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Meiosis is a specialized cell division process that generates gametes for sexual reproduction. However, the factors and underlying mechanisms involving meiotic progression remain largely unknown, especially in humans. Here, it is first showed that HSF5 is associated with human spermatogenesis. Patients with a pathogenic variant of HSF5 are completely infertile. Testicular histologic findings in the patients reveal rare postmeiotic germ cells resulting from meiotic prophase I arrest. Hsf5 knockout (KO) mice confirms that the loss of HSF5 causes defects in meiotic recombination, crossover formation, sex chromosome synapsis, and sex chromosome inactivation (MSCI), which may contribute to spermatocyte arrest at the late pachytene stage. Importantly, spermatogenic arrest can be rescued by compensatory HSF5 adeno-associated virus injection into KO mouse testes. Mechanistically, integrated analysis of RNA sequencing and chromatin immunoprecipitation sequencing data revealed that HSF5 predominantly binds to promoters of key genes involved in crossover formation (e.g., HFM1, MSH5 and MLH3), synapsis (e.g., SYCP1, SYCP2 and SYCE3), recombination (TEX15), and MSCI (MDC1) and further regulates their transcription during meiotic progression. Taken together, the study demonstrates that HSF5 modulates the transcriptome to ensure meiotic progression in humans and mice. These findings will aid in genetic diagnosis of and potential treatments for male infertility., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Cattleyaks are the crossbred offspring between cattle and yaks, exhibiting the prominent adaptability to the harsh environment as yaks and much higher growth performances than yaks around Qinghai-Tibet plateau. Unfortunately, cattleyak cannot be effectively used in yak breeding due to its male infertility resulted from spermatogenic arrest. In this study, we performed RNA sequencing (RNA-seq) and bioinformatics analysis to determine the expression profiles of long noncoding RNA (lncRNA) from cattleyak and yak testis. A total of 604 differentially expressed (DE) lncRNAs (135 upregulated and 469 downregulated) were identified in cattleyak with respect to yak. Through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, we identified several DE lncRNAs regulating the mitotic cell cycle processes by targeting the genes significantly associated with the mitotic cell cycle checkpoint and DNA damage checkpoint term and also significantly involved in p53 signaling pathway, mismatch repair and homologous recombination pathway (P < 0.05). The reverse transcription PCR (RT-PCR) and quantitative Real-Time PCR (qRT-PCR) analysis of the randomly selected fourteen DE lncRNAs and the seven target genes validated the RNA-seq data and their true expressions during spermatogenesis in vivo. Molecular cloning and sequencing indicated that the testis lncRNAs NONBTAT012170 and NONBTAT010258 presented higher similarity among different cattleyak and yak individuals. The downregulation of these target genes in cattleyak contributed to the abnormal DNA replication and spermatogenic arrest during the S phase of mitotic cell cycle. This study provided a novel insight into lncRNA expression profile changes associated with spermatogenic arrest of cattleyak., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

To achieve spermatogenesis in vitro , one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or impaired homologous recombination, can cause failure of crossover formation and subsequent chromosome nondisjunction, eventually leading to aneuploid sperm. These meiotic events are therefore strictly monitored by meiotic checkpoints that initiate apoptosis of aberrant spermatocytes and lead to spermatogenic arrest. However, we recently found that, in vitro derived meiotic cells proceeded to the first meiotic division (MI) stage, despite displaying incomplete chromosome synapsis, no discernible XY-body and lack of crossover formation. We therefore optimized our in vitro culture system of meiosis from male germline stem cells (mGSCs) in order to achieve full chromosome synapsis, XY-body formation and meiotic crossovers. In comparison to previous culture system, the in vitro -generated spermatocytes were transferred after meiotic initiation to a second culture dish. This dish already contained a freshly plated monolayer of proliferatively inactivated immortalized Sertoli cells supporting undifferentiated mGSCs. In this way we aimed to simulate the multiple layers of germ cell types that support spermatogenesis in vivo in the testis. We found that in this optimized culture system, although independent of the undifferentiated mGSCs, meiotic chromosome synapsis was complete and XY body appeared normal. However, meiotic recombination still occurred insufficiently and only few meiotic crossovers were formed, leading to MI-spermatocytes displaying univalent chromosomes (paired sister chromatids). Therefore, considering that meiotic checkpoints are not necessarily fully functional in vitro , meiotic crossover formation should be closely monitored when mimicking gametogenesis in vitro to prevent generation of aneuploid gametes., 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 © 2021 Lei, Zhang, van Pelt and Hamer.)

The current study assessed the cross-link between mitochondria-related apoptosis and cell cycle machinery systems during ischemia and reperfusion in a rat model of testicular torsion and detorsion. The Wistar male rats were divided into control, 1 h, 2 h, 4 h and 8 h testicular torsion-induced, and 1 h, 2 h, 4 h and 8 h testicular detorsion-induced groups. The Johnson's score was analyzed. The mRNA and protein contents of Bcl-2, Bax, Caspase-3, Cyclin D1, Cdk4, P21 and P53 were investigated by sqRT-PCR and immunohistochemical staining, respectively. The apoptosis index was analyzed by TUNEL staining. The mRNA levels of bax, p53, p21 and cyclin D1 were increased, and the mRNA levels of bcl-2 and cdk4 were decreased in torsion and reperfusion-induced groups, time-dependently. The caspase-3 mRNA was increased in torsion-induced and diminished in detorsion-induced groups. A time-dependent reduction in Bcl-2 + , Caspase-3 + , Cyclin D1 + , Cdk4 + and P53 + and increment in P21 + cells distribution per mm 2 of tissue were revealed after torsion and detorsion. The apoptosis index was increased after torsion and decreased after detorsion. In conclusion, torsion-induced severe DNA damage stimulates the cyclin D1, p53 and p21 mRNA expression while more than 8 h is needed to reveal them as protein content in testicular tissue. About detorsion, decreased Cyclin D1 and Cdk4 proteins and the P53-induced transcriptional effect on p21 expression, stimulates the p21 bind to cdk4 and consequent failure in Cyclin D1/Cdk4 complex formation. This situation in association with apoptotic genes results in spermatogenesis failure., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

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.)

