Non-obstructive Azoospermia Patients Research Articles

Gonadotropins treatment prior to microdissection testicular sperm extraction in non-obstructive azoospermia: a single-center cohort study

BackgroundMicrodissection testicular sperm extraction (micro-TESE) in combination with ICSI can make paternity possible for non-obstructive azoospermia (NOA) patients. Testicular sperm can be successfully retrieved in nearly half of NOA patients. Nevertheless, not many convincing protocols are established to improve sperm retrieval rate (SRR). The goal of this study was to evaluate whether gonadotropins therapy before micro-TESE could improve sperm retrieval rate and affect the ICSI outcomes in non-obstructive azoospermia patients with hypergonadotropic hypogonadism.MethodsThis retrospective cohort study included a total of 569 non-obstructive azoospermia men who underwent micro-TESE with or without 3-month of preoperative hCG / hCG plus highly purified urinary FSH (uFSH) between January 2016 and December 2019. The primary outcome was the sperm retrieval rate of micro-TESE.ResultsSperm was found in 27 patients among 395 NOA men who accepted preoperative gonadotropins treatment (6.8%, 27/395) in post-treatment semen analysis for ICSI. One hundred forty nine out of 542 patients could successfully obtain enough sperm for ICSI through the micro-TESE (overall SRR = 27.5%). There was a statistically significant difference in the SRR between the preoperative gonadotropins treatment and non-gonadotropins treatment groups (31.2%, 115/368 vs. 19.5%, 34/174, P = 0.006). In the multivariable analysis with IPTW according to the propensity score, there was a significant association between preoperative gonadotropins treatment and the SRR (OR, 1.59; 95% CI: 1.02–2.52; P = 0.042). No differences in the clinical pregnancy rate, live birth delivery rate, or miscarriage rate were observed between the two groups.ConclusionPreoperative gonadotropins therapy seems to have a role in improving SRR in NOA patients with hypergonadotropic hypogonadism. We found that gonadotropins therapy had no effect on ICSI clinical outcomes and live birth.

Intrasurgical Seminiferous Tubular Diameter Correlates with Total Motile Sperm Count in Azoospermia: a Prospective Cohort Study

We aimed to find a correlation between the intraoperative diameters of the seminiferous tubules evaluated at high magnification during micro-testicular sperm extraction (micro-TESE) and total motile sperm count (TMSC) in non-obstructive azoospermic (NOA) patients. Five hundred four consecutive NOA patients were included and underwent micro-TESE. The change in the mean TMSC and different seminiferous tubular diameters was of high statistical significance (p < 0.001). It should be noted that the highest mean TMSC was reported in the dilated tubules (DTs) group followed by the other study groups 941.72 ± 196.97, 487.37 ± 443.57, and 34.54 ± 60.79, respectively. Furthermore, 21 naïve cases had dilated tubules (DTs) and 18 (85.7%) of them had shown eventful micro-TESE. Conversely, 186 naïve cases had slightly dilated tubules (SDTs), and 101 (54.3%) of them had eventful micro-TESE. Only 8 (24.25%) cases of the 33 cases had non dilated tubules (NDTs) and showed eventful micro-TESE. The frequency of intrasurgical seminiferous tubular diameter and micro-TESE outcome among the naïve cases had demonstrated a highly statistical significance (p < 0.001). Interestingly, all salvaged cases (100%) with DTs and a previous eventful TESE had shown eventful TESE in the current study. The most dilated intrasurgical seminiferous tubular diameter is associated with the highest TMSC in NOA patients including SCO cases.

Multi-stage screening cell-free seminal mRNAs to diagnose completion of meiosis and predict testicular sperm retrieval in men with non-obstructive azoospermia.

