Idiopathic Nonobstructive Azoospermia Patients Research Articles

Identification of risk genes in Chinese nonobstructive azoospermia patients based on whole-exome sequencing.

Nonobstructive azoospermia (NOA) is a severe condition in infertile men, and increasing numbers of causative genes have been identified during the last few decades. Although certain causative genes can explain the presence of NOA in some patients, a proportion of NOA patients remain to be addressed. This study aimed to investigate potential high-risk genes associated with spermatogenesis in idiopathic NOA patients by whole-exome sequencing. Whole-exome sequencing was performed in 46 male patients diagnosed with NOA. First, screening was performed for 119 genes known to be related to male infertility. Next, further screening was performed to determine potential high-risk causative genes for NOA by comparisons with 68 healthy male controls. Finally, risk genes with high/specific expression in the testes were selected and their expression fluctuations during spermatogenesis were graphed. The frequency of cystic fibrosis transmembrane conductance regulator (CFTR) gene pathogenic variant carriers was higher in the NOA patients compared with the healthy controls. Potential risk genes that may be causes of NOA were identified, including seven genes that were highly/specifically expressed in the testes. Four risk genes previously reported to be involved in spermatogenesis (MutS homolog 5 [MSH5], cilia- and flagella-associated protein 54 [CFAP54], MAP7 domain containing 3 [MAP7D3], and coiled-coil domain containing 33 [CCDC33]) and three novel risk genes (coiled-coil domain containing 168 [CCDC168], chromosome 16 open reading frame 96 [C16orf96], and serine protease 48 [PRSS48]) were identified to be highly or specifically expressed in the testes and significantly different in the 46 NOA patients compared with 68 healthy controls. This study on clinical NOA patients provides further evidence for the four previously reported risk genes. The present findings pave the way for further functional investigations and provide candidate risk genes for genetic diagnosis of NOA.

Efficacy of stepwise mini-incision microdissection testicular sperm extraction for nonobstructive azoospermia with varied etiologies.

Stepwise mini-incision microdissection testicular sperm extraction (mTESE) is a procedure that attempts to minimize testicular damage. However, the mini-incision approach may vary in patients with different etiologies. Here, we performed a retrospective analysis of 665 men with nonobstructive azoospermia (NOA) who underwent stepwise mini-incision mTESE (Group 1) and 365 men who underwent standard mTESE (Group 2). The results showed that the operation time (mean ± standard deviation) for patients with successful sperm retrieval in Group 1 (64.0 ± 26.6 min) was significantly shorter than that in Group 2 (80.2 ± 31.3 min), with P <0.001. The total sperm retrieval rate (SRR) was 23.1% in our study, and there was no significant difference between Group 1 and Group 2 ( P >0.05), even when the etiologies of NOA were taken into consideration. The results of consecutive multivariate logistic regression analysis (odds ratio [OR]: 0.57; 95% confidence interval [CI]: 0.38-0.87; P =0.009) and receiver operating characteristic (ROC) analysis (area under the ROC curve [AUC]=0.628) showed that preoperative anti-Müllerian hormone (AMH) level in idiopathic NOA patients was a potential predictor for surgical outcomes after initial three small incisions made in the equatorial region without sperm examined under an operating microscope (Steps 2-4). In conclusion, stepwise mini-incision mTESE is a useful technique for NOA patients, with comparable SRR, less surgical invasiveness, and shorter operation time compared with the standard approach. Low AMH levels may predict successful sperm retrieval in idiopathic patients even after a failed initial mini-incision procedure.