In mammals, more than 2000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male fertility. Tesmin (Metallothionein-like 5; Mtl5) was originally reported as a testis-specific transcript that encodes a member of the cysteine-rich motif containing metallothionein family. Later studies showed that Tesmin has two splicing variants and both are specifically expressed in male and female germ cells. Herein, we clarified that the long (Tesmin-L) and short (Tesmin-S) transcript forms start expressing from spermatogonia and the spermatocyte stage, respectively, in testis. Furthermore, while Tesmin-deficient female mice are fertile, male mice are infertile due to arrested spermatogenesis at the pachytene stage. We were able to rescue the infertility with a Tesmin-L transgene, where we concluded that TESMIN-L is critical for meiotic completion in spermatogenesis and indispensable for male fertility., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Background: Cattleyak are the hybrid offspring between cattle and yak and combine yak hardiness with cattle productivity. Much attempt has been made to examine the mechanisms of male sterility caused by spermatogenic arrest, but yet there is no research systematically and precisely elucidated testis gene expression profiling between cattleyak and yak., Methods: To explore the higher resolution comparative transcriptome map between the testes of yak and cattleyak, and further analyze the mRNA expression dynamics of spermatogenic arrest in cattleyak. We characterized the comparative transcriptome profile from the testes of yak and cattleyak using high-throughput sequencing. Then we used quantitative analysis to validate several differentially expressed genes (DEGs) in testicular tissue and spermatogenic cells., Results: Testis transcriptome profiling identified 6477 DEGs (2919 upregulated and 3558 downregulated) between cattleyak and yak. Further analysis revealed that the marker genes and apoptosis regulatory genes for undifferentiated spermatogonia were upregulated, while the genes for differentiation maintenance were downregulated in cattleyak. A majority of DEGs associated with mitotic checkpoint, and cell cycle progression were downregulated in cattleyak during spermatogonial mitosis. Furthermore, almost all DEGs related to synaptonemal complex assembly, and meiotic progression presented no sign of expression in cattleyak. Even worse, dozens of genes involved in acrosome formation, and flagellar development were dominantly downregulated in cattleyak., Conclusion: DEGs indicated that spermatogenic arrest of cattleyak may originate from the differentiation stage of spermatogonial stem cells and be aggravated during spermatogonial mitosis and spermatocyte meiosis, which contributes to the scarcely presented sperms in cattleyak., Competing Interests: The authors have declared that no competing interests exist.

Objective: To establish which meiotic checkpoints are activated in males with severe spermatogenic impairment to improve phenotypic characterization of meiotic defects., Design: Retrospective observational study., Setting: University medical center research laboratory and andrology clinic., Patient(s): Forty-eight patients with confirmed spermatogenic impairment (Johnsen scores 3-6) and 15 controls (Johnsen score 10)., Intervention(s): None., Main Outcome Measure(s): Quantitative assessment of immunofluorescent analyses of specific markers to determine meiotic entry, chromosome pairing, progression of DNA double-strand break repair, crossover formation, formation of meiotic metaphases, metaphase arrest, and spermatid formation, resulting in a novel classification of human meiotic arrest types., Result(s): Complete metaphase arrest was observed most frequently (27%), and the patients with the highest frequency of apoptotic metaphases also displayed a reduction in crossover number. Incomplete metaphase arrest was observed in 17% of the patients. Only four patients (8%) displayed a failure to complete meiotic chromosome pairing leading to pachytene arrest. Two new types of meiotic arrest were defined: premetaphase and postmetaphase arrest (15% and 13%, respectively)., Conclusion(s): Meiotic arrest in men occurs most frequently at meiotic metaphase. This arrest can be incomplete, resulting in low numbers of spermatids, and often occurs in association with reduced crossover frequency. The phenotyping approach described here provides mechanistic insights to help identify candidate infertility genes and to assess genotype-phenotype correlations in individual cases., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Objective: To explore the effect of small interfering RNA (siRNA)-mediated CEP55 gene silencing on the proliferation of mouse spermatogonia., Methods: Six patients with azoospermia diagnosed to have maturation arrest (3 cases) or normal spermatogenesis (3 cases) based on testicular biopsy between January 1 and December 31, 2017 in our center were examined for differential proteins in the testicular tissue using isobaric tags for relative and absolute quantitation (iTRAQ), and CEP55 was found to differentially expressed between the two groups of patients. We constructed a CEP55 siRNA for transfection in mouse spermatogonia and examined the inhibitory effects on CEP55 expressions using Western blotting and qPCR. The effect of CEP55 gene silencing on the proliferation of mouse spermatogonia was evaluated with CCK8 assay., Results: In the testicular tissues from the 6 patients with azoospermia, iTRAQ combined with LC/MS/MS analysis identified over two hundred differentially expressed proteins, among which CEP55 showed the most significant differential expression between the patients with maturation arrest and those with normal spermatogenesis. The cell transfection experiment showed that compared with the cells transfected with the vehicle or the negative control sequence, the mouse spermatogonia transfected with CEP55 siRNA showed significantly lowered expressions of CEP55 mRNA and protein ( P &lt; 0.05) and significantly decreased proliferation rate as shown by CCK8 assay ( P &lt; 0.05)., Conclusions: CEP55 may play a key role in spermatogenesis and may serve as a potential therapeutic target for non-obstructive azoospermia with maturation arrest.

Male infertility affects approximately half of couples who have difficulty becoming pregnant, and its prevalence is continuously rising. Many studies have been performed using animal testes to reveal the mechanisms of male infertility, but few studies have investigated human testes due to various limitations. The aim of this study was to investigate the gene expression profile of impaired human testes through a meta-analysis of microarray data sets, which was accomplished by using 178 human testis samples and 7 microarray data sets. Impaired testes were categorised into four pathological phenotypes or the normal phenotype based on their Johnsen score. Then, a meta-analysis was performed to screen out the differentially expressed genes (DEGs) in each phenotype. The DEGs were used in a subsequent bioinformatics analysis. Our results identified several novel hub genes and pathways and suggested that G1 mitotic cell cycle arrest was a remarkable feature in pre-meiotic arrest. Furthermore, fifteen p53-interacting proteins, such as ABL1 and HDAC2, whose roles in spermatogenesis have not been well characterised, were selected from the DEGs through a strict screening procedure., (© 2019 Blackwell Verlag GmbH.)

Peroxisomes are involved in the degradation of very long-chain fatty acids (VLCFAs) by β-oxidation. Besides neurological defects, peroxisomal dysfunction can also lead to testicular abnormalities. However, underlying alterations in the testes due to a peroxisomal defect are not well characterized yet. To maintain all metabolic functions, peroxisomes require an import machinery for the transport of matrix proteins. One component of this translocation machinery is PEX13. Its inactivation leads to a peroxisomal biogenesis defect. We have established a germ cell-specific KO of Pex13 to study the function of peroxisomes during spermatogenesis in mice. Exon 2 of floxed Pex13 was specifically excised in germ cells prior to meiosis by using a transgenic mouse strain carrying a STRA8 inducible Cre recombinase. Germ cell differentiation was interrupted at the round spermatid stage in Pex13 KO mice with formation of multinucleated giant cells (MNCs) and loss of mature spermatids. Due to a different cellular content in the germinal epithelium of Pex13 KO testes compared to control, whole testes biopsies were used for the analyses. Thus, differences in lipid composition and gene expression are only shown for whole testicular tissue but cannot be limited to single cells. Gas chromatography revealed an increase of shorter fatty acids and a decrease of n-6 docosapentaenoic acid (C22:5n-6) and n-3 docosahexaenoic acid (C22:6n-3), the main components of sperm plasma membranes. Representative genes of the metabolite transport and peroxisomal β-oxidation were strongly down-regulated. In addition, structural components of the blood-testis barrier (BTB) were altered. To conclude, defects in the peroxisomal compartment interfere with normal spermatogenesis.

