Pathogenesis Of Non-obstructive Azoospermia Research Articles

An integrated approach for improving clinical management of non-obstructive azoospermia.

Non-obstructive azoospermia is the most severe form of male infertility. A testicular biopsy is required for the diagnosis of non-obstructive azoospermia, and the causal factors for non-obstructive azoospermia remain unknown. To reduce the risk of multiple biopsies and identify factors that contribute to non-obstructive azoospermia, we proposed an integrated approach for the preoperative diagnosis and clinical management of non-obstructive azoospermia by applying the chromosome-spreading technique and whole-exome sequencing. Between July 2020 and December 2022, after ruling out definitive obstructive azoospermia and non-obstructive azoospermia patients with testicular volume<6mL, 20 patients with non-obstructive azoospermia who underwent preoperative testicular diagnostic biopsy using testicular sperm aspiration were subjected to retrospective analysis. Microscopic examination identified four patients with sperm cells, and 16 without sperm cells. Routine pathological analysis classified one patient as normal spermatogenesis, three as hypospermatogenesis, five as maturation arrest, nine as Sertoli cell-only, and two as unable to judge. With chromosome-spreading technology using routine cell suspension samples for microscopic examination, 18 patient diagnoses were validated, and two patients without a definitive diagnosis were supplemented. Detection of the Y chromosome and a well-organized whole-exome sequencing analysis revealed potential genetic factors. The full use of testicular biopsy is beneficial for the diagnosis of azoospermia, as it avoids the risk of multiple biopsies. Moreover, in combination with whole-exome sequencing, clinicians can obtain more information regarding the pathogenesis of non-obstructive azoospermia, which may guide treatment.

Identifying potential biomarkers for non-obstructive azoospermia using WGCNA and machine learning algorithms.

The cause and mechanism of non-obstructive azoospermia (NOA) is complicated; therefore, an effective therapy strategy is yet to be developed. This study aimed to analyse the pathogenesis of NOA at the molecular biological level and to identify the core regulatory genes, which could be utilised as potential biomarkers. Three NOA microarray datasets (GSE45885, GSE108886, and GSE145467) were collected from the GEO database and merged into training sets; a further dataset (GSE45887) was then defined as the validation set. Differential gene analysis, consensus cluster analysis, and WGCNA were used to identify preliminary signature genes; then, enrichment analysis was applied to these previously screened signature genes. Next, 4 machine learning algorithms (RF, SVM, GLM, and XGB) were used to detect potential biomarkers that are most closely associated with NOA. Finally, a diagnostic model was constructed from these potential biomarkers and visualised as a nomogram. The differential expression and predictive reliability of the biomarkers were confirmed using the validation set. Furthermore, the competing endogenous RNA network was constructed to identify the regulatory mechanisms of potential biomarkers; further, the CIBERSORT algorithm was used to calculate immune infiltration status among the samples. A total of 215 differentially expressed genes (DEGs) were identified between NOA and control groups (27 upregulated and 188 downregulated genes). The WGCNA results identified 1123 genes in the MEblue module as target genes that are highly correlated with NOA positivity. The NOA samples were divided into 2 clusters using consensus clustering; further, 1027 genes in the MEblue module, which were screened by WGCNA, were considered to be target genes that are highly correlated with NOA classification. The 129 overlapping genes were then established as signature genes. The XGB algorithm that had the maximum AUC value (AUC=0.946) and the minimum residual value was used to further screen the signature genes. IL20RB, C9orf117, HILS1, PAOX, and DZIP1 were identified as potential NOA biomarkers. This 5 biomarker model had the highest AUC value, of up to 0.982, compared to other single biomarker models; additionally, the results of this biomarker model were verified in the validation set. As IL20RB, C9orf117, HILS1, PAOX, and DZIP1 have been determined to possess the strongest association with NOA, these five genes could be used as potential therapeutic targets for NOA patients. Furthermore, the model constructed using these five genes, which possessed the highest diagnostic accuracy, may be an effective biomarker model that warrants further experimental validation.