P-012 Investigating the potential role of microRNAs as biomarkers in idiopathic non-obstructive azoospermia: A systematic review and in-silico analysis of the affected pathways

Abstract Study question Is there an association between microRNA profiles in seminal plasma and microRNAs profiles in testicular tissue samples obtained from idiopathic non-obstructive azoospermia (NOA) patients? Summary answer This study identified common alterations in microRNA profiles and gene expression patterns between seminal plasma and testicular tissue samples obtained from NOA patients. What is known already Despite the significant advances in the field of reproductive medicine the exact infertility aetiology remains unidentified regarding 30-40% of infertile men. This fact highlights the need for more accurate and sensitive diagnostic tools and biomarkers, especially regarding the most severe cases of male infertility, such as idiopathic NOA patients. It has been voiced that microRNA profiling of seminal plasma and testicular tissue samples obtained from idiopathic NOA patients could be a powerful tool towards better understanding the underlined pathogenesis and towards developing novel diagnostic and individualized biomarkers. However, a collective analysis of these data has not been performed hitherto. Study design, size, duration A systematic review was performed in PubMed/Medline and Embase up to November 2020. Search strategy included a combination of keywords, namely non-coding RNA OR small non-coding RNA OR microRNA AND non-obstructive azoospermia. Following study selection, original data on altered microRNAs were analyzed aiming to underline differences between microRNA expression profiles in seminal plasma and testicular tissue samples of idiopathic NOA cases. Following this, in-silico analysis was performed to detect commonly affected gene expression pathways. Participants/materials, setting, methods Only full-length original retrospective or prospective human studies were included. Strict inclusion-exclusion criteria were applied. The studied population consisted of idiopathic NOA patients, while the control groups consisted of men with normal semen analysis. In-silico analysis was performed employing a combination of bioinformatic tools, namely the DIANA-TarBase, microT-CDS, the GTEx repository and the KEGG database. Statistical analysis was performed using the R-package-limma. The statistically significant threshold indicating altered gene pathways was set at 0.01 P-value. Main results and the role of chance Five studies were considered eligible, including 382 NOA cases and 412 controls (Finocchi et al., 2020; Song et al., 2017; Wu et al., 2013; Wu et al., 2012; Wang et al., 2011). Two studies co-evaluated the profile of microRNAs in both seminal plasma and testicular tissue samples (Wu et al., 2013; Wu et al., 2012), one study evaluated only testicular tissue (Song et al., 2017) and the other two only seminal plasma (Finocchi et al., 2020 and Wang et al., 2011). Data extraction revealed a total of 14 differentially expressed microRNAs between NOA patients and controls. The following microRNAs were found to be up-regulated in both seminal plasma and testicular tissue samples of NOA cases: hsa-miR-141-3p, hsa-miR-429, hsa-miR-7-1-3p, hsa-miR-19b-3p and hsa-let-7a-5p. The hsa-miR-188-3p was found to be down-regulated in testicular tissue samples. Finally, the following microRNAs were also downregulated only in seminal plasma: hsa-miR-34c-5p, hsa-miR-122-5p, hsa-miR-181a-5p, hsa-miR-146b-5p, hsa-miR-374b-5p, hsa-miR-509-5p, hsa-miR-513a-5p and hsa-miR-34b-3p. Despite the limited number of common microRNAs between seminal plasma and testicular tissue samples, in-silico analysis revealed 34 statistically significant dysregulated gene pathways, regarding both seminal plasma and testicular tissue samples, indicating that idiopathic NOA patients are sharing several common altered molecular mechanisms involved in NOA pathogenesis. Limitations, reasons for caution The limited number of the included studies as well as the small size population characterizing the great majority of them, constitute the main limitations of this systematic review. Moreover, great heterogeneity was observed among the studies regarding the molecular methods employed for microRNA profiling. Wider implications of the findings These collective findings indicate that microRNA profiling in seminal plasma could indeed be raised as a powerful non-invasive tool towards better understanding and diagnosing idiopathic NOA. Larger well-controlled studies employing state-of-the-art microRNAome techniques are needed to validate these conclusions. Moreover, the molecular network of targeted gene pathways also merits investigation. Trial registration number Not Applicable

A Homozygous Loss-of-Function Mutation in MSH5 Abolishes MutSγ Axial Loading and Causes Meiotic Arrest in NOA-Affected Individuals.