Study Question: What is the diagnostic potential of next generation sequencing (NGS) based on a 'mouse azoospermia' gene panel in human non-obstructive azoospermia (NOA)?, Summary Answer: The diagnostic performance of sequencing a gene panel based on genes associated with mouse azoospermia was relatively successful in idiopathic NOA patients and allowed the discovery of two novel genes involved in NOA due to meiotic arrest., What Is Known Already: NOA is a largely heterogeneous clinical entity, which includes different histological pictures. In a large proportion of NOA, the aetiology remains unknown (idiopathic NOA) and yet, unknown genetic factors are likely to play be involved. The mouse is the most broadly used mammalian model for studying human disease because of its usefulness for genetic manipulation and its genetic and physiological similarities to man. Mouse azoospermia models are available in the Mouse Genome Informatics database (MGI: http://www.informatics.jax.org/)., Study Design, Size, Duration: The first step was to design of a 'mouse azoospermia' gene panel through the consultation of MGI. The second step was NGS analysis of 175 genes in a group of highly selected NOA patients (n = 33). The third step was characterization of the discovered gene defects in human testis tissue, through meiotic studies using surplus testicular biopsy material from the carriers of the RNF212 and STAG3 pathogenic variants. The final step was RNF212 and STAG3 expression analysis in a collection of testis biopsies., Participants/materials, Setting, Methods: From a total of 1300 infertile patients, 33 idiopathic NOA patients were analysed in this study, including 31 unrelated men and 2 brothers from a consanguineous family. The testis histology of the 31 unrelated NOA patients was as follows: 20 Sertoli cell-only syndrome (SCOS), 11 spermatogenic arrest (6 spermatogonial arrest and 5 spermatocytic arrest). The two brothers were affected by spermatocytic arrest. DNA extracted from blood was used for NGS on Illumina NextSeq500 platform. Generated sequence data was filtered for rare and potentially pathogenic variants. Functional studies in surplus testicular tissue from the carriers included the investigation of meiotic entry, XY body formation and metaphases by performing fluorescent immunohistochemical staining and immunocytochemistry. mRNA expression analysis through RT-qPCR of RNF212 and STAG3 was carried out in a collection of testis biopsies with different histology., Main Results and the Role of Chance: Our approach was relatively successful, leading to the genetic diagnosis of one sporadic NOA patient and two NOA brothers. This relatively high diagnostic performance is likely to be related to the stringent patient selection criteria i.e. all known causes of azoospermia were excluded and to the relatively high number of patients with rare testis histology (spermatocytic arrest). All three mutation carriers presented meiotic arrest, leading to the genetic diagnosis of three out of seven cases with this specific testicular phenotype. For the first time, we report biallelic variants in STAG3, in one sporadic patient, and a homozygous RNF212 variant, in the two brothers, as the genetic cause of NOA. Meiotic studies allowed the detection of the functional consequences of the mutations and provided information on the role of STAG3 and RNF212 in human male meiosis., Limitations, Reasons for Caution: All genes, with the exception of 5 out of 175, included in the panel cause azoospermia in mice only in the homozygous or hemizygous state. Consequently, apart from the five known dominant genes, heterozygous variants (except compound heterozygosity) in the remaining genes were not taken into consideration as causes of NOA. We identified the genetic cause in approximately half of the patients with spermatocytic arrest. The low number of analysed patients can be considered as a limitation, but it is a very rare testis phenotype. Due to the low frequency of this specific phenotype among infertile men, our finding may be considered of low clinical impact. However, at an individual level, it does have relevance for prognostic purposes prior testicular sperm extraction., Wider Implications of the Findings: Our study represents an additional step towards elucidating the genetic bases of early spermatogenic failure, since we discovered two new genes involved in human male meiotic arrest. We propose the inclusion of RNF212 and STAG3 in a future male infertility diagnostic gene panel. Based on the associated testis phenotype, the identification of pathogenic mutations in these genes also confers a negative predictive value for testicular sperm retrieval. Our meiotic studies provide novel insights into the role of these proteins in human male meiosis. Mutations in STAG3 were first described as a cause of female infertility and ovarian cancer, and Rnf212 knock out in mice leads to male and female infertility. Hence, our results stimulate further research on shared genetic factors causing infertility in both sexes and indicate that genetic counselling should involve not only male but also female relatives of NOA patients., Study Funding/competing Interest(s): This work was funded by the Spanish Ministry of Health Instituto Carlos III-FIS (grant number: FIS/FEDER-PI14/01250; PI17/01822) awarded to CK and AR-E, and by the European Commission, Reproductive Biology Early Research Training (REPROTRAIN, EU-FP7-PEOPLE-2011-ITN289880), awarded to CK, WB, and AE-M. The authors have no conflict of interest., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The proper assessment of male fertility is essential for diagnosing and treating male infertility. Currently, spermiogram and Johnsen testicular biopsy score counts are used to assess male fertility. However, spermiogram is not a suitable option for non-obstructive azoospermia patients, and Johnsen testicular biopsy scores only represent localized and not the overall spermatogenesis. Whole-mount staining was a novel method for evaluating protein expression in the tissue. Thus, we explored its application in human seminiferous tubules. Testicular biopsies from 57 azoospermia patients were categorized as obstructive azoospermia (OA), maturation arrest (MA) and Sertoli-cells only syndrome (SCOS). We performed whole-mount staining of their seminiferous tubules and evaluated the spermatogonial stem cells (SSCs), differentiated spermatogonia (SG), spermatocytes (SPC) and spermatids (SD) with their respective markers (GFRA1, CD117, SYCP3, and PNA) to assess fertility. GFRA1, CD117, SYCP3, and PNA were not expressed in SCOS patients, whereas all of them were detected in OA patients. In MA patients with arrested spermatogenesis at the SPC stage, GFRA1, CD117, and SYCP3, but not PNA were expressed in the seminiferous tubules. In MA patients with arrested spermatogenesis at the spermatogonia stage, only GFRA1 was expressed in the seminiferous tubules. These results were consistent with the Johnsen testicular biopsy score counts except for one patient, where although only Sertoli cells were indicated by the score, SSCs were also detected in the whole-mounts. Collectively, whole-mount staining could be used to analyze the inherent spermatogenesis of seminiferous tubules through staining of germ cells at different stages. It offers a more accurate and promising faster method for assessing male fertility compared with traditional biopsy screening. And it could have potential value for the clinical purpose for male fertility management., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Testis tissue xenografting complemented with cryopreservation is a feasible technique for fertility preservation in children with malignancy receiving gonadotoxic therapy and for endangered species with high neonatal mortality rate. However, xenografted testis of human and most endangered species are known to undergo spermatogenic arrest. In this study, we xenografted immature rat testis onto immunodeficient male mice to investigate the plausible underlying causes of spermatogenic arrest. Histological analysis of xenografted testes collected 8-wk post-grafting showed incomplete spermatogenesis with pachytene-stage spermatocytes as the most advanced germ cells. Although the levels of serum luteinizing hormone and testosterone were normal in recipient mice, those of follicle stimulating hormone (FSH) were significantly high, and specific receptors of FSH were absent in the xenografts. The xenografts demonstrated dysregulated expression of Sertoli cell-transcriptional regulators (WT1 and SOX9) and secretory proteins (SCF and GDNF). In conclusion, results from our study suggested that an altered hormonal milieu in recipients and dysregulated protein expression in xenografts could be a potential cause of spermatogenic arrest in xenografted immature rat testis. Further stereological analysis of xenografts can demonstrate precise cellular composition of xenografts to decipher interactions between germ and somatic cells to better understand spermatogenic arrest in xenografted testis.