Clinical management of nonobstructive azoospermia: An update.

Approximately 1% of the general male population has azoospermia, and nonobstructive azoospermia accounts for the majority of cases. The causes vary widely, including chromosomal and genetic abnormalities, varicocele, drug-induced causes, and gonadotropin deficiency; however, the cause is often unknown. In azoospermia caused by hypogonadotropic hypogonadism, gonadotropin replacement therapy can be expected to produce sperm in the ejaculate. In some cases, upfront varicocelectomy for nonobstructive azoospermia with varicocele may result in the appearance of ejaculated spermatozoa; however, the appropriate indication should be selected. Each guideline recommends microdissection testicular sperm extraction for nonobstructive azoospermia in terms of successful sperm retrieval and avoidance of complications. Sperm retrieval rates generally ranged from 20% to 70% but vary depending on the causative disease. Various attempts have been made to predict sperm retrieval and improve sperm retrieval rates; however, the evidence is insufficient. Further evidence accumulation is needed for salvage treatment in cases of failed sperm retrieval. In Japan, there is inadequate provision on the right to know the origin of children born from artificial insemination of donated sperm and the rights of sperm donors, as well as information on unrelated family members, and the development of these systems is challenging. In the future, it is hoped that the pathogenesis of nonobstructive azoospermia with an unknown cause will be elucidated and that technology for omics technologies, human spermatogenesis using pluripotent cells, and organ culture methods will be developed.

P-021 A novel SNP in HUWE1 promoter confers increased risk of NOA by affecting the RA/RARα pathway in Chinese individuals

Abstract Study question The present study aimed to investigate the role of HUWE1 in non-obstructive azoospermia (NOA) etiology and germ cell differentiation. Summary answer An SNP mutation in HUWE1 promoter region downregulates HUWE1 expression in NOA patients, the genetic polymorphisms of HUWE1 are related to the pathogenesis of NOA. What is known already HUWE1, a ubiquitin ligase, is crucial to the development of male reproductive system and embryonic development. However, the specific role of HUWE1 in regulating germ cell differentiation is unclear, and clinical evidence for the relationship between HUWE1 and the pathogenesis of male infertility is lacking. Study design, size, duration 190 NOA Chinese patients were recruited and analyzed single nucleotide polymorphisms (SNPs) in HUWE1 gene. Participants/materials, setting, methods Regulation of HUWE1 gene expression by RARα factor was evaluated by performing ChIP assay, EMSA, and siRNA-mediated RARα knockdown. We used C18-4 cells to determine whether HUWE1 participated in RA-mediated RARα signaling and performed luciferase assay, CCK-8 assay, immunofluorescence staining, RT-qPCR, and western blot analysis. The expression level of HUWE1 and RARa was evaluated in clinical samples obtained from NOA or obstructive azoospermia (OA) patients using RT-qPCR. Main results and the role of chance We explored the mechanism by which polymorphism of the HUWE1 promoter leads to spermatogenesis disorders, and focused on the identification of mutations in the SNP site of the HUWE1 regulatory sequence among patients with NOA, which may influence HUWE1 expression. The results of in vitro experiments showed that RARα binds to the HUWE1 promoter region and regulates its expression. However, the regulatory effect of RARα weakened following introduction of the mutation into the HUWE1 regulatory sequence. The expression of spermatogonial differentiation-related gene STRA8 was impaired, and γH2AX foci accumulated following inhibition of HUWE1 gene expression using siRARα. Finally, to determine whether HUWE1 expression was important in patients with NOA, its expression was verified using testicular puncture samples collected from patients with NOA and the results revealed that HUWE1 expression was abnormal (P &amp;lt; 0.01). In addition, RARα expression was significantly downregulated (P &amp;lt; 0.001). Overall, our result support the hypothesis that HUWE1 expression is induced by the RA/RARα signaling pathway and it inhibit γH2AX foci accumulation, thereby considerably promoting sperm differentiation and meiotic prophase. Limitations, reasons for caution The effect of SNPs in HUWE1 on male infertility is further studied in a wider population. Wider implications of the findings Our study of HUWE1 and RA/RARα signaling pathway provides new information on the regulatory mechanism of spermiogenesis, suggesting that SNPs in HUWE1 promoter are etiological factors in patients with NOA and providing a rationale for further drug development to target HUWE1 in these patients. Trial registration number the National Key Research and Development Program of China (2018YFC1003603)

Characterization of Non-Obstructive Azoospermia in Men Using Gut Microbial Profiling.