Differential diagnosis of men with subtypes of non-obstructive azoospermia (NOA) is important for their treatment. Many genes are transcripted during meiosis. We hypothesized that some of these genes can be detected in cell-free seminal message RNA (mRNAs) (cfs-mRNA) and be developed as non-invasive biomarkers for diagnosing NOA subtypes. To screen cfs-mRNA to diagnose the completion of meiosis and predict successful sperm retrieval (SR) in men with NOA. NOA patients who visited our institutes from September 2018 to December 2020 for testicular histopathological diagnosis (n = 109) or testicular SR (n = 92) were screened for participation in the study. Microarray and real-time quantitative polymerase chain reaction were used in five stages to obtain candidate cfs-mRNAs for comparisons between patients with early maturation arrest (eMA, meiosis not completed) and late MA or hypospermatogenesis (meiosis completed) and between NOA patients with successful SR and SR failure. Twelve cfs-mRNAs were selected based on this comparison between men with eMA and hypospermatogenesis and their gene expression and function information. Of these, AKAP1, BOLL, TCP11 and SETX predominantly derived from testes and germ cells were proposed as candidate cfs-mRNAs. Further quantification in men with NOA demonstrated significantly higher levels of BOLL cfs-mRNA (p < 0.0001) in men with late MA or hypospermatogenesis (n = 23), compared with men with eMA (n = 51); and significantly higher levels (p < 0.0001) in patients with successful SR (n = 44) when compared with patients with SR failure (n = 37). Interestingly, with a similar cutoff value, BOLL cfs-mRNA showed high sensitivity and specificity in diagnosing late MA and hypospermatogenesis (>404 copies/ml) and predicting successful SR (>415 copies/ml). Correlation of BOLL mRNA levels was observed in paired semen and testicular tissues. We propose that BOLL cfs-mRNA is a promising non-invasive marker for diagnosing the completion of meiosis and predicting successful testicular SR in men with NOA.

Bi-allelic SPATA22 variants cause premature ovarian insufficiency and nonobstructive azoospermia due to meiotic arrest.

The genetic causes of idiopathic premature ovarian insufficiency (POI) and nonobstructive azoospermia (NOA) remain unclear. We performed whole-exome sequencing (WES) in members of a consanguineous family with two POI and two NOA patients to screen for potential pathogenic variants for familial POI and NOA. And a homozygous variant in SPATA22 (c.400C>T:p.R134X) was identified. Histological analysis and spermatocyte spreading assay demonstrated that the spermatogenesis was arrested at a zygotene-like stage in the proband with NOA. The candidate gene was further screened in the in-house WES database of idiopathic POI-affected patients. One additional compound heterozygous variant in SPATA22 (c.900+1G>A and c.31C>T:p.R11X) was found in one patient with sporadic POI and validated by minigene assay. Thus, this is the first report identifying SPATA22 as the causative gene for human POI. Combined with the observations in the familial patient with NOA, our findings highlighted the essential role of meiotic HR genes in gametogenesis and gonadal function maintenance.

Recombinant gonadotropin therapy to improve spermatogenesis in nonobstructive azoospermic patients - A proof of concept study.

Nonobstructive azoospermia (NOA) associated with primary spermatogenic failure is a common cause of male infertility usually considered untreatable; however, some reports have suggested that hormonal stimulation to boost the intra-testicular testosterone level and spermatogenesis might increase the chance of achieving pregnancy using homologous sperm. We report a series of eight NOA males who received long-term treatment with recombinant human chorionic gonadotropin twice a week for spermatogenesis stimulation. Six males received additional recombinant follicle-stimulating hormone (FSH) supplementation 150-225 IU twice weekly. After recombinant gonadotropin therapy, viable spermatozoa were retrieved from the ejaculate in two patients and by testicular sperm aspiration (TESA) in another two subjects. Singleton spermatozoon retrieved from testes were frozen by vitrification on Cell-Sleeper devices. Two live births were obtained after intracytoplasmic sperm injection with ejaculated spermatozoa and one live birth and an ongoing pregnancy using thawed spermatozoa from TESA. Our proof-of-concept study indicates that hormonal therapy with recombinant gonadotropins could be considered in infertile men with NOA as an alternative to sperm donation. Large-scale studies are needed to substantiate hormone stimulation therapy with recombinant gonadotropins in routine clinical practice for this severe form of male infertility.

