NOA Patients Research Articles

The mutation c.346-1G > A in SOHLH1 impairs sperm production in the homozygous but not in the heterozygous condition.

Non-obstructive azoospermia (NOA) is an important cause of male infertility, and the genetic pathogenesis is still incompletely understood. The previous study reported that heterozygous mutation of c.346-1G > A in spermatogenesis and oogenesis specific basic helix–loop–helix 1 (SOHLH1) was identified in two NOA patients and suggested it is the pathogenic factor for NOA. However, in our research, this heterozygous mutation was confirmed in three Chinese infertile patients who suffered from teratozoospermia, but they had normal sperm number. Intriguingly, a homozygous mutation of c.346-1G > A in SOHLH1 was detected in a severe oligozoospermia (SOZ) patient, characterized with severely decreased sperm count. Notably, we unprecedently revealed that this homozygous mutation of c.346-1G > A in SOHLH1 leads to the sharp decrease in various germ cells and spermatogenesis dysfunction, which is similar to the phenotype of SOHLH1 knockout male mice. Moreover, western blotting confirmed that the homozygous mutation declined SOHLH1 protein expression. Additionally, we correlated the good prognosis of intracytoplasmic sperm injection (ICSI) in the patients carrying the mutation of c.346-1G > A in SOHLH1. Thus, we suggested that the heterozygous mutation of c.346-1G > A in SOHLH1 is responsible for teratozoospermia, and this homozygous mutation in SOHLH1 impairs spermatogenesis and further leads to the reduced sperm count, eventually causing male infertility, which unveils a new recessive-inheritance pattern of SOHLH1-associated male infertility initially.

Novel variants in helicase for meiosis 1 lead to male infertility due to non-obstructive azoospermia

BackgroundNon-obstructive azoospermia (NOA) is the most severe form of male infertility; more than half of the NOA patients are idiopathic. Although many NOA risk genes have been detected, the genetic factors for NOA in majority of the patients are unknown. In addition, it is difficult to retrieve sperm from these patients despite using the microsurgical testicular sperm extraction (microTESE) method. Therefore, we conducted this genetic study to identify the potential genetic factors responsible for NOA and investigate the sperm retrieval rate of microTESE for genetically deficient NOA patients.MethodsSemen analyses, sex hormone testing, and testicular biopsy were performed to categorize the patients with NOA. The chromosome karyotypes and Y chromosome microdeletion analyses were used to exclude general genetic factors. Whole exome sequencing and Sanger sequencing were performed to identify potential genetic variants in 51 patients with NOA. Hematoxylin and eosin staining (H&E) and anti-phosphorylated H2AX were used to assess the histopathology of spermatogenesis. Quantitative real time-polymerase chain reaction, western blotting, and immunofluorescence were performed to verify the effects of gene variation on expression.ResultsWe performed whole exome sequencing in 51 NOA patients and identified homozygous helicase for meiosis 1(HFM1) variants (NM_001017975: c.3490C > T: p.Q1164X; c.3470G > A: p.C1157Y) in two patients (3.9%, 2/51). Histopathology of the testis showed that spermatogenesis was completely blocked at metaphase in these two patients carrying the HFM1 homozygous variants. In comparison with unaffected controls, we found a significant reduction in the levels of HFM1 mRNA and protein expression in the testicular tissues from these two patients. The patients were also subjected to microTESE treatment, but the sperms could not be retrieved.ConclusionsThis study identified novel homozygous variants of HFM1 that are responsible for spermatogenic failure and NOA, and microTESE did not aid in retrieving sperms from these patients.

O-119 Evaluating the reproductive potential of azoospermic men by germline mutation profiling