(1) Background: Non-obstructive azoospermia (NOA) is a complex multifactorial disease and the causes of most NOA cases remain unknown. (2) Methods: Here, we performed comprehensive clinical analyses and gut microbial profiling using shotgun metagenomic sequencing in patients with NOA and control individuals. (3) Results: The gut microbial alpha and beta diversity significantly differed between patients with NOA and controls. Several microbial strains, including Bacteroides vulgatus and Streptococcus thermophilus, were significantly more abundant in the NOA group, whereas Bacteroides thetaiotaomicron and Parabacteroides sp. CT06 were enriched in the control group. Moreover, functional pathway analysis suggested that the altered microbiota in NOA suppressed the carbohydrate metabolism pathway, while amino acid metabolism and methane metabolism pathways were enhanced. We observed that the differential microbial species, such as Acinetobacter johnsonii, had a strong correlation with clinical parameters, including age, body mass index, testosterone, and follicle-stimulating hormone. Communication and interplay among microbial genera were significantly increased in NOA than in the control group. (4) Conclusions: Altered microbial composition and functional pathways in the NOA group were revealed, which highlight the utility of gut microbiota in understanding microbiota-related pathogenesis of NOA and might be helpful to the clinical management of NOA.

OIP5 Interacts with NCK2 to Mediate Human Spermatogonial Stem Cell Self-Renewal and Apoptosis through Cell Cyclins and Cycle Progression and Its Abnormality Is Correlated with Male Infertility

Spermatogonial stem cells (SSCs) have important applications in both reproduction and regenerative medicine. Nevertheless, specific genes and signaling transduction pathways in mediating fate decisions of human SSCs remain elusive. Here, we have demonstrated for the first time that OIP5 (Opa interacting protein 5) controlled the self-renewal and apoptosis of human SSCs. RNA sequencing identified that NCK2 was a target for OIP5 in human SSCs, and interestingly, OIP5 could interact with NCK2 as shown by Co-IP (co-immunoprecipitation), IP-MS (mass spectrometry), and GST pulldown assays. NCK2 silencing decreased human SSC proliferation and DNA synthesis but enhanced their apoptosis. Notably, NCK2 knockdown reversed the influence of OIP5 overexpression on human SSCs. Moreover, OIP5 inhibition decreased the numbers of human SSCs at S and G2/M phases, while the levels of numerous cell cycle proteins, including cyclins A2, B1, D1, E1 and H, especially cyclin D1, were remarkably reduced. Significantly, whole-exome sequencing of 777 patients with nonobstructive azoospermia (NOA) revealed 54 single-nucleotide polymorphism mutations of the OIP5 gene (6.95%), while the level of OIP5 protein was obviously lower in testes of NOA patients compared to fertile men. Collectively, these results implicate that OIP5 interacts with NCK2 to modulate human SSC self-renewal and apoptosis via cell cyclins and cell cycle progression and that its mutation and/or lower expression is correlated with azoospermia. As such, this study offers novel insights into molecular mechanisms underlying the fate determinations of human SSCs and the pathogenesis of NOA, and it provides new targets for treating male infertility.