Non-obstructive azoospermia (NOA), characterized by spermatogenesis failure and the absence of sperm in ejaculation, is the most severe form of male infertility. However, the etiology and pathology between meiosis-associated monogenic alterations and human NOA remain largely unknown. A homozygous MSH5 mutation (c.1126del) was identified from two idiopathic NOA patients in the consanguineous family. This mutation led to the degradation of MSH5 mRNA and abolished chromosome axial localization of MutSγ in spermatocytes from the affected males. Chromosomal spreading analysis of the patient’s meiotic prophase I revealed that the meiosis progression was arrested at a zygotene-like stage with extensive failure of homologous synapsis and DSB repair. Therefore, our study demonstrates that the MSH5 c.1126del could cause meiotic recombination failure and lead to human infertility, improving the genetic diagnosis of NOA clinically. Furthermore, the study of human spermatocytes elucidates the meiosis defects caused by MSH5 variant, and reveals a conserved and indispensable role of MutSγ in human synapsis and meiotic recombination, which have not previously been well-described.

Potential of testis-derived circular RNAs in seminal plasma to predict the outcome of microdissection testicular sperm extraction in patients with idiopathic non-obstructive azoospermia.

Do testis-derived circular RNAs (circRNAs) in seminal plasma have potential as biomarkers to predict the outcome of microdissection testicular sperm extraction (micro-TESE) in patients with idiopathic non-obstructive azoospermia (NOA)? Testis-derived circRNAs in the seminal plasma can indeed be used for predicting the outcome of micro-TESE in patients with idiopathic NOA. Micro-TESE is an effective method to obtain sperm samples from patients with idiopathic NOA. However, its success rate is only 40-50% in such patients. Six idiopathic NOA patients with different micro-TESE results were included as the discovery cohort. Their testicular tissues were used for extracting and sequencing circRNAs. Five circRNAs with the most significantly different expression levels were selected for further verification. Fifty-two patients with idiopathic NOA were included as the validation cohort. Preoperative seminal plasma samples of 52 patients with idiopathic NOA and 25 intraoperative testicular tissues were collected and divided into 'success' and 'failure' groups according to the results of micro-TESE. Quantitative real-time polymerase chain reaction was performed to verify differences in the expression levels of the selected circRNAs between the two groups in the testicular tissues and seminal plasma. Whether at the seminal plasma or testicular tissue level, the differences in the expression levels of the three circRNAs (hsa_circ_0000277, hsa_circ_0060394 and hsa_circ_0007773) between the success and failure groups were consistent with the sequencing results. A diagnostic receiver operating curve (ROC) analysis of the AUC indicated excellent diagnostic performance of these circRNAs in seminal plasma in predicting the outcome of micro-TESE (AUC values: 0.920, 0.928 and 0.891, respectively). On the basis of least absolute shrinkage and selection operator (LASSO) logistic regression, the three circRNAs were combined to construct a new prediction model. The diagnostic ROC curve analysis of the model showed an AUC value of 0.958. The expression levels of these circRNAs in seminal plasma using three normospermic volunteer samples remained stable after 48 h at room temperature. NA. This was a single-center retrospective study with relatively few cases. The functions of these circRNAs, as well as their relationship with spermatogenesis, have not yet been established. Testis-derived circRNAs in seminal plasma can reflect the microenvironment of the testis and can be used as reliable biomarkers to screen patients with idiopathic NOA who might be suitable for micro-TESE. This article was funded by the National Natural Science Foundation of China (Grant no. 81871151). There were no competing interests.

A panel of extracellular vesicle long noncoding RNAs in seminal plasma for predicting testicular spermatozoa in nonobstructive azoospermia patients.