Abstract Study question Can whole exome sequencing (WES) of spermatozoa from azoospermic men identify mutations related to the etiology of their infertility and ability to support a pregnancy? Summary answer Key de novo germline mutations that affect sperm production and/or embryo developmental competence may explain reproductive failure in azoospermic men, regardless of the etiology. What is known already Azoospermia accounts for approximately 15% of male factor infertility cases. Although it can be caused by pre-testicular factors, the most recognized forms are testicular and post-testicular. While post-testicular azoospermia is mainly due to a mechanical obstruction, testicular azoospermia, the most severe form, is characterized by scattered functional germinal epithelia that strive to support the meiotic process during gamete development. To shed light on the etiology of this condition, genetic studies have been performed, albeit exclusively on peripheral blood. We chose to perform a genomic assessment of spermatozoa to preferentially detect germline mutations that may be passed to the progeny. Study design, size, duration In a 2-year period, we recruited infertile men undergoing epididymal aspiration for acquired obstructive azoospermia (OA; n = 19) or testicular retrieval for nonobstructive azoospermia (NOA; n = 10). Four additional men were included as fertile controls. Following WES, copy number variants (CNVs) and gene mutation profiles were compared between the OA and NOA patients, and within those two categories, in relation to whether they generated a clinical pregnancy (fertile) or not (infertile). Participants/materials, setting, methods Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens of consenting men (DNA concentration, 762±492 ng/ul; quality, 1.7±0.1 nm). CNVs, gene mutations, duplications, and deletions were detected using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when the read depth was >1.5 or < 0.5 times the median read depth in the control. Common mutations in the OA and NOA cohorts were assessed according to the couples’ clinical outcome. Main results and the role of chance Of 29 couples (maternal age, 41.9±7yrs; paternal age, 42.5±7yrs), 19 OA men underwent epididymal sperm retrieval (1.1±4x106/ml concentration, 9±12% motility) while 10 NOA men underwent testicular biopsy (0.03±0.2x106/ml concentration, 0.5±1% motility). WES did not reveal a significant difference in sperm aneuploidy between the two etiologies (OA, 1.8%; NOA, 1.9%). In OA patients, only 3 genes were deleted, mainly housekeeping-related, while in the NOA cohort, 5 genes were deleted, involved in RNA transcription (POLR2L) and apoptosis (AP5M1), in addition to spermiogenic functions (AP1S2, AP1G2, APOE). OA patients and their partners (maternal age, 36.8±4yrs) underwent 19 ICSI cycles that resulted in a pregnancy and delivery rate of 47.4% (9/19). Those able to reproduce (n = 9) shared a mutation in ZNF749, a gene affecting only sperm production. The infertile individuals (n = 10) all had a deletion on PRB1, controlling essential DNA replication. NOA men and their partners (maternal age, 38.2±2yrs) underwent 10 ICSI cycles, yielding a clinical pregnancy rate of 70% (7/10). The fertile men (n = 7) had a concurrent gene deletion involved in stem cell lineage differentiation (MPIG6B). Their infertile counterparts (n = 3) had deleted genes involved in spermato/spermio-genesis (n = 6) and, most importantly, in early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC9, REPIN1, MAPRE3, and ARL4C). Limitations, reasons for caution This is a novel study with limited observations. The presence of housekeeping-related mutations in fertile OA men as well as the DNA replication mutation in infertile OA patients, considering the acquired condition, remains puzzling. Although maternal age was controlled for, confounding factors related to the female partner cannot be excluded. Wider implications of the findings Screening men for germline mutations provides valuable information on their ability to reproduce, regardless of the etiology of azoospermia. Genome profiling was able to identify reasons for failed reproductive performance in azoospermic men, particularly those individuals with secretory azoospermia (NOA). Genomic profiling may identify gametes with retained embryo developmental competence. Trial registration number n/a

RNA-binding protein ELAVL2 plays post-transcriptional roles in the regulation of spermatogonia proliferation and apoptosis.

ObjectivesRNA‐binding proteins (RBPs) play essential post‐transcriptional roles in regulating spermatogonial stem cells (SSCs) maintenance and differentiation. We identified a conserved and SSCs‐enriched RBP ELAVL2 from our single‐cell sequencing data, but its function and mechanism in SSCs were unclear.Materials and methodsExpressions of ELAVL2 during human and mouse testis development were validated. Stable C18‐4 and TCam‐2 cell lines with overexpression and knockdown of ELAVL2 were established, which were applied to proliferation and apoptosis analysis. RNA immunoprecipitation and sequencing were used to identify ELAVL2 targets, and regulatory functions of ELAVL2 on target mRNAs were studied. Proteins interacting with ELAVL2 in human and mouse testes were identified using immunoprecipitation and mass spectrometric, which were validated by in vivo and in vitro experiments.ResultsELAVL2 was testis‐enriched and preferentially expressed in human and mouse SSCs. ELAVL2 was down‐regulated in NOA patients. ELAVL2 promoted proliferation and inhibited apoptosis of C18‐4 and TCam‐2 cell lines via activating ERK and AKT pathways. ELAVL2 associated with mRNAs encoding essential regulators of SSCs proliferation and survival, and promoted their protein expression at post‐transcriptional level. ELAVL2 interacted with DAZL in vivo and in vitro in both human and mouse testes.ConclusionsTaken together, these results indicate that ELAVL2 is a conserved SSCs‐enriched RBP that down‐regulated in NOA, which regulates spermatogonia proliferation and apoptosis by promoting protein expression of targets.

