round spermatid (RS) fractions obtained from men with various types of NOA including hypospermatogenesis (hyposperm), RS arrest, SC arrest, and Sertoli cell-only syndrome (SCO)., Results: In the testicular biopsy samples, both PABPC1 and PABPC3 mRNA expressions were gradually decreased from hyposperm to SCO groups (P < 0.05), whereas there was no remarkable difference for the EPAB expression among groups. The expression levels of cytoplasmically localized PABPC1 and PABPC3 proteins dramatically reduced from hyposperm to SCO groups (P < 0.05). In the isolated SC and RS fractions, the EPAB, PABPC1, and PABPC3 mRNA expressions were gradually decreased from hyposperm to SC arrest groups (P < 0.05). Similarly, both PABPC1 and PABPC3 proteins were expressed at higher levels in the SC and RS fractions from hyposperm group when compared to the SC and RS fractions from either RS arrest or SC arrest group (P < 0.05)., Conclusion: Our findings suggest that observed significant alterations in the PABPs expression may have an implication for development of different NOA forms.

Sertoli cells play critical roles in regulating spermatogenesis and they can be reprogrammed to the cells of other lineages, highlighting that they have significant applications in reproductive and regenerative medicine. The fate determinations of Sertoli cells are regulated precisely by epigenetic factors. However, the expression, roles, and targets of microRNA (miRNA) in human Sertoli cells remain unknown. Here we have for the first time revealed that 174 miRNAs were distinctly expressed in human Sertoli cells between Sertoli-cell-only syndrome (SCOS) patients and obstructive azoospermia (OA) patients with normal spermatogenesis using miRNA microarrays and real time PCR, suggesting that these miRNAs may be associated with the pathogenesis of SCOS. MiR-133b is upregulated in Sertoli cells of SCOS patients compared to OA patients. Proliferation assays with miRNA mimics and inhibitors showed that miR-133b enhanced the proliferation of human Sertoli cells. Moreover, we demonstrated that GLI3 was a direct target of miR-133b and the expression of Cyclin B1 and Cyclin D1 was enhanced by miR-133b mimics but decreased by its inhibitors. Gene silencing of GLI3 using RNA inference stimulated the growth of human Sertoli cells. Collectively, miR-133b promoted the proliferation of human Sertoli cells by targeting GLI3. This study thus sheds novel insights into epigenetic regulation of human Sertoli cells and the etiology of azoospermia and offers new targets for treating male infertility.

Sertoli cell-only syndrome is defined by the complete absence of germ cells in testicular tissues and always results in male infertility. The aetiology often remains unknown. In this paper, we have investigated possible causes of Sertoli cell-only syndrome with a special focus on genetic causes. Our results show that, for a large part of the patients (>23% in an unselected group), the sex chromosomes are involved. The majority of patients had a Klinefelter syndrome, followed by patients with Yq microdeletions. Array comparative genomic hybridization in a selected group of "idiopathic patients" showed no known infertility related copy number variations.

About 15% of couples wishing to have children are infertile; approximately half these cases involve a male factor. Polo-like kinase 4 (PLK-4) is a member of the polo protein family and a key regulator of centriole duplication. Male mice with a point mutation in the Plk4 gene show azoospermia associated with germ cell loss. Mutational analysis of 81 patients with azoospermia and Sertoli cell-only syndrome (SCOS) identified one man with a heterozygous 13-bp deletion in the Ser/Thr kinase domain of PLK4. Division of centrioles occurred in wild-type PLK4-transfected cells, but was hampered in PLK-4-mutant transfectants, which also showed abnormal nuclei. Thus, this PLK4 mutation might be a cause of human SCOS and nonobstructive azoospermia., (© 2015 American Society of Andrology and European Academy of Andrology.)

A loss of function of the murine Sin3A gene resulted in male infertility with Sertoli cell-only syndrome (SCOS) phenotype in mice. Here, we investigated the relevance of this gene to human male infertility with azoospermia caused by SCOS. Mutation analysis of SIN3A in the coding region was performed on 80 Japanese patients. However, no variants could be detected. This study suggests a lack of association of SIN3A gene sequence variants with azoospermia caused by SCOS in humans., (© 2014 Blackwell Verlag GmbH.)