Whether the testis-specific extracellular vesicle (EV) long noncoding RNAs (lncRNAs) in seminal plasma could be utilized to predict the presence of testicular spermatozoa in nonobstructive azoospermia (NOA) patients? Our findings indicate that the panel based on seminal plasma EV lncRNAs was a sensitive and specific method in predicting the presence of testicular spermatozoa and may improve clinical decision-making of NOA. The adoption of sperm retrieval techniques, especially microdissection testicular sperm extraction (mTESE), in combination with ICSI has revolutionized treatment for NOA. However, there are no precise and noninvasive methods for predicting whether there are testicular spermatozoa in NOA patients before mTESE. RNA sequencing was performed on seminal plasma EVs from 6 normozoospermic men who underwent IVF due to female factor and 5 idiopathic NOA patients who failed to obtain testicular spermatozoa by mTESE and were diagnosed as having Sertoli cell-only syndrome by postoperative pathology. A biomarker panel of lncRNAs was constructed and verified in 96 NOA patients who underwent mTESE. Decision-making process was established based on the panel in seminal plasma EVs from 45 normozoospermia samples, 43 oligozoospermia samples, 62 cryptozoospermia samples, 96 NOA samples. RNA sequencing was done to examine altered profiles of EV lncRNAs in seminal plasma. Furthermore, a panel consisting of EV lncRNAs was established and evaluated in training set and validation sets. A panel consisting of nine differentially expressed testis-specific lncRNAs, including LOC100505685, SPATA42, CCDC37-DT, GABRG3-AS1, LOC440934, LOC101929088 (XR_927561.2), LOC101929088 (XR_001745218.1), LINC00343 and LINC00301, was established in the training set and the AUC was 0.986. Furthermore, the AUC in the validation set was 0.960. Importantly, the panel had a unique advantage when compared with models based on serum hormones from the same group of NOA cases (AUC, 0.970 vs 0.723; 0.959 vs 0.687, respectively). According to the panel of lncRNAs, a decision-making process was established, that is when the score of an NOA case exceeds 0.532, sperm retrieval surgery may be recommended. In the future, the sample size needs to be further expanded. Meanwhile, the regulatory functions and mechanism of lncRNAs in spermatogenesis also need to be elucidated. When the score of our panel is below 0.532, subjecting the NOA patients to ineffective surgical interventions may not be recommended due to poor sperm retrieval rate. This work was supported by the National Natural Science Foundation of China (81871110, 81971314 and 81971759); the Guangdong Special Support Plan-Science and Technology Innovation Youth Top Talents Project (2016TQ03R444); the Science and Technology Planning Project of Guangdong Province (2016B030230001 and 201707010394); the Key Scientific and Technological Program of Guangzhou City (201604020189); the Pearl River S&T Nova Program of Guangzhou (201806010089); the Transformation of Scientific and Technological Achievements Project of Sun Yat-sen University (80000-18843235) and the Youth Teacher Training Project of Sun Yat-sen University (17ykpy68 and 18ykpy09). There are no competing interests related to this study. N/A.

Preliminary investigation of the function of hsa_circ_0049356 in nonobstructive azoospermia patients.

Nonobstructive azoospermia (NOA), which is considered the most severe form of male infertility, has placed a heavy burden on families and society. As vital regulators of transcriptional and post-transcriptional levels, Noncoding RNAs (ncRNAs) are closely related to all the pathophysiological processes involved in infertility in males, especially spermatogenesis. Our study explored the expression levels of circ_0049356 in both the whole blood and seminal plasma samples of idiopathic NOA patients via quantitative real-time PCR. Furthermore, the relative expression of its host gene (CARM1) was also determined using the same methods. In addition, as circRNAs have been demonstrated to regulate gene expression as miRNAs sponge, we predicted a total of five miRNAs and 101 mRNAs as putative downstream targets and constructed a circRNA-miRNA-mRNA network. Based on the predictions, Gene Ontology and KEGG pathway analyses were performed for further bioinformatics analysis to explore the potential function and investigate the circ_0049356-miRNA-mRNA interactions. Our results show target mRNAs that have been predicted to regulate guanyl-nucleotide exchange factor activity to mediate the GTP/GDP exchange, and downstream targets possibly involved in the regulation of the actin cytoskeleton, which play a significant role in cytoskeleton rearrangement of germ cells during spermatogenesis.