Comparison of the Effects of Different Testicular Sperm Extraction Methods on the Embryonic Development of Azoospermic Men in Intracytoplasmic Sperm Injection (ICSI) Cycles: A Retrospective Cohort Study.

Background The effects of different testicular sperm extraction methods on the embryonic development and clinical outcome of azoospermic men in intracytoplasmic sperm injection (ICSI) cycles have not been researched. Our goal was to evaluate the effect of different sperm retrieval methods used for patients with OA or NOA on the embryonic development and clinical outcomes during the ICSI cycles. Methods This was a retrospective cohort study. A total of 530 azoospermic patients who underwent 570 ICSI cycles met the study criteria. ICSI was performed using testicular sperm by TESA in 282 cycles (TESA group); ICSI with testicular sperm by mTESE was performed due to NOA in 90 cycles (mTESE group); ICSI with testicular sperm by MESA was performed in 198 cycles (MESA group). The embryonic development and clinical outcomes of the three groups were counted. Results The general characteristics of the three groups were comparable. Our findings showed that the three groups were matched in terms of infertility durations and age. The mean age and the mean BMI of the female partners were similar in the three groups. Also, our findings showed there were no significant differences in the three groups regarding day 3 of the menstrual cycle FSH and days of stimulation. The research results showed that the total dose of FSH and E2 on the HCG administration day was also not statistically different in the three groups. The number of oocytes retrieved had no significant differences in the three groups. However, the number of 2PNs per cycle and the number of cleavages per cycle were higher in the MESA group than in the other two groups; the TESA group and mTESE group were similar. The number of good quality D3 embryos and the number of good quality D5 embryos were significantly decreased in the mTESE group as compared to the other two groups. Good quality D3 embryos and the rate of good quality D5 embryos in the mTESE group were lower than those in the other two groups. Moreover, the clinical pregnancy rates of the TESA group (50.71%) and the MESA group (51.52%) were similar, but both were much higher than that of the mTESE group (32.22%). Conclusions The mTESE provides a good clinical outcome for NOA patients with severe spermatogenic impairment, including the rate of good quality D3 embryos, the rate of good quality D5 embryos, and the clinical pregnancy rate. However, our data suggested that both the TESA and MESA groups had better embryonic development and clinical outcomes than the mTESE group.

Effect and in silico characterization of genetic variants associated with severe spermatogenic disorders in a large Iberian cohort.

Severe spermatogenic failure (SpF) represents the most extreme manifestation of male infertility, as it decreases drastically the semen quality leading to either severe oligospermia (SO, <5 million spermatozoa/mL semen) or non-obstructive azoospermia (NOA, complete lack of spermatozoa in the ejaculate without obstructive causes). The main objective of the present study is to analyze in the Iberian population the effect of 6 single-nucleotide polymorphisms (SNPs) previously associated with NOA in Han Chinese through genome-wide association studies (GWAS) and to establish their possible functional relevance in the development of specific SpF patterns. We genotyped 674 Iberian infertile men (including 480 NOA and 194 SO patients) and 1058 matched unaffected controls for the GWAS-associated variants PRMT6-rs12097821, PEX10-rs2477686, CDC42BPA-rs3000811, IL17A-rs13206743, ABLIM1-rs7099208, and SOX5-rs10842262. Their association with SpF, SO, NOA, and different NOA phenotypes was evaluated by logistic regression models, and their functional relevance was defined by comprehensive interrogation of public resources. ABLIM1-rs7099208 was associated with SpF under both additive (OR=0.86, p=0.036) and dominant models (OR=0.78, p=0.026). The CDC42BPA-rs3000811 minor allele frequency was significantly increased in the subgroup of NOA patients showing maturation arrest (MA) of germ cells compared to the remaining NOA cases under the recessive model (OR=4.45, p=0.044). The PEX10-rs2477686 SNP was associated with a negative testicular sperm extraction (TESE) outcome under the additive model (OR=1.32, p=0.034). The analysis of functional annotations suggested that these variants affect the testis-specific expression of nearby genes and that lincRNA may play a role in SpF. Our data support the association of three previously reported NOA risk variants in Asians (ABLIM1-rs7099208, CDC42BPA-rs3000811, and PEX10-rs2477686) with different manifestations of SpF in Iberians of European descent, likely by influencing gene expression and lincRNA deregulation.