Purpose: Sertoli-cell only syndrome is one of the reasons for male infertility but its pathogenesis remains unclear. PRPS2, a subset of PRS, is reported to be a potential protein associated with Sertoli-cell only syndrome. In this study we further investigated the correlation between PRPS2 and Sertoli-cell only syndrome, and evaluated the effect of PRPS2 expression on apoptosis of TM4 Sertoli cells., Materials and Methods: PRPS2 expression was detected in patients with Sertoli-cell only syndrome and normal spermatogenesis, and in Sertoli-cell only syndrome mouse models by immunohistochemistry, quantitative reverse transcription-polymerase chain reaction and Western blot. PRPS2 expression in TM4 Sertoli cells was then down-regulated and up-regulated by lentivirus vectors. The effect of PRPS2 expression on cell apoptosis and cell cycle transition was evaluated by flow cytometry., Results: PRPS2 expression in patients with Sertoli-cell only syndrome was significantly greater than in those with normal spermatogenesis. A significant increase in PRPS2 expression was observed in Sertoli-cell only syndrome mouse models. PRPS2 over expression significantly inhibited cell apoptosis and promoted cell cycle transition in TM4 Sertoli cells. However, PRPS2 down-regulation showed a reverse effect. Moreover, results revealed that PRPS2 over expression inhibited cell apoptosis via the p53/Bcl-2/caspase-9/caspase-3/caspase-6/caspase-7 signaling pathway., Conclusions: PRPS2 expression correlates with Sertoli-cell only syndrome and inhibits the apoptosis of TM4 Sertoli cells via the p53/Bcl-2/caspases signaling pathway., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Objective: To study a potential association between male infertility and DMRT1 mutations., Design: Retrospective sequencing study., Setting: University hospital., Patient(s): 171 patients with cryptozoospermia (sperm concentration<0.1 million/mL, n=40) or nonobstructive azoospermia (n=131), and 215 normozoospermic controls., Intervention(s): Sequence analysis of DMRT1., Main Outcome Measure(s): Identification of rare variants in DMRT1 that are associated with male infertility., Result(s): In total, we detected four putative pathogenic mutations in six patients (3.5%) and less frequently in two controls (0.9%)., Conclusion(s): Point mutations of DMRT1 may be rarely associated with male infertility., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The molecular basis of spermatogenic failure (SpF) is still largely unknown. Accumulating evidence suggests that a series of specific events such as meiosis, are determined at the early stage of spermatogenesis. This study aims to assess the expression profile of pre-meiotic genes of infertile testicular biopsies that might help to define the molecular phenotype associated with human deficiency of sperm production. An accurate quantification of testicular mRNA levels of genes expressed in spermatogonia was carried out by RT-qPCR in individuals showing SpF owing to germ cell maturation defects, Sertoli cell-only syndrome or conserved spermatogenesis. In addition, the gene expression profile of SpF was compared with that of testicular tumour, which is considered to be a severe developmental disease of germ cell differentiation. Protein expression from selected genes was evaluated by immunohistochemistry. Our results indicate that SpF is accompanied by differences in expression of certain genes associated with spermatogonia in the absence of any apparent morphological and/or numerical change in this specific cell type. In SpF testicular samples, we observed down-regulation of genes involved in cell cycle (CCNE1 and POLD1), transcription and post-transcription regulation (DAZL, RBM15 and DICER1), protein degradation (FBXO32 and TM9SF2) and homologous recombination in meiosis (MRE11A and RAD50) which suggests that the expression of these genes is critical for a proper germ cell development. Interestingly, a decrease in the CCNE1, DAZL, RBM15 and STRA8 cellular transcript levels was also observed, suggesting that the gene expression capacity of spermatogonia is altered in SpF contributing to an unsuccessful sperm production. Altogether, these data point to the spermatogenic derangement being already determined at, or arising in, the initial stages of the germ line., (© 2014 American Society of Andrology and European Academy of Andrology.)