The gene regulatory role of non-coding RNAs in non-obstructive azoospermia

Non-coding RNAs are classified as small non-coding RNAs, long non-coding RNAs and circular RNAs, which are involved in a variety of biological processes, including cell differentiation, proliferation, apoptosis and pathological conditions of various diseases. Many studies have shown that non-coding RNAs are related to spermatogenesis, maturation, apoptosis, function, etc. In addition, the expression of non-coding RNAs in testicular tissue and semen of patients with non-obstructive azoospermia was different. However, the role of non-coding RNAs in the pathogenesis of non-obstructive azoospermia has not been fully elucidated, and the role of non-coding RNAs in non-obstructive azoospermia is rarely reviewed. Here we summarize the research progress of non-coding RNAs in the pathogenesis of non-obstructive azoospermia.

Identification and Potential Value of Candidate Genes in Patients With Non-obstructive Azoospermia

ObjectivesTo explore the candidate genes involved in the pathogenesis of non-obstructive azoospermia (NOA) using bioinformatics analysis and experimental verification. MethodsThe gene expression profiles (GSE9210) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified. We performed function enrichment analyses, constructed protein–protein interaction (PPI) network and identified hub genes. Further, the miRNA-hub genes regulatory network was constructed. Finally, the expression level of CEP55 was verified using RT-qPCR and Western blot, and its diagnostic value was analyzed by the receiver operating characteristic (ROC) curve. Results626 DEGs were identified, including 11 upregulated and 615 downregulated genes. Function enrichment analyses showed that these DEGs were significantly enriched in spermatogenesis, fertilization, meiotic cell cycle, flagellated sperm motility, sperm capacitation, spermatid nucleus differentiation and male meiotic nuclear differentiation. The top 10 hub genes were identified including CCNB2, BUB1, TOP2A, BIRC5, CENPF, PBK, NCAPG, DLGAP5, NUF2 and CEP55. In the miRNAs prediction, the hsa-miRNA-449a, hsa-miRNA-34c-5p and hsa-miRNA-34b-5p may be implicated in NOA. In the validation stage, the expression level of CEP55 was significantly decreased in patients with NOA compared to patients with OA. ROC analysis showed that CEP55 had a good diagnostic value for NOA and the combination of CEP55, FSH and mean testicular volume enhanced the prediction performance. ConclusionsThis study identified key genes associated with NOA and their biological functions. Furthermore, CEP55 might play an important role in the pathogenesis of NOA, which will provide novel insights into the targeting therapy of NOA.

Infertility network and hub genes for nonobstructive azoospermia utilizing integrative analysis.

Non-obstructive azoospermia (NOA) is the most severe form of male infertility owing to the absence of sperm during ejaculation as a result of failed spermatogenesis. The molecular mechanisms of NOA have not been well studied. Here, we revealed the dysregulated differentially expressed genes in NOA and related signaling pathways or biological processes. Cluster features of biological processes include spermatogenesis, fertilization, cilium movement, penetration of zona pellucida, sperm chromatin condensation, and being significantly enriched metabolic pathways in proximal tubule bicarbonate reclamation, aldosterone synthesis and secretion, glycolysis and glycogenesis pathways in NOA using Gene Ontology analysis and pathway enrichment analysis. The NOA gene co-expression network was constructed by weighted gene co-expression network analysis to identify the hub genes (CHD5 and SPTBN2). In addition, we used another Gene Expression Omnibus dataset (GSE45887) to validate these hub genes. Furthermore, we used the Seurat package to classify testicular tissue cells from NOA patients and to characterize the differential expression of hub genes in different cell types from different adult males based on the scRNA-seq dataset (GSE106487). These results provide new insights into the pathogenesis of NOA. Of particular note, CHD5 and SPTBN2 may be potential biomarkers for the diagnosis and treatment of NOA.

MicroRNA expression profiles in the seminal plasma of nonobstructive azoospermia patients with different histopathologic patterns

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. Sequencing and validation using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Reproductive center and research institute. 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. None. 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. 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. 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.

Testicular biopsies microarray analysis reveals circRNAs are involved in the pathogenesis of non-obstructive azoospermia.