TG12-T5-V470 haplotype in the CFTR gene is associated with non-obstructive azoospermia in Iranian infertile men

IntroductionRecent studies have shown that some variations and polymorphisms of CFTR gene can be involved in other types of infertility apart from CBAVD. In this study, we decided to survey the poly-T, TG repeats and M470V of the CFTR gene in infertile men with non-obstructive azoospermia in Iran. Material and methodThis case-control study was performed on 203 NOA patients and a control group (n = 200), the peripheral blood samples were collected in EDTA vacuum tubes. Then genomic DNA was extracted and PCR-sequencing was carried out on the 3′ ends of intron 8 CFTR gene to identify poly-T and TG repeats. Genotyping of M470V was carried out using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). ResultThe mean age of the infertile men and in control group was 31 ± 3.7, 30 ± 2.6 years, respectively. Frequency of T5 allele in males with non-obstructive azoospermia was found higher than that of the control group (OR 2.5798, 95% CI 1.3013–5.1143, P < 0.001). The allelic frequency of TG12-5T in males with non-obstructive azoospermia was significantly higher than that observed in the control group (OR 15.7143, 95% CI 2.7576–89.5497, P < 0.001) and also the TG12-5T-V470 haplotype was significantly associated with NOA as compared to normal controls, (OR 19.2857, 95% CI 1.0458–355.6390, P < 0.0001). ConclusionIt seems that T5, TG12-T5 and also TG12-T5-V470 haplotype can be associated with the risk of NOA in men, but further researches are necessary to clarify accurate role CFTR gene in NOA.

Methylation patterns of methylenetetrahydrofolate reductase gene promoter in infertile males.

Errors of folate/homocysteine pathways which are critical for transferring methyl groups have been suggested to affect male fertility. We aimed to evaluate the methylation patterns of the promoter of methylenetetrahydrofolate reductase (MTHFR) gene in infertile males and to investigate the association between MTHFR promoter methylation and success of sperm retrieval. Thirty-five nonobstructive azoospermic and 46 severe oligozoospermic patients constituted the study group and were compared with 49 fertile and/or normozoospermic men. The methylation status was analysed by methylation-specific polymerase chain reaction. MTHFR promoter methylation was detected in infertile men with NOA and SO in the ratio of 48.6% and 58.7%, respectively. Methylation was also observed in 51% of controls. MTHFR promoter was methylated in 65% of men with viable spermatozoon during TESE. No association was found regarding to the profile of MTHFR promoter methylation between both NOA and SO patients and controls (p=.621). There was no relation between the methylation status of MTHFR promoter and low motility and poor morphology (p=.682 and p=.413, respectively). No association was found between MTHFR promoter methylation and presence of viable spermatozoa (p=.382). Our data indicate that the promoter methylation of MTHFR gene may not be associated with male infertility.

EVALUATING THE QUALITY OF REPORTING IN OUTCOMES OF MICROSURGICAL-TESE IN MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA (NOA): A METHODOLOGICAL ANALYSIS

Microsurgical testicular sperm extraction (micro-TESE), since its introduction in 1999, has been demonstrated to be the optimal approach to surgical sperm retrieval for men with NOA. However, there is heterogeneity in the methodological quality of published micro-TESE approaches and outcomes. We conducted a systematic methodological analysis of all published literature on the use of micro-TESE in men with NOA from 1999 to the present. A PubMed search was performed using the search terms “microdissection TESE” or “microsurgical TESE”. Only studies on NOA patients were considered. Studies on obstructive azoospermia, case studies, and review articles were excluded. We included only the most recent publication from a given academic medical center’s cohort. Adapted from previously published quality reporting criteria for prospective trials (CONSORT), we evaluated publications on their reporting of preoperative factors, intraoperative techniques, surgical and clinical outcomes, and adverse events. Two reviewers evaluated each publication for inclusion. 182 articles were identified in our initial search, of which 55 met inclusion criteria. Specific reporting criteria are listed in Table 1. Surgical technique and sperm retrieval rates were the most commonly reported criteria. Reporting on the presence of an embryologist intraoperatively was observed in approximately 25% of articles, while other procedural details including method of sperm quantification, quantity retrieved, and number of cryopreserved vials were observed in fewer than 10% of articles. Clinical outcomes, including surgical complications, pregnancy rates, and live birth rates were reported in fewer than half of the articles. Fetal outcomes were rarely reported. There are many inconsistencies in reporting quality of micro-TESE outcomes, specifically a lack of information on the quantity of sperm retrieved, the role of embryology intraoperatively, and clinical and fetal outcomes. Development of a reporting guideline may help standardize reported results to better assess clinical outcomes across institutions.Table 1Reporting CriteriaPREOPERATIVE FACTORSINTRAOPERATIVE TECHNIQUESSURGICAL OUTCOMESFERTILIZATION TECHNIQUESCLINICAL OUTCOMESGenetic TestingSurgical TechniqueSperm Retrieval RateFresh/Frozen SpermClinical PregnancyDiagnosis of AzoospermiaOperative Time# of Sperm RetrievedMethod of CryopreservationLive Birth RateHormonal TherapyAnesthesiaMethod of Quantification# of Cryopreserved VialsFetal/Neonatal AnomalyLateralityAdverse EventsFertilization RateGrowth DefectsEmbryologist in ORUse of intracytoplasmic sperm injectionPreterm DeliveryNICU Admission Open table in a new tab