The reported sperm retrieval rate (SRR) in patients with nonobstructive azoospermia (NOA) due to spermatogenic arrest (SA) is highly variable in the literature. This discrepancy could be explained by the heterogeneity of testicular tissues. Surprisingly, even though inhibin B levels reflect directly Sertoli cell function; no studies have evaluated this parameter in SA. We aimed to clarify the morphological and biological profile in 158 men with SA. From the total population, patients whose seminiferous tubules diameter was below 165 µm have higher SRR (46.9% vs. 27.4%, p < 0.05), lower inhibin levels and a higher frequency of nonuniform SA (71.9% vs. 38.7%, p < 0.001). On multivariate analysis, patients with late SA and a history of cryptorchidism were positively associated with successful sperm extraction. Patients with successful SRR and uniform SA exhibited inhibin levels twofold lower than those with failed TESE (45 pg/ml vs. 95 pg/ml, p < 0.05), whereas FSH levels were similar in the two groups. In this study, we showed for the first time that the mean diameter of the seminiferous tubules may be of value in the diagnosis of SA. Our results suggest that inhibin levels could be useful in the management of NOA with SA, along with FSH levels and testicular volume., (© 2018 Blackwell Verlag GmbH.)

For the reason that adult Sertoli cell specific connexin 43 knockout (SCCx43KO) mice show arrested spermatogenesis at spermatogonial level or Sertoli cell only tubules and significantly reduced germ cell (GC) numbers, the aims of the present study were (1) to characterize the remaining GC population and (2) to elucidate possible mechanisms of their fading. Apoptosis was analyzed in both, KO and wild type (WT) male littermates during postnatal development and in adulthood using TUNEL. Although GC numbers were significantly reduced in KO at 2 and 8 days postpartum (dpp) when compared to WT, no differences were found concerning apoptotic incidence between genotypes. From 10 dpp, the substantial GC deficiency became more obvious. However, significantly higher apoptotic GC numbers were seen in WT during this period, possibly related to the first wave of spermatogenesis, a known phenomenon in normal pubertal testes associated with increased apoptosis. Characterization of residual spermatogonia in postnatal to adult KO and WT mice was performed by immunohistochemical reaction against VASA (marker of GCs in general), Lin28 and Fox01 (markers for undifferentiated spermatogonia) and Stra8 (marker for differentiating spermatogonia and early spermatocytes). During puberty, the GC component in SCCx43KO mice consisted likely of undifferentiated spermatogonia, few differentiating spermatogonia and very few early spermatocytes, which seemed to be rapidly cleared by apoptosis. In adult KOs, spermatogenesis was arrested at the level of undifferentiated spermatogonia. Overall, our data indicate that Cx43 gap junctions in SCs influence male GC development and differentiation rather than their survival., (Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Although meiotic arrest in males is observed in about 25% of azoospermic patients, pure homogeneous arrest in all seminiferous tubules is less frequent, and may be due to mutation of a single gene. However, given the large number of genes involved in meiosis, this gives rises to extensive genetic heterogeneity. Only two genetic abnormalities have been reported on a regular basis: the X-linked exonic TEX11 deletion, and the AZFb microdeletion on the Y chromosome. Other single gene defects were private and found in consanguineous families. Here, we report on a homozygous missense mutation in the gene coding for meiotic double-stranded break formation protein 1 (MEI1; c.C3307T:p.R1103W) observed in two brothers (from a consanguineous Tunisian family) with non-obstructive azoospermia and meiotic arrest. A fertile brother was heterozygous for the mutation. All the queried databases predicted that this mutation is damaging, and it has previously been reported that Mei1 knock-out is associated with meiotic arrest in a murine model. Hence, meiotic arrest in the two brothers was probably caused by an alteration in a gene known to be fundamental for chromosome synapsis.

Cattleyak are interspecific hybrids between cattle and yak, exhibiting the same prominent adaptability as yak and much higher performances than yak. However, male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted their effective utilization in yak breeding. In past decades, much work has been done to investigate the mechanisms of spermatogenic arrest, but little is known about the differences of the post-transcriptional regulators between cattleyak and yak, which may contribute to the impaired spermatogenesis. MiRNAs, a class of endogenous non-coding small RNA, were revealed to play crucial roles in regulating gene expression at post-transcriptional level. In the present study, we identified 50 differentially expressed (DE) known miRNAs and 11 novel miRNAs by using Illumina HISeq and bioinformatic analysis. A total of 50 putative target sites for the 13 DE known miRNAs and 30 for the 6 DE novel miRNAs were identified, respectively. GO and KEGG analyses were performed to reveal the functions of target genes for DE miRNAs. In addition, RT-qPCR was performed to validate the expression of the DE miRNAs and its targets. The identification of these miRNAs may provide valuable information for a better understanding of spermatogenic arrest in cattleyak.

Spermatogenesis is a multifactorial process that forms differentiated sperm cells in a complex microenvironment. This process involves the genome, epigenome, transcriptome, and proteome to ensure the stability of the spermatogonia and supporting cells. The identification of signaling pathways linked to infertility has been hampered by the inherent complexity and multifactorial aspects of spermatogenesis. Systems biology is a promising approach to unveil underlying signaling pathways and genes and identify putative biomarkers. In this study, we analyzed thirteen microarray libraries of infertile humans and mice, and different classes of male infertility were compared using differentially expressed genes and functional enrichment analysis. We found regulatory processes, immune response, glutathione transferase and muscle tissue development to be among the most common biological processes in up-regulated genes, and genes involved in spermatogenesis were down-regulated in maturation arrest (MArrest) and oligospermia cases. We also observed the overexpression of genes involved in steroid metabolism in post-meiotic and meiotic arrest. Furthermore, we found that the infertile mouse model most similar to human MArrest was the Dazap1 mutant mouse. The results of this study could help elucidate features of infertility etiology and provide the basis for diagnostic markers.