Circular RNAs (circRNAs) have been reported to be involved in many diseases. But there is no report on circRNAs in non-obstructive azoospermia (NOA). The purpose of this paper is to explore the circular RNA expression profile and potential functions of circRNAs in NOA patients. We first preformed circRNA expression profiling analysis using a circRNA microarray in testicular samples from NOA and obstructive azoospermia (OA) patients. CircRNAs were validated by qRT-PCR. Bioinformatics analysis were used to construct the ceRNA network. GO and KEGG enrichment analysis were performed by using DAVID. Microarray analysis identified 82 differentially expressed circRNAs in NOA specimens. The differential expression of hsa_circRNA_402130, hsa_circRNA_072697, hsa_circRNA_030050, hsa_circRNA_100812 and hsa_circRNA_406168 was confirmed by qRT-PCR. Enrichment analysis revealed the association of hsa_circRNA_402130 and hsa_circRNA_072697 with multiple signaling pathways. The data indicated that circRNAs were significantly dysregulated in NOA specimens and might involve in the pathogenesis of NOA.

Epigenetic Inactivation of SOX30 Is Associated with Male Infertility and Offers a Therapy Target for Non-obstructive Azoospermia

Non-obstructive azoospermia (NOA) is the most severe form of male infertility. However, the etiology of NOA is largely unknown, resulting in a lack of clinical treatments. Here, we performed a comparative genome-wide profiling of DNA methylation and identified SOX30 as the most notably hyper-methylated gene at promoter in testicular tissues from NOA patients. This hyper-methylation at promoter of SOX30 directly causes its silencing of expression in NOA. The reduced levels of SOX30 expression are correlated with severity of NOA disease. Deletion of Sox30 in mice uniquely impairs testis development and spermatogenesis with complete absence of spermatozoa in testes leading to male infertility, but does not influence ovary development and female fertility. The pathology and testicular size of Sox30 null mice highly simulate those of NOA patients. Re-expression of Sox30 in Sox30 null mice at adult age reverses the pathological damage of testis and restores the spermatogenesis. The re-presented spermatozoa after re-expression of Sox30 in Sox30 null mice have the ability to start a pregnancy. Moreover, the male offspring of Sox30 re-expression Sox30 null mice still can father children, and these male offspring and their children can live normally more than 1 year without significant difference of physical appearance compared with wild-type mice. In summary, methylated inactivation of SOX30 uniquely impairs spermatogenesis, probably causing NOA disease, and re-expression of SOX30 can successfully restore the spermatogenesis and actual fertility. This study advances our understanding of the pathogenesis of NOA, offering a promising therapy target for NOA disease.

Human mitochondrial DNA haplogroup M8a influences the penetrance of m.8684C>T in Han Chinese men with non-obstructive azoospermia.

What is the role of mitochondrial DNA (mtDNA) in the pathogenesis of non-obstructive azoospermia (NOA)? mtDNA genome sequencing followed by an independent population validation were performed in 628 NOA cases and 584 healthy controls. Antioxidant capacity of serum was evaluated in 54 randomly selected cases out of 536 and 49 out of 489 controls. In the screening stage, 13 mtDNA haplogroups (hg) were ascertained, and 10 susceptible variants were observed. In the validation stage, hg M8* in individuals was found to be associated with increased risk of NOA [odds ratio (OR) 2.61, 95% confidence interval (CI) 1.47-4.61] (P=0.001). Unexpectedly, the frequency of m.8684C>T, the defining marker for hg M8a, was also higher in NOA (OR 4.14, 95% CI 1.56-11.03) (P=0.002). Subsequently, the frequency distributions were compared among the sub-hg of hg M8* (including hg M8a, C and Z) and, intriguingly, no significance was found in hg C and Z. Additionally, the level of total antioxidant capacity was significantly decreased (P<0.05) compared with the control group. hg M8a background in general played an active role in the penetrance of 8684C>T in NOA, and mtDNA genetic variants (causing low antioxidant levels) might increase mtDNA damage and impair normal spermatogenesis.