Acute effect of microdissection testicular sperm extraction on blood total testosterone and luteinising hormone levels.

Testicular sperm extraction is a common surgical procedure to retrieve spermatozoa from men with azoospermia which may affect the tissue and cause hypogonadism (Human Reproduction Update, 24, 2018, 442). We aimed to evaluate the acute effect of micro-TESE on blood total testosterone (TT) and luteinising hormone (LH) levels. Between April 2018 and September 2019, 60 selected NOA (i.e. with normal TT and LH) patients were included in the study. After procedure, blood samples were collected to evaluate the levels of TT and LH at 1st and 24th hour and first week. Values were compared. In addition, patients were divided into two groups according to their body mass index. Mean age of patients was 27.37±4.52years, and mean of body mass index was 24.49±3.78kg/m2 . Mean TT levels at the 1st hour, 24th hour and the 1st week were 3.81, 3.50 and 3.05ng/ml respectively. Mean LH levels at the 1st hour, 24th hour and the 1st week were 5.03, 5.15 and 5.46IU/L respectively. Statistically significant differences between pre- and post-operative 24th hour and 1st week values were detected (p=.001, p<.001 respectively). No statistical differences were found between two BMI's groups. TT decreased in acute period after the procedure, whereas the increase of LH levels was observed after 24th hour.

Unraveling epigenomic abnormality in azoospermic human males by WGBS, RNA-Seq, and transcriptome profiling analyses.

To determine associations between genomic DNA methylation in testicular cells and azoospermiain human males. This was a case-control study investigating the differences and conservations in DNA methylation, genome-wide DNA methylation, and bulk RNA-Seq for transcriptome profiling using testicular biopsy tissues from NOA and OA patients. Differential methylation and different conserved methylation regions associated with azoospermia were identified by comparing genomic DNA methylation of testicular seminiferous cells derived from NOA and OA patients. The genome methylation modification of testicular cells from NOA patients was disordered, and the reproductive-related gene expression was significantly different. Our findings not only provide valuable knowledge of human spermatogenesis but also paved the way for the identification ofgenes/proteins involved in male germ cell development. The approach presented in this report provides a powerful tool to identify responsible biomolecules, and/or cellular changes (e.g., epigenetic abnormality) thatinduce male reproductive dysfunction such as OA and NOA.

Identification of KISS1R gene mutations in disorders of non-obstructive azoospermia in the northeast population of China.

BackgroundNon‐obstructive azoospermia (NOA), a serious phenotype of male spermatogenesis failure, is a multifactorial disease which is regulated by genetic, epigenetic, and environmental factors. Some gene structural variants have been demonstrated to be related to NOA. Loss‐of‐function mutations of KISS1R cause normosmic idiopathic hypogonadotropic hypogonadism (IHH) which result in azoospermia at the pre‐testicular level. The objective of this research was to investigate genetic variants of KISS1R in NOA patients.MethodsThe entire coding region of 52 spermatogenesis‐associated genes (KISS1R included) was sequenced from 200 NOA patients. Mutation screening was performed to identify genetic variations of these genes by targeted exome sequencing. Sequencing data analysis was carried out by a series of bioinformatics tools. Candidate variants confirmation was performed by Sanger sequencing. Functional analysis of candidate variants was evaluated using SIFT and PolyPhen‐2.ResultsThree heterozygous missense variants in KISS1R were identified in three patients, respectively. No deleterious variations in other candidate genes were found in the three patients. Two of these three variants, p.A211T and p.G186E, had been reported in the ExAC and dbSNP database, respectively, while the other variant p.A301D was novel. These variants were all predicted to be likely pathogenic by in silico analysis.ConclusionOur study revealed three heterozygous missense variants in KISS1R which expanded the mutation spectrum of KISS1R in infertile men with NOA in the northeast of China.