Testicular maturation arrest is characterized by interruption of germ cell development and differentiation. Genetic factors play important role in the causation of human disease, including male infertility. The objective was to study copy number variations in testicular maturation arrest using single nucleotide polymorphism (SNP) microarray technique. Conventional cytogenetics, targeted fluorescence in situ hybridization (FISH) and sequence-tagged site (STS) polymerase chain reaction (PCR) were used to confirm some of the SNP microarray findings. SNP microarray on 68 cases of testicular maturation arrest detected copy number variations (CNVs) mostly on sex chromosomes involving pseudoautosomal regions (PAR) 1, 2 and 3 as well as azoospermic factors (AZFs) besides three cases of chromosomal abnormalities (two Klinefelter syndromes and one case of dicentric Y). The AZF deletion was observed in 14 (20.6%) cases and the AZFc gain was observed in 6 (8.8%) cases. PAR 1 and 2 CNVs was observed in 5 (7.3%) cases. PAR 3 CNVs was detected in 19 cases and 2 controls. The TSPY2 gene gain (within PAR 3 CNVs) was observed in 16 cases and 1 control. CNV containing autosomal genes possibly associated with male infertility in this study was SPATA31A2-A5 (9p12) in five cases. In this study, SNP microarray identified possible underlying aetiology in 55.9% (38/68) cases besides identifying minimal critical region of AZFc deletion as 0.51 mb (Y:24356128-24873665) involving TTY5, RBMY2FP, RBMY1F, RBMY1J, TTY6 and PRY genes. SNP microarray seems superior, sensitive, specific as well as cost-effective method and has potential to be the first tier investigations to explore underlying genomic factors of testicular maturation arrest. The present study is an attempt to find out probable genomic factors with idiopathic testicular maturation arrest., (© 2017 American Society of Andrology and European Academy of Andrology.)

Imprinting errors have been described in spermatozoa from infertile patients with oligozoospermia and azoospermia. However, little is known about methylation of imprinted genes in other spermatogenic cells from azoospermic patients. Therefore, we aimed to evaluate the methylation status of single CpGs located in the differentially methylated regions (DMRs) of two imprinted genes, one paternally (H19) and one maternally (MEST) methylated, in primary spermatocytes of azoospermic patients presenting complete (MAc, n = 7) and incomplete (MAi, n = 8) maturation arrest, as well as in other spermatogenic cells from MAi patients that presented focus of complete spermatogenesis in some seminiferous tubules. We observed H19 imprinting errors in primary spermatocytes from one MAi patient and MEST imprinting errors in one MAi and two MAc patients. Additionally, H19 imprinting errors were observed in elongated spermatids/spermatozoa from one MAi patient. Nevertheless, no statistical differences were found for H19 and MEST global methylation levels (percentage of methylated and unmethylated CpGs, respectively) between patients with complete and incomplete MA and also between MA groups and a control group. These results provide further evidence that imprinting errors occur in spermatogenic cells from patients presenting impaired spermatogenesis, as we and others have previously described in ejaculated and testicular spermatozoa. As paternal imprinting errors can be transmitted to the embryo by the sperm cell, they can provide a possible explanation for poor embryo development and/or low pregnancy rates as correct expression of imprinted genes is crucial for embryo and placental development and function. Therefore, in cases with male factor infertility where unsuccessful in vitro fertilization (IVF) treatments are recurrent, analysis of imprinting marks in spermatozoa might be a useful diagnostic tool., (© 2017 American Society of Andrology and European Academy of Andrology.)

To explore that it is necessary to routinely detect chromosomes in infertile patients, we investigated peripheral blood lymphocyte karyotype in 16,294 male infertile patients in the north-east of China and analysed the incidence and type of chromosomal anomaly and polymorphism. G-banding karyotype analysis of peripheral blood lymphocytes was performed in 16,294 cases. Semen analysis was performed three times in all the men. PCR and FISH confirmed the presence of the SRY gene. The rate of chromosomal anomaly in the 16,294 male infertile patients was 4.15% (677/16,294). The rates of chromosomal anomaly were 0.24% in normal semen group, 12.6% in light oligoasthenospermia group, 4.7% in moderate-to-severe oligoasthenospermia group and 9.59% in azoospermia group. There are two male infertile patients with 45,X chromosome karyotype. One X male patient had confirmed the presence of the SRY gene and FISH analysis demonstrated its location on the p arm of chromosome 13. The other X male patient had not found SRY gene in its whole-genome DNA. Meanwhile, sperm motility is slightly oligo-asthenozoospermic at the age of 35-39 and nearly azoospermic at the age of 40-45. As the rates of chromosomal anomaly are 0.24% and 12.6% even in normal semen group and light oligoasthenospermia group, the rates of chromosomal polymorphism are 5.36% and 25.51% in normal semen group and light oligoasthenospermia group, respectively; it is necessary to explore peripheral blood lymphocyte karyotype in all infertile couples. We mentioned that Y, 1, 2, 9 and 12 chromosomes were quite important about male infertility. These findings demonstrate that autosomal retention of SRY can be submicroscopic and emphasise the importance of PCR and FISH in the genetic workup of the monosomic X male. At the same time, it suggested that male infertility might be related to meiotic disturbances with spermatogenetic arrest in Y-autosome translocations, which could result in infertility by reduction of sperm production. Last but not least, ageing is one of the factors that could reduce sperm motility and quality., (© 2016 Blackwell Verlag GmbH.)

Few sex-autosome chromosome abnormalities have been documented in domestic animal species. In humans, Y-autosome chromosome abnormalities may occur at a rate of 1/2,000 live births, whereas in the domestic pig only 2 Y-autosome reciprocal translocations have been previously described. During a routine cytogenetic screening of young boars, we identified a new Y-autosome translocation carrier, which after puberty showed semen devoid of sperm and testicular hypoplasia with spermatogenesis arrest. Whole chromosome painting by FISH analysis corroborated the reciprocal nature of the chromosomal exchanges between the Y chromosome and SSC13. The possible causes for the observed meiotic arrest of the carrier are reviewed., (© 2016 S. Karger AG, Basel.)

Purpose: Piwi-interacting RNAs (piRNAs) are a broad group of noncoding small RNAs that have important biological functions in germline cells and can maintain genome integrity via silencing of retrotransposons. In this study, we aimed to explore the associations between genetic variants of important genes involved in piRNA biogenesis and male infertility with spermatogenic impairment., Methods: To this end, five single-nucleotide polymorphisms (SNPs) in the ASZ1, PIWIL1, TDRD1, and TDRD9 genes were genotyped by TaqMan allelic discrimination assays in 342 cases of nonobstructive azoospermia (NOA) and 493 controls., Results: The SNP rs77559927 in TDRD1 was associated with a reduced risk of spermatogenic impairment. The genotypes TC and TC + CC showed odds ratios and 95 % confidence intervals of 0.73 (0.55-0.98, P = 0.034) and 0.73 (0.56-0.97, P = 0.030), respectively, in patients with NOA compared with those in the controls., Conclusion: Thus, our results provided the first epidemiological evidence supporting the involvement of TDRD1 genetic polymorphisms in piRNA processing genes in determining the risk of spermatogenic impairment in a Han Chinese population.