Correlation between high Choline metabolite signal in spectroscopy and sperm retrieval chance at micro-TESE.

To determine the relationship between choline (Cho) signal intensity measured before micro-dissection testicular sperm extraction (micro-TESE) and sperm retrieval rates in mTESE in non-obstructive azoospermia (NOA) patients. A total of 20 patients who underwent testicular MR spectroscopy were included in the study. Participants consisted of 10 patients diagnosed with NOA and 10 fertile cases with normal sperm counts. Both groups of participants underwent bilateral testis MR spectroscopy. Ten patients in the NOA group underwent mTESE after spectroscopy. The signal intensities of Cho, creatine (Cr), lactate, and lipids were analyzed and compared with the results of fertile control. Cho signal intensity detected before mTESE in the NOA group and sperm retrieval rates were compared. Sperm was found in 5 of 10 patients who underwent mTESE. No sperm was found in five NOA cases. The main metabolites detected in NOA cases with sperm in mTESE were Cho and Cr. Cho and Cr signals were found to be significantly lower in NOA cases where no sperm could be found in mTESE. Cho and Cr signal intensities of the fertile group were similar to NOA patients with sperm in mTESE but were significantly higher than those with NOA without sperm. While the cut-off value of Cho was 1.24 ppm (AUC 0.665, p = 0.01 [95% CI: 0.722-1.00]) the cut-off value of Cr was 1.18 ppm (AUC 0.887, p = 0.02 [95% CI]): 0.620-1.00]) for positive sperm retrieval. Detection of high Cho metabolite in the spectra before TESE in NOA patients increases sperm retrieval rates in mTESE.

Azoospermic men with isolated elevation of follicle-stimulating hormone represent a specific subpopulation of patients with poor reproductive outcomes

ObjectiveThis study aimed to describe a distinct subpopulation of azoospermic patients with isolated elevation of follicle-stimulating hormone (iFSH) and poor outcomes of microdissection testicular sperm extraction (microTESE).Methods A retrospective analysis of microTESE outcomes was conducted among 565 patients with non-obstructive azoospermia (NOA). Testicular pathology was assessed by the dominant histological pattern and Bergmann-Kliesch score (BKS). Descriptive statistics were presented for the iFSH subgroup. Inhibin B levels, the sperm retrieval rate (SRR), and BKS were compared in iFSH patients and other NOA patients.Results The overall SRR was 33.3% per microTESE attempt. The median BKS was 0.6 (interquartile range, 0–2). Of all NOA patients, 132 had iFSH, and microTESE was successful only in 11 of those cases, with an SRR of 8.3%, while the total SRR in other NOA patients was 38.1% (p<0.001). iFSH had a sensitivity of 32.1% (95% confidence interval [CI], 27.4%–36.8%) and specificity of 94.1% (95% CI, 90.8–97.5%) as a predictor of negative microTESE outcomes.Conclusion Patients with iFSH may harbor a distinct testicular phenotype with total loss of the germ cell population and poor outcomes of surgical sperm retrieval.

Repeated microdissection testicular sperm extraction in patients with non-obstructive azoospermia: Outcome and predictive factors