CircRNA expression profile and functional analysis in testicular tissue of patients with non-obstructive azoospermia

BackgroundNon-obstructive azoospermia (NOA) is a multifactorial disorder whose molecular basis remains largely unknown. Circular RNAs (CircRNAs), a novel class of endogenous RNAs, have been recognized to play important roles in many biological processes. However, little is known about the expression patterns and functions of circRNAs in human testes involved in NOA.MethodsIn this study, the testicular circRNA expression profile were explored in NOA patients and the controls by high-throughput circRNA microarray. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to confirm the microarray data. Bioinformatics analyses including the circRNA/miRNA/mRNA interaction network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to predict the functions of differentially expressed circRNAs.ResultsA total of 368 differentially down-regulated and 526 up-regulated circRNAs were detected in NOA patients. These findings have been verified by qRT-PCR on 6 selected circRNAs. Among these differentially expressed circRNAs, the hsa_circRNA_0023313 was obviously up-regulated in testicular tissue of NOA patients. The most likely potential target miRNA for hsa_circRNA_0023313 include hsa-miR-520d-3p, hsa-miR-373-3p, hsa-miR-372-3p, hsa-miR-302c-3p and hsa-miR-130b-5p. Function analysis indicated that hsa_circRNA_0023313 was ubiquitin-protein transferase activity and chromatin binding. KEGG analysis revealed that the top five pathways related to hsa_circRNA_0023313 were endocytosis, meiosis, FoxO signaling pathway, ubiquitin mediated proteolysis and AMPK signaling pathway.ConclusionsThis is the first report that the testicular circRNA expression profile is altered in NOA patients indicating circRNAs might play important roles in regulating spermatogenesis and be potential biomarkers for the diagnosis and treatment of NOA.

Effects and mechanism of lncRNA serving as ceRNA in non-obstructive azoospermia

Objective: To investigate the mechanisms of lncRNA on the occurrence and development of NOA by constructing ceRNA regulation network of lncRNA, miRNA and mRNA. Methods: Samples of adult human testis were obtained from NOA patients and OA patients with normal spermatogenesis (controls), recruited from the Reproductive Medicine Center of Nanfang Hospital from June 2017 to June 2018. Differentially expressed lncRNAs and mRNAs in testicular tissues from patients with NOA were identified by microarray analysis in previous association study. In this study, differentially expressed lncRNAs and mRNA were used to construct the ceRNA regulatory network in NOA and clarify the interaction relationship among lncRNA, miRNA and mRNA. GeneMANIA database was used to construct Protein-Protein Interaction (PPI) of the mRNAs in ceRNA regulatory network. WebGestalt toolkit was employed to perform gene function and pathway enrichment analyses of those coding genes. Finally, qRT-PCR and dual luciferase reporter system were employed for further experimental validation. Results: The ceRNA regulatory network of lncRNA, miRNA and mRNA consists of 21 nodes and 26 edges, of which 4 lncRNAs, 13 miRNAs and 4 mRNAs. 19 proteins were found to interact with the mRNA coding proteins in ceRNA regulatory network by PPI analysis. Gene oncology and KEGG pathway enrichment analyses indicate these coding genes were significantly enriched in pentose metabolic process and pentose phosphate pathway. Furthermore, lncRNA ANXA2P3 was found binding with miR-613 and miR-206 to inhibit mRNA TKT expression. Conclusion: lncRNAs exert an important role in the occurrence and development of NOA via ceRNA regulatory network, which could be used as new biomarkers for NOA treatment.