Corrigendum. Sequencing of a 'mouse azoospermia' gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest.

Corrigendum. Sequencing of a 'mouse azoospermia' gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest.

Sequencing of a 'mouse azoospermia' gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest.

What is the diagnostic potential of next generation sequencing (NGS) based on a 'mouse azoospermia' gene panel in human non-obstructive azoospermia (NOA)? 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. 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/). 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. 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. 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. 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. 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. 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.

Clinical, Hormonal, and Genetic Evaluation of Idiopathic Nonobstructive Azoospermia and Klinefelter Syndrome Patients

To investigate the clinical, hormonal, and genetic factors in infertile men with idiopathic nonobstructive azoospermia (NOA) or azoospermic Klinefelter syndrome (KFS), a total of 556 and 96 patients, respectively, were included in this study. All patient samples were analyzed cytogenetically. Serum reproductive hormone levels were measured. Microdeletions in the azoospermia factor (AZF) region of the Y chromosome were detected by multiplex PCR using 16 specific sequence-tagged sites. FSH and LH levels in both NOA and KFS patients were significantly higher than the normal range, and the testosterone level in KFS patients was significantly lower. Ninety-two (95.8%) of the KFS patients showed non-mosaic 47,XXY karyotypes and 47,XXY,inv(9)(p11.1q13); the other KFS patients had mosaic karyotypes of 47,XXY/46,XY, 47,XXY/46,XX, and 47,XXY/48,XXXY/46,XX. Among the 556 idiopathic NOA patients with normal karyotypes, 67 (12.05%) had microdeletions in the AZF region of the Y chromosome. Microdeletions were most frequently detected in the AZFc region, followed by AZFa, AZFb, AZFbc, and partial AZFc deletions. However, Y chromosome microdeletions were not found in any of the azoospermic KFS patients. In view of the hormonal and genetic abnormalities in infertile men with idiopathic NOA and with azoospermic KFS, genetic testing for karyotype, Y chromosome microdeletions, and hormonal parameters is advocated.

A novel functional variant in Wilms' Tumor 1 (WT1) is associated with idiopathic non-obstructive azoospermia.

Idiopathic nonobstructive azoospermia (INOA) is one of the most severe forms of male infertility, yet its pathophysiology remains unclear. WT1 (Wilms' tumor 1) regulates the polarity of Sertoli cells, thereby playing a critical, indirect role in spermatogenesis. Here, we evaluated WT1 gene variation associates with INOA by assessing its promoter and coding regions in 200 patients diagnosed with INOA and 200 proven-fertile men. Three novel variants in the WT1 coding region were detected only in INOA patients, including two synonymous variants and one missense variant, p.Phe435Leu (p.F435L), which was predicted to be deleterious to protein function. The results of dual luciferase reporter showed that the WT1 p.F435L variant decreases transcription of COL4A1 and WNT4 promoters through a dominant-negative effect. Furthermore, chromatin immunoprecipitation assays revealed that COL4A1 and WNT4 promoter is directly bound by wild-type WT1 protein, but not the p.F435L WT1 variant. Thus, we identified a novel functional variant of WT1 functionally associated with INOA. Mol. Reprod. Dev. 84: 222-228, 2017. © 2017 Wiley Periodicals, Inc.

Copy number gain of VCX, X-linked multi-copy gene, leads to cell proliferation and apoptosis during spermatogenesis.