MOLECULAR GENETIC AND LABORATORY FINDINGS IN INFERTILE MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA

Objectives:To study therelationship between Y-chromosome microdeletions withclinicaland laboratory findings in infertile men with non-obstructive azoospermia (NOA). Design: Cross-sectionalstudy. Patients: Infertile men withnon-obstructive azoospermia (n = 146). Methods: Clinical evaluation and scrotal colour Doppler ultrasonography were evaluated. Standard semen analysis andserum levels of hormones(FSH, LH, total testosterone and prolactin) were performed. Multiplex PCR was done for detection of Y chromosome microdeletions. Results:AZF deletions were detected in 9.59% of azoospermic men.CompleteAZFc was detected in 2.05% of azoospermic men.Partial AZFc deletions were found in 7.5% of azoospermic men, with gr/gr deletion in 6.85% and b2/b3 deletion in 0.69%.There was no significant difference between patients with AZF deletions and azoospermic men without deletions as regards testicular volume and serum levels of reproductive hormones. Conclusions:Microdeletions of Y chromosome may play a role in pathogenesis of NOA. The testicular volumes as well as levels of reproductive hormoneswere not correlated with the finding of Y chromosome microdeletions.

Y-Chromosome Microdeletion Analysis in Infertile Men from Upper Egypt

Objectives:To study the prevalence and patterns of Y-chromosome microdeletions in infertile men from Upper Egypt and to determine the relationship between Y-chromosome microdeletions with clinicalandlaboratoryfindings in these patients. Design: Cross-sectionalstudy. Patients: Infertile men (n = 210) and a control group of fertile men with normal semen analysis (n = 30). Methods: Clinical evaluation, standard semen analysis according to the WHO guidelines (WHO, 2010)andserumlevels of reproductive hormones were evaluated. Multiplex PCR was done for detection of Y chromosome microdeletions. Results:AZF deletions were present in 7.14% of infertile men (9.59% in azoospermic and 1.56% in oligo-zoospermic men) with no deletions in the fertile normo-zoospericmen.CompleteAZFc was the detected in 2.05% of azoospermicmen.PartialAZFc deletions were found in 5.7% of infertile men, with gr/gr deletion in 5.24% and b2/b3 deletion in 0.48%.There was no significant difference between patients with AZF deletions and azoospermic men without deletions as regards testicular volume and serum levels of FSH, LH, testosterone and prolactin. Conclusions:Microdeletions of Y chromosome may play a role in pathogenesis of non-obstructive azoospermia. The testicular volumes as well as levels of FSH, LH, testosterone and prolactin were not correlated with the finding of Y chromosome microdeletions.

X chromosome-wide identification of SNVs in microRNA genes and non-obstructive azoospermia risk in Han Chinese population.

Human X chromosome has higher densities of microRNAs (miRNAs) compared to the average densities on autosomes. Given that numbers of X-linked miRNAs can escape from meiotic sex chromosome inactivation (MSCI) silencing, it is proposed that X-linked miRNAs may play critical roles in the process of spermatogenesis. To test the hypothesis, we performed DNA capture sequencing of human X-linked miRNAs, which was followed by a two-stage case-control study to identify the non-obstructive azoospermia (NOA) related single nucleotide variants (SNVs) in 1107 NOA cases and 1191 fertile healthy controls. Eventually, we found rs5951785, located near hsa-miRNA-506/507, increased the risk of NOA, while rs1447393, near hsa-miRNA-510, decreased the risk of NOA. Functional analysis revealed that rs5951785 significantly inhibited cell proliferation and induced cell apoptosis. Taken together, our results demonstrated that X-linked miRNAs played important roles in the pathogenesis of NOA.