XRCC1 deficiency correlates with increased DNA damage and male infertility

High fidelity DNA repair is critical to sustain the genomic integrity and quality of developing germ cells. Deficiencies in DNA repair machinery may result in increased DNA damage in germ cell leading to abnormal spermatogenesis and infertility. X-ray repair cross-complementing group 1 (XRCC1) is a testis enriched protein that plays a crucial role in the DNA base excision repair (BER) pathway. The aim of this study was to analyze the level of XRCC1 transcript and protein in infertile men and its association with DNA damage in sperm. A total of eighty infertile patients with different infertile phenotypes (Azoospermia, n = 30; Severe oligozoospermia, n = 25; Severe oligoasthenozoospermia, n = 25) and age-matched controls (normal spermatogenesis [NS], n = 15 and fertile controls, n = 10) were recruited. γ-H2 AX protein levels were analyzed to estimate the DNA damage in sperm. XRCC1 transcript levels in cases and controls were determined by qRT-PCR. XRCC1 and γ-H2 AX proteins were immunohistochemically analyzed in testicular biopsy sections obtained from NOA patients and OA controls. The determination of XRCC1 and γ-H2 AX protein levels was performed with Western blots. The results revealed reduced expression of XRCC1 mRNA and protein in infertile individuals as compared to controls (p < 0.001). γ-H2 AX levels were significantly increased in infertile cases as compared to controls, indicating increased DNA damage in infertile men. The results indicate decreased expression of the XRCC1 gene in infertile patients which may be one of the factors associated with impaired spermatogenesis and infertility.

Results of intracytoplasmic sperm injection performed with sperm retrieved by microscopic testicular sperm extraction in azoospermic patients.

The absence of any sperm in the ejaculate is called azoospermia and it is detected in 1% of males and 10-15% of those with infertility complaints. Azoospermia may be due to obstructive (OA) and non-obstructive (NOA) causes. Today, healthy pregnancies can be achieved in azoospermic patients by intracytoplasmic sperm injection (ICSI) performed using sperm retrieved from microscopic testicular sperm extraction (m-TESE). In this study, we examined the sperm retrieval rates with m-TESE in azoospermic patients, the results of ICSI in OA and NOA patients with sperm and the underlying testicular pathologies in patients without sperm. Patients who underwent m-TESE at IVF unit of our hospital between January 2005 and April 2017 were retrospectively reviewed. A total of 342 azoospermic patients (117 OA and 225 NOA cases) with regular follow-up were included in the study. In these cases, sperm retrieval and clinical pregnancy rates after ICSI were compared. In the m-TESE procedure, motile sperm was found in all of the OA patients and in 52.4% (118/225) of the NOA patients. Clinical pregnancy rate in the OA group was 29.9% (35/117) and live birth rate was 25.6% (30/117). In the NOA group, the clinical pregnancy rate was 27.1% (32/118) and the live birth rate was 23.7% (27/118). Histopathologic evaluation was made in 107 cases in the NOA group with no testicular sperm, revealing that 59 cases with germ-cell aplasia (sertoli-cell only syndrome), 42 cases with maturation arrest, and 6 cases with hypospermatogenesis. Postoperative hematoma developed in 3 of m-TESE cases and subsided with conservative treatment. If motile sperm is retrieved with m-TESE application in azoospermic patients, pregnancy resulting in one live birth in about 4 couples who undergo ICSI application can be achieved. In the presence of motile sperm, live birth rates are similar between OA and NOA case with very low complication rates.

P115 - Time to first infertility diagnosis and sperm retrieval rate: Results after 30 consecutive micro tese in NOA patients

P115 - Time to first infertility diagnosis and sperm retrieval rate: Results after 30 consecutive micro tese in NOA patients

532 Time to first infertility diagnosis and sperm retrieval rate: results after 30 consecutive micro TESE in NOA patients

532 Time to first infertility diagnosis and sperm retrieval rate: results after 30 consecutive micro TESE in NOA patients

ADAMTS1 and ADAMTS5 metalloproteases produced by Sertoli cells: a potential diagnostic marker in azoospermia