ABSTRACT Objective To assess the feasibility of repeated sperm recovery in patients with non-obstructive azoospermia (NOA), as little is known about the extraction rate in repeated microdissection testicular sperm extraction (microTESE) in these patients. Patients and Methods A total of 134 men with NOA had their first sperm recovery between January 2013 and February 2020. Repeated microTESE had been done mostly for patients with a successful initial retrieval. Results In the 323 procedures performed on the 134 men with NOA, sperm could be retrieved in 236 procedures (73.1%). A total of 88, 61 and 40 men underwent two, three and four sperm retrievals, respectively. In these cycles, sperm could be extracted in 65 (73.9%), 53 (86.9%) and 37 (92.5%) men, respectively. During the first microTESE procedure, sperm could be extracted in 81 (60.4%) men with NOA. In all, the success rate was significantly different between subgroups, showing highest rate in hypospermatogenesis cases (95.6%), followed by maturation arrest (58.5%), and Sertoli cell-only syndrome (56.0%). However, this difference was not significant at the third and fourth repeated microTESE. The FSH levels and testicular volume were among the noticeable factors affecting success of sperm retrieval. The duration between the first and second biopsies significantly increased the success rate by a factor of 1.3-fold/month; however, afterwards, the duration did not play any role in the success of microTESE. The success of previous trial significantly increased the probability of success by 10.1-fold in the second trial, 5.6-fold in the third trial, and 16.5 folds in the fourth. Conclusion Repeated MD -TESE ensures a high sperm recovery rate in patients with NOA. These data also show that when no spermatozoa can be obtained after thawing cryopreserved testicular sperm for ICSI in NOA patients, a repeat microTESE procedure can be planned. Abbreviations ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilisation; MA: maturation arrest; (N)OA: (non-)obstructive azoospermia; OR: odds ratio; SCOS, Sertoli cell-only syndrome; SRR: spermatozoa retrieval rate; (micro)TESE: (microdissection) testicular sperm extraction

Endocrine aberrations of human nonobstructive azoospermia.

Nonobstructive azoospermia (NOA) refers to the failure of spermatogenesis, which affects approximately 1% of the male population and contributes to 10% of male infertility. NOA has an underlying basis of endocrine imbalances since proper human spermatogenesis relies on complex regulation and cooperation of multiple hormones. A better understanding of subtle hormonal disturbances in NOA would help design and improve hormone therapies with reduced risk in human fertility clinics. The purpose of this review is to summarize the research on the endocrinological aspects of NOA, especially the hormones involved in hypothalamic–pituitary–testis axis (HPTA), including gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, testosterone, estradiol, sex hormone binding globulin, inhibin B, anti-Müllerian hormone, and leptin. For the NOA men associated with primary testicular failure, the quality of currently available evidence has not been sufficient enough to recommend any general hormone optimization therapy. Some other NOA patients, especially those with hypogonadotropic hypogonadism, could be treated with hormonal replacement. Although these approaches have succeeded in resuming the fertility in many NOA patients, the prudent strategies should be applied in individuals according to specific NOA etiology by balancing fertility benefits and potential risks. This review also discusses how NOA can be induced by immunization against hormones.

Identification and characterization of circular RNAs in the testicular tissue of patients with non-obstructive azoospermia.

Circular RNAs (circRNAs) are highly conserved and ubiquitously expressed noncoding RNAs that participate in multiple reproduction-related diseases. However, the expression pattern and potential functions of circRNAs in the testes of patients with non-obstructive azoospermia (NOA) remain elusive. In this study, according to a circRNA array, a total of 37 881 circRNAs were identified that were differentially expressed in the testes of NOA patients compared with normal controls, including 19 874 upregulated circRNAs and 18 007 downregulated circRNAs. Using quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we confirmed that the change tendency of some specific circRNAs, including hsa_circ_0137890, hsa_circ_0136298, and hsa_circ_0007273, was consistent with the microarray data in another larger sample. The structures and characteristics of these circRNAs were confirmed by Sanger sequencing, and fluorescence in situ hybridization revealed that these circRNAs were primarily expressed in the cytoplasm. Bioinformatics analysis was used to construct the competing endogenous RNA (ceRNA) network, and numerous miRNAs that could be paired with circRNAs validated in this study were reported to be vital for spermatogenesis regulation. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that genes involved in axoneme assembly, microtubule-based processes, and cell proliferation were significantly enriched. Our data suggest that there are aberrantly expressed circRNA profiles in patients with NOA and that these circRNAs may help identify key diagnostic and therapeutic molecular biomarkers for NOA patients.

Different group of non-obstructive intermittent azoospermias among non-obstructive azoospermia patients: A ten-year retrospective cohort study with control group.