Unlabelled: Male infertility of cattleyak due to spermatogenic arrest greatly restricts their effective utilization in yak breeding. Although much work has been done to investigate the mechanisms of spermatogenic arrest, there is no information regarding the differences of protein composition between cattleyak and yak testis. Comparative investigation of testis proteomes between cattleyak and yak using iTRAQ proteomics identified 256 differentially abundant proteins with fold change values higher than ±1.5. Most of the differentially abundant proteins were involved in extracellular matrix organization, response to stimulus, metabolic and cellular process, in which a large number of the cattleyak predominant proteins were associated with various stresses, cell adhesion and germ cell migration. Such upregulated proteins as integrins and their ligands in the extracellular matrix involved in ECM-receptor interaction pathway may help germ cells to endure pulling forces and impede their migration. In contrast, down-regulated proteins in cattleyak were associated with defects in various metabolic processes and cellular processes during spermatogenesis. Such Downregulated proteins as the subunits of mitochondrial cytochrome Bc1 complex involved in the Alzheimer's disease and oxidative phosphorylation pathways may lead to mitochondrial dysfunction and cell death in cattleyak testis., Biological Significance: Spermatogenic arrest of cattleyak involves defects of both germ cells and their micro-environment in testis. In this study, Dozens of proteins possibly associated with spermatogenic arrest of cattleyak were identified by comparative iTRAQ proteomics, in which a large number of the cattleyak predominant proteins may act in response to various stresses (especially inflammatory stresses), enhance cell adhesion and impeded germ cell migration, while down-regulated proteins in cattleyak were associated with defects in various metabolic processes and cellular processes during spermatogenesis. Therefore, germ cells in testis of cattleyak may be constantly in a stress state (similar to inflammation) due to accumulation of some toxic intermediate products resulted from metabolic disturbances. Metabolic defects of germ cells and their deleterious micro-environment in testis of cattleyak may be the root of all other problems during spermatogenesis., (Copyright © 2016. Published by Elsevier B.V.)

Male infertility is often associated with a decreased sperm count. The Pygo2 gene is expressed in the elongating spermatid during chromatin remodeling; thus impairment in PYGO2 function might lead to spermatogenic arrest, sperm count reduction, and subsequent infertility. The aim of this study was to identify mutations in Pygo2 that might lead to idiopathic oligospermia and azoospermia. DNA was isolated from venous blood from 77 men with normal fertility and 195 men with idiopathic oligospermia or azoospermia. Polymerase chain reaction-sequencing analysis was performed for the three Pygo2 coding regions. Non-synonymous single nucleotide polymorphisms (SNPs) were detected and analyzed using SIFT, Polyphen-2, and Mutation Taster softwares to identify possible changes in protein structure that could affect phenotype. Pygo2 sequencing was successful for 178 patients (30 with mild or moderate oligospermia, 57 with severe oligospermia, and 91 with azoospermia). Three previously reported non-synonymous SNPs were identified in patients with azoospermia or severe oligospermic but not in those with mild or moderate oligozoopermia or normozoospermia. SNPs rs61758740 (M141I) and rs141722381 (N240I) cause the replacement of one hydrophobic or hydrophilic amino acid, respectively, with another, and SNP rs61758741 (K261E) causes the replacement of a basic amino acid with an acidic one. The software predictions demonstrated that SNP rsl41722381 would likely result in disrupted tertiary protein structure and thus could be involved in disease pathogenesis. Overall, this study demonstrated that SNPs in the coding region of Pygo2 might be one of the causative factors in idiopathic oligospermia and azoospermia, resulting in male infertility.

Dmrt7 is a member of the DM domain family of genes. Dmrt7 deficiency is also a strong candidate as a cause for male cattle-yak infertility, as it is regarded as essential for male spermatogenesis, between the pachynema and diplonema stages. In our study, the coding region sequence of yak and cattle-yak Dmrt7 was cloned by molecular cloning techniques, and the sequence, conserved domains, functional sites, and secondary and tertiary structures of the Dmrt7-encoded protein were predicted and analyzed using bioinformatics methods. The coding region sequences of the Dmrt7 gene, encoding 370 amino acids, were consistent in yak and cattle-yak. The protein encoded by yak and cattle-yak Dmrt7 contains a DM domain. We detected Dmrt7 mRNA expression in testis, but not in any other tissue. Dmrt7 mRNA and protein expression was significantly higher in testis of cattle and yak than that in cattle-yak (p < 0.01). Histological analysis indicated that seminiferous tubules in male cattle-yak were highly vacuolated and contained primarily Sertoli cells and spermatogonia, while those of cattle and yak contained abundant primary spermatocytes. Male cattle-yak testis contained a significantly larger number of apoptotic cells than those in cattle and yak assessed by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) analysis. The accumulation of SCP3-positive spermatocytes indicated the arrest of spermatogenesis at the pachynema stage in the cattle-yak. These results suggest low levels of Dmrt7 expression lead to male sterility in cattle-yak. The molecular function of Dmrt7 and the regulation of its expression warrant need to be examined in future studies.

Purpose: The present research was undertaken to study probable genetic variations of MOV10L1 in 30 infertile men that had complete maturation arrest in their spermatocyte levels and 70 fertile men as the control group., Methods: We performed polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) on extracted DNAs and sequencing was used to confirm the results. Identified polymorphisms in the MOV10L1 were further subjected to a haplotype analysis., Results: We identified eight single nucleotide polymorphisms (SNPs): one missense (rs2272837) and four nonsense polymorphisms (rs2272836, rs11704548, rs2272838, rs138271) in the exonic sequences and three polymorphisms (rs12170772, rs2272840, rs17248147) in the intronic regions. With the exception of rs2272838, there was a statistically significant association in all polymorphisms between study population (P < 0.05). The result of haplotyping analysis showed ten possible haplotypes, from which five were significantly increased in infertile patients compared with the control group., Conclusions: Our results suggest that MOV10L1 gene polymorphisms in the studied infertile males with complete maturation arrest are linked to infertility.

The blood-testis barrier (BTB) is found between adjacent Sertoli cells in the testis where it creates a unique microenvironment for the development and maturation of meiotic and postmeiotic germ cells in seminiferous tubes. It is a compound proteinous structure, composed of several types of cell junctions including tight junctions (TJs), adhesion junctions and gap junctions (GJs). Some of the junctional proteins function as structural proteins of BTB and some have regulatory roles. The deletion or functional silencing of genes encoding these proteins may disrupt the BTB, which may cause immunological or other damages to meiotic and postmeiotic cells and ultimately lead to spermatogenic arrest and infertility. In this review, we will summarize the findings on the BTB structure and function from genetically-modified mouse models and discuss the future perspectives.