MP70-01 IFT140 IS A NOVEL CANDIDATE GENE FOR IMPAIRED SPERMATOGENESIS: IDENTIFICATION BY WHOLE EXOME SEQUENCING AND VALIDATION WITH SANGER SEQUENCING

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology1 Apr 2016MP70-01 IFT140 IS A NOVEL CANDIDATE GENE FOR IMPAIRED SPERMATOGENESIS: IDENTIFICATION BY WHOLE EXOME SEQUENCING AND VALIDATION WITH SANGER SEQUENCING Amin Herati, Peter Butler, cenk Cengiz, Matthew Bainbridge, James Lupski, Richard Gibbs, Larry Lipshultz, and Dolores Lamb Amin HeratiAmin Herati More articles by this author , Peter ButlerPeter Butler More articles by this author , cenk Cengizcenk Cengiz More articles by this author , Matthew BainbridgeMatthew Bainbridge More articles by this author , James LupskiJames Lupski More articles by this author , Richard GibbsRichard Gibbs More articles by this author , Larry LipshultzLarry Lipshultz More articles by this author , and Dolores LambDolores Lamb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1426AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES One third of male factor infertility cases are idiopathic. Many of these cases are likely caused by genetic factors. Non-obstructive azoospermia (NOA) is characterized by the absence of sperm in the ejaculate secondary to the lack of sperm production in the testicle. Advances in genetic and genomic analysis have allowed for the rapid identification of candidate genes in NOA men. The aim of our study was to identify novel variants implicated in the pathogenesis of NOA through whole-exome sequencing (WES). METHODS Homozygosity mapping with whole-exome sequencing was performed using the NimbleGen SeqCap EZ Exome V3 sample preparation kit and the Illumina HiSeq 2000 next-generation sequencing platform to sequence the exomes of two siblings with NOA from a consanguineous family. Genetic mutations that passed filter criteria were then validated using Sanger sequencing. We identified one candidate gene, Intraflagellar Transport Protein 140 (IFT140), based on its expression pattern in testes according to the Human Protein Atlas, as well the mutation′s damage prediction based on two online bioinformatic resources. We subsequently performed Sanger sequencing on 95 NOA men and 26 fertile men, all of whom were treated at our institution. RESULTS WES identified 442 variants in the index patients. Four (0.9%) of these variants passed filter criteria, one of which was a six-nucleotide deletion (HG38, chr16:1,519,961 TCTTGGC>T) in exon 22 of IFT140. Family segregation showed that this mutation was homozygous in the two brothers who underwent WES, as well as in a third brother with NOA and their sister. This mutation was not identified in the 1000 Genome Project and was predicted to be deleterious by the bioinformatics tools, MutationTaster and PROVEAN. Sanger sequencing validated the mutation in this family and identified it as a heterozygous mutation in 1 (1.1%) of the 95 NOA men and none of the 26 fertile controls in our cohort. Pathologic correlation of this unrelated patient showed hypospermatogenesis with a clinical history significant for three failed cycles of in vitro fertilization (IVF). CONCLUSIONS We have identified through WES a small indel mutation in IFT-140 as a novel, disease-causing variant. This mutation was detected in our cohort of NOA in addition to the index family assessed with a prevalence of 1.1%. Further studies are required to determine the role of IFT140 in spermatogenesis. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e905-e906 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Amin Herati More articles by this author Peter Butler More articles by this author cenk Cengiz More articles by this author Matthew Bainbridge More articles by this author James Lupski More articles by this author Richard Gibbs More articles by this author Larry Lipshultz More articles by this author Dolores Lamb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Genome-wide microRNA expression profiling in idiopathic non-obstructive azoospermia: significant up-regulation of miR-141, miR-429 and miR-7-1-3p