We have identified patients among non-obstructive azoospermia (NOA) cases in whom spermatozoa could not be detected despite treatment, but intermittent ejaculatory spermatozoon was found in their follow-up. NOA was observed in the retrospective screening at a rate of 15.35% among infertile men (n=1976/12,871), while non-obstructive intermittent azoospermia (NO-IA) was detected at a rate of 6.8% among NO-IA (n=135/1976) and 1.1% among all infertile men (n=135/12,871). Spermatozoon was identified in the form of cryptospermia or extreme oligospermia in 58.13 (13.6-92.3) weeks on average in n=55/135 patients among NO-IA. Pregnancy and live birth were achieved at a rate of 43.6% (n=24/55) and 29% (n=16/55), respectively, in intracytoplasmic sperm injection. NO-IA was composed of a group with no genetic pathological diagnosis, with lower follicle-stimulating hormone, lutenizing hormone and clinical varicocele rates compared with those of NOA (<0.05) and higher testicular volumes and pathological scores (<0.05). A major activity was observed in total testosterone, lutenizing hormone, testicular volumes and the logistic regression of pathological scores (<0.05).

Leydig Cells in Patients with Non-Obstructive Azoospermia: Do They Really Proliferate?

Background: Non-obstructive azoospermia (NOA) is a form of male infertility caused by disorders of the testicular parenchyma and impaired spermatogenesis. This study aimed to investigate the nature of Leydig cell changes in patients with NOA, especially whether their actual proliferation occurred. Methods: 48 testicular biopsies from infertile patients with NOA and 24 testicular biopsies originating from azoospermic patients suffering from obstructive azoospermia (OA) were included in the study. Leydig cells and their possible proliferative activity were analysed by immunohistochemistry and morphometry (stereology). Results: Unlike in the OA group, Leydig cells in NOA patients were sometimes organised into larger clusters and displayed an abundant cytoplasm/hypertrophy. Moreover, significant fibrosis of the interstitial compartment was demonstrated in some NOA samples, often accompanied by inflammatory cells. Stereological analysis showed no increase/proliferation of Leydig cells; on the contrary, these cells decreased in number in the NOA group. Conclusions: The decrease in the number of Leydig cells can be explained by previous inflammatory changes within the testicular interstitium and consequent interstitial fibrosis. The interstitial fibrosis might have a deteriorating effect on Leydig cells.

Follicle-stimulating hormone (FSH) receptor gene polymorphisms in Iraqi patients with non-obstructive azoospermia

Background: Follicle-stimulating hormone (FSH) is a pivotal hormone for male fertility, and its action on gonads is exerted by FSH receptors (FSHRs). Objectives: To examine whether the presence of FSHR gene single nucleotide polymorphisms (SNPs), G919A and A2039G, involved in non-obstructive azoospermia (NOA) in Iraqi infertile men. Methods: Two common SNPs, A919G and A2039G, in the FSHR gene were analyzed in 104 subjects (70 infertile patients with NOO: 33 NOA patients were not receiving treatment and 37 were on infertility treatment, and 34 normozoospermic fertile men as controls). Results: The results revealed that the homozygous wild genotype (AA) of rs6165 FSHR gene SNP was more abundant than (AG) and (GG) genotypes in both groups of infertile NOA patients with a frequency of 49% in those who untreated, 81% in patients undergoing treatment and in the control group 41%. Whereas, the highest percentage of heterozygous genotype (AG) in the fertile control group was 41% when compared to NOApatients with a genotype frequency of 24% (for those who untreated) and 11% (for patients on treatment), respectively; with (A) allele frequency of 86% and the observed frequency of (G) allele was only 14% in the patients’ group as compared to that of controls that were (65 %) and (35 %), respectively. The rs6166 genotyping revealed that the homozygous wild genotype (GG) of FSHR gene was more abundant than (AG) and (AA) genotypes in NOA patients receiving infertility treatment with a frequency of (68%), in NOA patients who didn’t receive treatment 49%, while the lowest frequency was detected in the healthy fertile control group (47%). Conclusions: These results support the evidence that rs6165 and rs6166, FSHR SNPs, might be involved in the pathogenesis and protection against NOA, respectively.