Male factor infertility affects one-sixth of couples worldwide, and non-obstructive azoospermia (NOA) is one of the most severe forms. In recent years there has been increasing evidence to implicate the participation of X chromosome in the process of spermatogenesis. To uncover the roles of X-linked multi-copy genes in spermatogenesis, we performed systematic analysis of X-linked gene copy number variations (CNVs) and Y chromosome haplogrouping in 447 idiopathic NOA patients and 485 healthy controls. Interestingly, the frequency of individuals with abnormal level copy of Variable charge, X-linked (VCX) was significantly different between cases and controls after multiple test correction (p = 5.10 × 10−5). To discriminate the effect of gain/loss copies in these genes, we analyzed the frequency of X-linked multi-copy genes in subjects among subdivided groups. Our results demonstrated that individuals with increased copy numbers of Nuclear RNA export factor 2 (NXF2) (p = 9.21 × 10−8) and VCX (p = 1.97 × 10−4) conferred the risk of NOA. In vitro analysis demonstrated that increasing copy number of VCX could upregulate the gene expression and regulate cell proliferation and apoptosis. Our study establishes a robust association between the VCX CNVs and NOA risk.

Alterations of testis-specific promoter methylation in cell-free seminal deoxyribonucleic acid of idiopathic nonobstructive azoospermic men with different testicular phenotypes

To explore the feasibility of quantification of testicular DNA methylation from cell-free seminal DNA (cfsDNA), and analyze promoter methylation alterations in men with idiopathic nonobstructive azoospermia (NOA). Comparison between testicular DNA and paired cfsDNA, and among NOA patients with different testicular phenotypes. Academic research institute and andrology practice. Eighty-eight idiopathic NOA patients with different testicular phenotypes and 24 normozoospermic men. Testicular biopsies and semen analysis. Five testis-specific methylated promoters were selected. Promoter methylation was quantified using MethyLight in testicular DNA and paired cfsDNA, and the mRNA level was determined by real-time quantitative polymerase chain reaction. Correlations of methylation of the selected five promoters between testicular DNA and paired cfsDNA were observed; and promoter methylation was negatively related to the messenger RNA level in testis. The cfsDNA methylation of these promoters showed different dynamic changes among the subtypes of NOA and normozoospermia. Among them, CCNA1 and DMRT1 promoter methylation in the hypospermatogenesis group was higher than in other groups and showed diagnostic potential for the patient with hypospermatogenesis. Cell-free seminal DNA could be a novel, noninvasive biomarker for the detection of testicular epigenetic aberrations. Epigenetic information in cfsDNA related to spermatogenesis may serve to predict successful testicular sperm retrieval in NOA patients.

Rs189037, a functional variant in ATM gene promoter, is associated with idiopathic nonobstructive azoospermia

To investigate the relationship between a functional variant rs189037(G>A) in ATM promoter and idiopathic nonobstructive azoospermia (INOA) in a Chinese population. Case-control study. Medical academy and hospital. Two hundred twenty-nine INOA patients and 236 fertile male controls. None. Genotyping was performed by polymerase chain reaction-based restriction fragment length polymorphism and subsequently confirmed by DNA sequencing. Odds ratio (ORs) and 95% confidence intervals (95% CIs) were calculated for the risk genotype and allele. Bioinformatic analysis was also performed to predict the biological function of rs189037(G>A). The AA genotype and A allele at rs189037(G>A) locus were both associated with an increased risk of INOA, with OR 1.90 (95% CI 1.214-3.007) for AA and 1.41 (95% CI 1.112-1.775) for A allele. The heterozygous GA and GA+AA had no relationship with INOA risk, with OR 1.06 (95% CI 0.761-1.472) and 1.28 (95% CI 0.954-1.708), respectively. Meanwhile, stratification by genotype showed that INOA patients with AA had higher FSH level, lower total T level, and smaller testicular size than those patients with GG. Furthermore, bioinformatic analysis predicted that the rs189037(G>A) variant was located in a well-conserved region in ATM promoter and that the transition of allele G to allele A might lead to differential allelic expression of ATM gene via modifying of the DNA-binding ability of transcription factor E2F1. The genetic variant rs189037(G>A) in ATM gene promoter contributes to an increased risk of INOA in a Chinese population, possibly through affecting the DNA-binding ability of E2F1 and subsequent ATM expression.