Objective: To investigate whether the normal expression of metastasis-associated protein 1 (MTA1) in Sertoli cells (SCs) is associated with adjacent germ cells (GCs) and to provide the functional relevance of MTA1 in this somatic cell., Methods: The expression pattern of MTA1 in the SCs of impaired human spermatogenesis was determined using immunohistochemistry. The effect of the depletion of GCs on the expression of MTA1 in isolated SCs was evaluated using reverse transcriptase polymerase chain reaction in murine testes treated with busulphan. Finally, using multiple assays, the functional investigation of MTA1 by its specific knockdown was performed in SC-GC co-cultures., Results: SCs were negatively immunolabeled in the tubules with impaired spermatogenesis. Depletion of murine GCs by treatment with busulphan resulted in a dramatic decrease of the MTA1 transcripts level in the isolated SCs on the 15th day of treatment and thereafter had totally abolished MTA1 expression by the 30th day of treatment, respectively. The addition of isolated round spermatids into SC culture could partially elevate MTA1 expression in the latter. Furthermore, MTA1 is crucial to maintain the GC nursery function and normal anchoring junction formation in SCs because ablation of MTA1 by siRNA induced extensive defects of genes related to SC homeostasis., Conclusion: We propose a novel role for SC-expressing MTA1, which is determined by the presence of surrounding GCs, in mediating the crosstalk between SCs and GCs by influencing a broad spectrum of gene changes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The meiotic program initiation pathway genes (CYP26B1, NANOS1 and STRA8) have been proposed to play key roles in spermatogenesis., Objective: To elucidate the exact role of the genetic variants of the meiosis initiation genes in spermatogenesis, we genotyped the potential functional genetic variants of CYP26B1, NANOS1 and STRA8 genes, and evaluated their effects on spermatogenesis in our study population., Design, Setting, and Participants: In this study, all subjects were volunteers from the affiliated hospitals of Nanjing Medical University between March 2004 and July 2009 (NJMU Infertile Study). Total 719 idiopathic infertile cases were recruited and divided into three groups according to WHO semen parameters: 201 azoospermia patients (no sperm in the ejaculate even after centrifugation), 155 oligozoospermia patients (sperm counts <20×10(6)/ml) and 363 infertility/normozoospermia subjects (sperm counts >20×10(6)/ml). The control group consisted of 383 subjects with normal semen parameters, all of which had fathered at least one child without assisted reproductive technologies., Measurements: Eight single nucleotide polymorphisms (SNPs) in CYP26B1, NANOS1 and STRA8 genes were determined by TaqMan allelic discrimination assay in 719 idiopathic infertile men and 383 healthy controls., Results and Limitations: The genetic variant rs10269148 of STRA8 gene showed higher risk of spermatogenic impairment in the groups of abnormospermia (including azoospermia subgroup and oligozoospermia subgroup) and azoospermia than the controls with odds ratios and 95% confidence intervals of 2.52 (1.29-4.94) and 2.92 (1.41-6.06), respectively (P = 0.006, 0.002 respective). Notably, larger sample size studies and in vivo or in vitro functional studies are needed to substantiate the biological roles of these variants., Conclusions: Our results provided epidemiological evidence supporting the involvement of genetic polymorphisms of the meiotic program initiation genes in modifying the risk of azoospermia and oligozoospermia in a Han-Chinese population.

Objective: To explore the possible mechanisms of spermatogenic arrest in severe oligoasthenoteratozoospermia induced by supernumerary, ring-neocentric 13q12.3 --> 13q22 chromosome and reciprocal deletion., Methods: We performed a genomic-wide high-density oaCGH analysis for a case of oligoasthenoteratozoospermia with abnormal chromosome 13 to characterize the breakpoints of the chromosome involved or the gene deletion caused by the rearrangement. We also conducted a fluorescence in situ hybridization analysis on the germ cells using probes of 13q14/13qter to observe the pairing condition of homologous chromosome 13., Results: We identified by oaCGH analysis a microdeletion of 4 consecutive probes (A&#95;16&#95;P19757882, A&#95;16&#95;P02744617, A&#95;14&#95; P108858 and A&#95;16&#95;P02744687 at chr13q12.3: 27979261 --> 28039191) with 59.93 kb between the FLT1 and POMP genes, with no annotated genes in the deleted region. The signals of 13q14 and 13qter were separated from each other in 90% of all the primary spermatocytes examined, indicating the unpairing of homologous chromosome 13 or synapse failure., Conclusion: Chromosomal rearrangement-induced spermatogenesis failure is caused by the unpairing of the homologous chromosomes involved in the first meiotic division of germ cells.

Background: Male infertility is an increasing problem in all domestic species including man. Localization and identification of genes involved in defects causing male infertility provide valuable information of specific events in sperm development. Sperm development is a complex process, where diploid spermatogonia develop into haploid, highly specialized spermatozoa. Correct expression and function of various genes and their protein products are required for production of fertile sperm. We have identified an infertility defect in Finnish Yorkshire boars caused by spermatogenic arrest. The aim of this study was to locate the disease associated region using genome wide screen with the PorcineSNP60 Beadchip and identify the causal mutation by candidate gene approach., Results: In the Finnish Yorkshire pig population the spermatogenic arrest (SA) defect appears to be of genetic origin and causes severe degeneration of germ cells and total absence of spermatozoa. Genome wide scan with the PorcineSNP60 Beadchip localized the SA defect to porcine chromosome 12 in a 2 Mbp region. Sequencing of a candidate gene Tex14 revealed a 51 bp insertion within exon 27, which caused differential splicing of the exon and created a premature translation stop codon. The expression of Tex14 was markedly down regulated in the testis of a SA affected boar compared to control boars and no protein product was identified by Western blotting. The SA insertion sequence was also found within intron 27 in all analyzed animals, thus the insertion appears to be a possible duplication event., Conclusion: In this study we report the identification of a causal mutation for infertility caused by spermatogenic arrest at an early meiotic phase. Our results highlight the role of TEX14 specifically in spermatogenesis and the importance of specific genomic remodeling events as causes for inherited defects.

DNA methylation events during spermatogenesis have important implications for gamete integrity and transmission of epigenetic information to the next generation. However, the role of DNA methyltransferases in the disorders of human spermatogenesis has not been elucidated. The aim of the present study was to evaluate the expression of DNMT3B, crucial for full germ cell methylation, in testicular germ cells of patients with spermatogenic arrest and to determine whether or not there is an association with the global methylation status. In order to determine the DNMTs expression status at various stages of spermatogenesis, immunohistochemical localization was performed on 16 fertile controls having normal spermatogenesis and 11 patients with bilateral spermatogenic arrest. DNMT3B was expressed in most of the germ cell types in both controls and patients with bilateral spermatogenic arrest. The number of DNMT3B positive preleptotene/zygotene cells and pachytene spermatocytes was significantly lower in patients with bilateral arrest. However, evaluation of 5-methylcytosine, a global methylation marker, in the few matured germ cells of these patients did not reveal altered methylation. In conclusion, the global methylation status of germ cells is not affected by spermatogenic defects in spite of aberrant DNMT3B expression indicating the necessity of proper methylation for full spermatogenesis., (© The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.)