Valproic acid promotes the in vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells

BackgroundStudying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on SSCLC induction.MethodsWe examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. Haploid cells and cells expressed meiotic genes were also detected. RNA-seq analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols.ResultsThe combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. The high concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-seq analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and decreased expression of genes in Wnt signaling pathway.ConclusionsVPA robustly promoted the differentiation of human pluripotent stem cells into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that SSC depletion in azoospermia testes might be associated with inactivation of Wnt signaling pathway. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.

A Comparative Analysis of the Altered Levels of Human Seminal Plasma Constituents as Contributing Factors in Different Types of Male Infertility.

(1) Background: The relationships between the biochemical and immunological components in seminal plasma and their physiological effects on male reproductive system have been underreported. In this study, we evaluated the potential of several seminal plasma biochemical and immunological markers in the pathophysiological developments of the infertile male patients. The study was designed to identify and assess different markers that may be associated with semen functions in different types of male infertility. (2) Methods: A total of 50 infertile male patients who underwent checkup for fertility assessment and 50 fertile controls were included in this study. The complete medical history of each recruited participant was reviewed. The infertile sub-groups (non-obstructive azoospermia (NOA), asthenozoospermia (AS), normozoospermic infertile (NI), and oligozoospermia (OZ)) were characterized based on sperm motility and concentration, while NI patients were included after a thorough check up of their female partners as well. We investigated each sample for 21 different analytes, enzymes, trace elements, and immunological markers to find crucial markers posing as contributing factors to a specific type of male infertility. (3) Results: The levels of 15 out of 21 markers, assayed from the seminal plasma of infertile males, were significantly altered in comparison to fertile controls (p < 0.05). For the first time, microprotein levels were also analyzed. The presence of monocytes, lymphocytes, and granulocytes was limited to semen from NOA patients, while a significant increase in the level of platelets was observed in AS. Hierarchical clustering and ROC-AUC analysis identified the three most significant markers (zinc, LDH, and TG) for the healthy control group and asthenozoospermic group (AUC, of 0.92 and 0.81, respectively). (4) Conclusions: The altered levels of biochemical and immunological markers in seminal plasma might be associated with the different male infertility profiles and could be required for the sperm metabolism and maintenance. However, a larger sample size and follow up analysis is required for establishing the hypothesized panel of markers as biomarkers at clinical stage.

Genetic Factors of Non-Obstructive Azoospermia: Consequences on Patients' and Offspring Health.

Non-Obstructive Azoospermia (NOA) affects about 1% of men in the general population and is characterized by clinical heterogeneity implying the involvement of several different acquired and genetic factors. NOA men are at higher risk to be carriers of known genetic anomalies such as karyotype abnormalities and Y-chromosome microdeletions in respect to oligo-normozoospermic men. In recent years, a growing number of novel monogenic causes have been identified through Whole Exome Sequencing (WES). Genetic testing is useful for diagnostic and pre-TESE prognostic purposes as well as for its potential relevance for general health. Several epidemiological observations show a link between azoospermia and higher morbidity and mortality rate, suggesting a common etiology for NOA and some chronic diseases, including cancer. Since on average 50% of NOA patients has a positive TESE outcome, the identification of genetic factors in NOA patients has relevance also to the offspring’s health. Although still debated, the observed increased risk of certain neurodevelopmental disorders, as well as impaired cardiometabolic and reproductive health profile in children conceived with ICSI from NOA fathers may indicate the involvement of transmissible genetic factors. This review provides an update on the reproductive and general health consequences of known genetic factors causing NOA, including offspring’s health.

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.