What is the profile of miRNAs in seminal plasma of patients with non-obstructive azoospermia (NOA)? miR-141, miR-429 and miR-7-1-3p are significantly increased in seminal plasma of patients with NOA compared with fertile controls. There is currently an urgent need to develop a noninvasive diagnostic test for NOA. Altered microRNA (miRNA) profiles have been proposed as potential biomarkers for the diagnosis of disease states. A total of 200 subjects (n = 100 for NOA, n = 100 for fertile control) were recruited to participate in this study. Recruitment took place from May 2008 to June 2010. We employed a strategy consisting of initial screening by TaqMan Low Density Array then further validation with a TaqMan quantitative RT-PCR assay. Validation of the profiling results was conducted in two independent phases. In addition, the expression of the three validated seminal plasma miRNAs (sp-miRNAs) was examined in testicular tissues of patients with NOA and of fertile controls. Methylation status and functional analyses were also performed for the identified sp-miRNAs. miR-141, miR-429 and miR-7-1-3p were significantly increased in seminal plasma of patients with NOA compared with fertile controls. As sensitive and specific biomarkers, the profiling of these three identified sp-miRNAs provides a novel noninvasive, semen-based test for NOA diagnosis. The methylation status of these sp-miRNAs was inversely associated with their expression patterns. Additionally, we found that Cbl and Tgfβ2 were down-regulated by miR-141, while Rb1 and Pik3r3 were down-regulated by miR-7-1-3p. miRNA expression profile was investigated in seminal plasma samples from only a small number of NOA patients. In future investigations, a larger sample size should be adopted and the functional role of the three sp-miRNAs should be further characterized in animal models. Given that sp-miRNAs show reproducible and stable expression levels, they are potentially novel noninvasive biomarkers for the diagnosis of NOA. We propose that the three sp-miRNAs described above may participate in a methylation-miRNA-gene network related to NOA development. This work provides a foundation for interpretation of miRNA changes associated with pathogenesis of NOA and extends the current understanding of human NOA pathogenesis.

A Genome-wide Association Study Reveals that Variants within the HLA Region Are Associated with Risk for Nonobstructive Azoospermia

A genome-wide association study of Han Chinese subjects was conducted to identify genetic susceptibility loci for nonobstructive azoospermia (NOA). In the discovery stage, 802 azoospermia cases and 1,863 controls were screened for genetic variants in the genome. Promising SNPs were subsequently confirmed in two independent sets of subjects: 818 azoospermia cases and 1,755 controls from northern China, and 606 azoospermia cases and 958 controls from central and southern China. We detected variants at human leukocyte antigen (HLA) regions that were independently associated with NOA (HLA-DRA, rs3129878, p(combine) = 3.70× 10(-16), odds ratio [OR]= 1.37; C6orf10 and BTNL2, rs498422, p(combine) = 2.43× 10(-12), OR = 1.42). These findings provide additional insight into the pathogenesis of NOA.

Genome-wide expression of azoospermia testes demonstrates a specific profile and implicates ART3 in genetic susceptibility.

Infertility affects about one in six couples attempting pregnancy, with the man responsible in approximately half of the cases. Because the pathophysiology underlying azoospermia is not elucidated, most male infertility is diagnosed as idiopathic. Genome-wide gene expression analyses with microarray on testis specimens from 47 non-obstructive azoospermia (NOA) and 11 obstructive azoospermia (OA) patients were performed, and 2,611 transcripts that preferentially included genes relevant to gametogenesis and reproduction according to Gene Ontology classification were found to be differentially expressed. Using a set of 945 of the 2,611 transcripts without missing data, NOA was further categorized into three classes using the non-negative matrix factorization method. Two of the three subclasses were different from the OA group in Johnsen's score, FSH level, and/or LH level, while there were no significant differences between the other subclass and the OA group. In addition, the 52 genes showing high statistical difference between NOA subclasses (p < 0.01 with Tukey's post hoc test) were subjected to allelic association analyses to identify genetic susceptibilities. After two rounds of screening, SNPs of the ADP-ribosyltransferase 3 gene (ART3) were associated with NOA with highest significance with ART3-SNP25 (rs6836703; p = 0.0025) in 442 NOA patients and 475 fertile men. Haplotypes with five SNPs were constructed, and the most common haplotype was found to be under-represented in patients (NOA 26.6% versus control 35.3%, p = 0.000073). Individuals having the most common haplotype showed an elevated level of testosterone, suggesting a protective effect of the haplotype on spermatogenesis. Thus, genome-wide gene expression analyses were used to identify genes involved in the pathogenesis of NOA, and ART3 was subsequently identified as a susceptibility gene for NOA. These findings clarify the molecular pathophysiology of NOA and suggest a novel therapeutic target in the treatment of NOA.