Purpose: There is a paucity of data characterizing infertile men with maturation arrest. We hypothesized that men with early stage maturation arrest could be clinically distinguished from men with late maturation arrest and would have worse reproductive outcomes., Materials and Methods: We retrospectively reviewed the records of all patients with nonobstructive azoospermia and cryptozoospermia who underwent testis mapping and sperm extraction from 2002 to 2009 and for whom histopathological findings were available. Patients had uniform maturation arrest if multiple biopsies revealed maturation arrest at the spermatogonia/spermatocyte (early maturation arrest) or the spermatid (late maturation arrest) stage. Clinical parameters and pregnancy outcomes of in vitro fertilization/intracytoplasmic sperm injection were examined. Statistical analysis consisted of univariate and multivariate analysis., Results: Uniform maturation arrest was identified in 49 of 219 men (22.3%) undergoing testicular sperm extraction. On multivariate analysis men with maturation arrest had significantly larger testes (p=0.01), decreased follicle-stimulating hormone (p=0.05) and more detectable genetic abnormalities (p=0.01) than men with other histopathological conditions. Men with late maturation arrest had decreased follicle-stimulating hormone (p=0.02), increased testosterone (p=0.03) and a higher sperm retrieval rate at testicular sperm extraction (p=0.01) than men with early maturation arrest. Predictors of successful sperm retrieval were larger testes, cryptozoospermia, late maturation arrest and hypospermatogenesis (each p≤0.05). Pregnancy outcomes for men with maturation arrest were not significantly different from those for men with other histopathological conditions., Conclusions: Maturation arrest is a common, diverse histopathological subtype of severe male infertility. Compared to men with late maturation arrest those with early maturation arrest have increased follicle-stimulating hormone, decreased testosterone and a decreased probability of mature spermatozoa. In vitro fertilization/intracytoplasmic sperm injection outcomes were similar when spermatozoa were discovered during testicular sperm extraction., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Working Hypothesis: Mutations in the CYP2R1 gene, highly expressed in the testis and encoding vitamin D 25-hydroxylase, result in a vitamin D deficiency and a defective calcium homeostasis leading to rickets., Objective: Our aim was to investigate CYP2R1 expression in pathological testis samples and relate this to vitamin D metabolism in testiculopathic patients. DESIGN, PATIENTS, SETTING: Testis samples for in vitro study and 98 young men were transversally evaluated at Padova's Center for Male Gamete Cryopreservation., Methods: CYP2R1 mRNA expression and protein production were evaluated by quantitative RT-PCR, Western blot analysis, and immunofluorescence. Hormonal and bone-marker levels, and bone densitometry by dual-energy x-ray absorptiometry, were determined in patients with Sertoli-cell-only syndrome and severe hypospermatogenesis., Results: We found a lower gene and protein expression of CYP2R1 in samples with hypospermatogenesis and Sertoli-cell-only syndrome (P < 0.05) and a colocalization with INSL-3, a Leydig cell marker, at immunofluorescence. In all testiculopathic patients 25-hydroxyvitamin D levels were significantly lower and PTH levels higher compared to controls (P < 0.05). Furthermore, testiculopathic patients showed osteopenia and osteoporosis despite normal testosterone levels compared with controls both with increased bone-marker levels and altered dual-energy x-ray absorptiometry in the femoral neck and lumbar spine (for all parameters, P < 0.05)., Conclusions: Our data show an association between testiculopathy and alteration of the bone status, despite unvaried androgen and estrogen levels and no other evident cause of vitamin D reduction. Further studies in larger cohorts are needed to confirm our results.

The primary aim of this study was to gain more insight into maturation arrest of spermatogenesis (MA) and its relationship with mutations in genes essential for meiosis. The study also investigated the possibility that mutations in human meiosis genes cause a milder phenotype and that, in such cases, meiosis could potentially be completed with the production of mature germ cells having an abnormal chromosomal constitution causing miscarriage. Among 40 patients with MA, five changes were observed that also predicted alterations at the amino acid level. However, since these changes were also present in men with normozoospermia in equal frequencies, it was assumed that these changes are single nucleotide polymorphisms. Among 46 patients with recurrent miscarriages, two additional changes were detected predicting an alteration at the amino acid level. One change was detected in controls. However, the second heterozygous change, detected in a conserved functional domain of the SYCP3 gene, was absent in >200 controls. These preliminary results stress the need to further investigate the relationship between abnormalities in meiosis genes and the formation of gametes with abnormal chromosomal constitution. More research is also necessary to determine the impact and frequency of such changes before implementing mutation screening in genetic counselling., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: To investigate the ultrasonographic features of epididymides in congenital obstructive azoospermia (COA) and acquired obstructive azoospermia (AOA)., Methods: A total of 211 infertile men with obstructive azoospermia were observed by scrotal ultrasonography, and the features of the epididymal ultrasonograms were compared between COA and AOA., Results: COA exhibited significantly higher rates of ectasia in the epididymal head, cord-like changes, abrupt tapering and absence of the epididymal body and tail than AOA (P < 0.05), while AOA showed markedly higher rates of epididymal body and tail duct ectasia and epididymal inflammatory mass than COA (P < 0.01). Tubular ectasia of the epididymal duct in the head, body and tail were markedly higher in the COA (14 [5.9%], 41 [17.2%] and 20 [8.4%] cases in 236 epididymides) than in the AOA (P < 0.05). Retiform ectasia were markedly higher in the AOA (119 [64.0%], 142 [76.3%] and 109 [58.6%] cases in 186 epididymides) than in the COA (P < 0.05), with statistically significant differences between the two groups (P < 0.05). Ultrasonographically, the epididymides of the COA patients were characterized by irregular ectasia of the epididymal tube with decreased and unclear wall echoes (P < 0.05), and those of the AOA patients by regular ectasia with enhanced wall echoes (P < 0.01)., Conclusion: The ultrasonographic epididymal features of COA are obviously different from those of AOA, which is of important clinical application value for distinguishing the two conditions from each other.

Guinea pig testicles were extracted with acetic acid; the extract was purified by removing material in consecutive precipitations with 30 per cent saturated ammonium-sulfate, trichloracetic acid, and chloroform. The solution so purified, when administered with complete adjuvants, was highly active in inducing impairment of spermatogenesis in guinea pigs. The activity resisted autoclaving at 15 pounds' pressure for 20 minutes, proteolytic enzymes, and formamide. Anaphylactic shock and cutaneous reaction to the purified homologous extract occurred in guinea pigs sensitized by the extract combined with adjuvants. For the production of aspermatogenesis it was essential to incorporate killed mycobacteria into the water-in-oil emulsion containing the antigen; but anaphylactic sensitization did not require the presence of mycobacteria.