ABSTRACTIn this study, our aim was to detect protein levels of A Disintegrin and Metalloproteinase with Thrombospondin Motifs 1 and 5 (ADAMTS1 and ADAMTS5) proteases and to examine the effect of in vitro FSH supplementation on protease production in cultured Sertoli cells. The expression of metalloproteases, ADAMTS1, and ADAMTS5 were investigated in Sertoli cell cultures as well as in ejaculate of azoospermic men which then were compared with ejaculates of the fertile control group. A total of 15 azoospermic men, diagnosed as obstructive (OA, n = 5) and nonobstructive (NOA, n = 10) azoospermia were included in the study. ADAMTS1, ADAMTS5 and FSH receptors (FSHR) were found to be expressed 2.56, 2.10, and 2.66-fold less in Sertoli cells of NOA patients, than those of OA (p < 0.05). After rFSH was added onto Sertoli cell cultures of NOA patients, their expression did not increase significantly and did not reach to levels of control group. Evaluation of ejaculates revealed that the expression of ADAMTS1 and ADAMTS5 were insignificantly 1.03 and 1.1-fold higher in OA group (p > 0.05), respectively; however, in the NOA group, their expression were 1.70 and 1.96-fold lower, respectively, when compared with the fertile control group (p < 0.05) which was statistically significant. As a conclusion, the present study has revealed that insufficiency of ADAMTS1 and ADAMTS5 expression in Sertoli cells may have an important role in the etiology of male infertility. As expected due to low FSHR expression, rFSH response is impaired in NOA patients with relatively low ADAMTS expression response; therefore, such patients might hardly benefit from rFSH treatment. Further studies with larger cohorts may reveal ADAMTSs’ potential use as a predictive marker for positive sperm retrieval in azoospermic patients who are scheduled to undergo testicular sperm extraction.Abbreviations: ADAM: A Disintegrin and Metalloproteinase; ADAMTS1 and ADAMTS5: A Disintegrin and Metalloproteinase with 10 Thrombospondin Motifs 1 and 5; ADAMTS: A Disintegrin and Metalloproteinase with Thrombospondin; ABP: androgen binding protein; CAMs: cell adhesion molecules; ECM: extracellular matrix; FSH: follicle stimulating hormone; FSHR: FSH receptors; HRP: horseradish peroxidase; MMP: matrix metalloproteinases; MP: metalloproteinases; NOA: nonobstructive azoospermia; OA: obstructive azoospermia; TIMP-1: tissue inhibitor of metalloproteinase-1

Sperm retrieval rates (SRR) and clinical outcomes after testicular sperm extraction (TESE) in the etiology of azoospermia

In assisted reproductive techniques, it is necessary to collect clinical outcomes in a separate group of patient background and provide it for patients. Sperm retrieval rates (SRR) and clinical outcomes after intracytoplasmic sperm injection (ICSI) with testicular sperm in the etiology azoospermia have hardly been investigated. We report that ICSI outcomes in men undergoing TESE have performed in our clinic latest three years. A retrospective study in the etiology of azoospermia. The studies applied conventional TESE (n=165) for OA patients, ED patients, and oligozoospermic patients and microdissection TESE (micro TESE) (n=843) for NOA patients performed in our clinic between September 2013 and March 2017 were used to analyze for SRR. Azoospermia in a patient with normal-size testes (>16mL) and normal FSH suggested obstructive etiology. The diagnosis of OA also required confirmation of normal spermatogenesis by testicular biopsy. The etiology of NOA included unexplained NOA, Klinefelter syndrome, azoospermia factor (AZF) c microdeletion of the Y chromosome, and post chemotherapy NOA. In addition, a total of 396 couples had 727 TESE-ICSI cycles (94 couples and 170 TESE-ICSI cycles for OA and 302 couples and 557 cycles for NOA) were evaluated in embryonic development rates and clinical pregnancy rates (CPR). Spermatozoa were retrieved 100% (118/118) of obstructive azoospermia (OA), 40.6% (43/106) of Klinefelter syndrome (KS), 20.5% (98/479) of unexplained NOA (NOA), 81.8% (27/33) of AZFc microdelection, and 40.9% (18/44) of post chemotherapy NOA. SRR of OA was significantly higher and unexplained NOA were lower than any other groups. SRR with the primary TESE (49.7%) is significantly higher than with the TESE after who had previously undergone with no sperm found (31.0%) (P<0.001). Especially, the significant difference was observed in KS (48.8% and 15.4%, p<0.01) and unexplained NOA (23.9% and 12.5%, p<0.01), respectively. Two pronuclei (2PN) rates in OA (64.0%) and post chemotherapy NOA (69.3%) were significantly higher, but in AZFc microdelection (47.0%) was significantly lower than any other groups. Blastocyst development rate in AZFc microdelection (33.9%) was significantly lower than any other groups and the rates in KS (41.9%) was lower than in OA (50.8%). Good-quality blastocyst rates in OA (42.6%) and post chemotherapy NOA (45.2%) were significantly higher, but the rate in AZFc microdelection (33.9%) was lower than any other groups. CPR per embryo transfer cycle was lower in unexplained NOA (30.0%), KS (28.6%) and AZFc microdelection (26.6%) than in OA (42.4%). Micro TESE is particularly helpful for successful sperm retrieval in KS and AZFc microdeletion cases, however, the motile and better sperm retrieval from testicular tissue seems to be a critical key to succeed and rationale for good embryonic development and clinical pregnancy.