PD39-02 SINGLE CELL TRANSCRIPTOME ANALYSIS AND BIOINFORMATICS ANALYSIS OF ISOLATED HUMAN SPERMATOGONIA OBTAINED FROM MEN WITH NONOBSTRUCTIVE AZOOSPERMIA

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (PD39)1 Sep 2021PD39-02 SINGLE CELL TRANSCRIPTOME ANALYSIS AND BIOINFORMATICS ANALYSIS OF ISOLATED HUMAN SPERMATOGONIA OBTAINED FROM MEN WITH NONOBSTRUCTIVE AZOOSPERMIA Koji Shiraishi, Masanori Tabara, Haruka Matsukuma, and Hideyasu Matsuyama Koji ShiraishiKoji Shiraishi More articles by this author , Masanori TabaraMasanori Tabara More articles by this author , Haruka MatsukumaHaruka Matsukuma More articles by this author , and Hideyasu MatsuyamaHideyasu Matsuyama More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002049.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Transcriptome sequencing and bioinformatics analysis of spermatogonia obtained from men with nonobstructive azoospermia (NOA) were performed to identify the key genes and signaling pathways related to spermatogenic disorders. METHODS: Morphologically based isolation of spermatogonia was performed in an IVF laboratory, and approximately 400-500 cells per patient were isolated from testicular samples. Testicular tissues were obtained during testicular sperm extraction (TESE) from patients with NOA whose pathological diagnosis was early maturation arrest (n=25) and obstructive azoospermia (OA, n=5), and the purity of isolated cells was evaluated by immunostaining for DDX4, UTF1 and SCP3. Genome-wide RNA sequencing using a next-generation sequencer (Illumina HiSeq2500) was performed to obtain transcriptional data. Differentially expressed genes (DEGs) were identified, and quantitative real-time PCR was used to verify the accuracy of the expression profile of key DEGs. Gene ontology (GO) analysis and pathway analysis using the Kyoto Encyclopedia of Gene and Genomes (KEGG) were performed to evaluate the biological significance of the gene expression profiles. RESULTS: A mean of 96% of the isolated cells showed DDX4, UTF1 and SCP expression, indicating that the isolated cells had a high purity of spermatogonia. Transcriptome analysis showed that 5,675 DEGs were upregulated and 12,596 DEGs were downregulated in the spermatogonia from NOA patients. The functional clusters of DNA synthesis, cell division, stress response and glucose metabolism were disrupted in the spermatogonia from NOA patients. KEGG analysis revealed that strong connections between cell division and glucose metabolism were the most enriched pathways in normal spermatogonia, whereas there were a variety of pathway changes with sparse connections for each gene in spermatogonia from NOA patients. CONCLUSIONS: Our genome-wide analysis has shown that spermatogenic arrest is caused by a complex association among a variety of gene expression profiles, but not a single gene disruption, which may help us to obtain a precise and comprehensive understanding of the pathophysiology of NOA. The results of GO and KEGG network analyses suggest that NOA is characterized by several clusters (e.g., low DNA synthesis, androgen signal impairment, impaired stress response and somatic cell-like clusters) that could be used for the establishment of personalized NOA diagnosis and treatment. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e668-e668 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Koji Shiraishi More articles by this author Masanori Tabara More articles by this author Haruka Matsukuma More articles by this author Hideyasu Matsuyama More articles by this author Expand All Advertisement Loading ...

Microdissection testicular sperm extraction (micro-TESE) in men with infertility due to nonobstructive azoospermia: summary of current literature.

Nonobstructive azoospermia (NOA) is associated with intrinsic testicular defects that severely impair sperm production. Although NOA invariably leads to infertility, focal sperm production may exist in the testicles of affected patients, which canbe retrieved and usedforintracytoplasmic sperm injection (ICSI) to generate healthy offspring. However, geographic locations of testicular sperm producing-areas are uncertain, making microsurgical-guided sperm retrieval (microdissection testicular sperm extraction; micro-TESE) an attractive method to identify and retrieve sperm in patients with NOA due to spermatogenic failure. Given the widespread use of micro-TESE, its effectiveness in harvesting sperm and related potential complications need to be clarified. We queried PubMed/MEDLINE for studies published in English, from inception to May 2021, concerning the effect of micro-TESE on sperm retrieval rate (SRR), complication rate and ICSI pregnancy rate-using retrieved testicular sperm in subfertile couples where the male had NOA. We found 116 articles, including 70 original papers, 32 review articles, and 14 systematic reviews. The evidence accounted for 4895 patients. Micro-TESE retrieved sperm in 46.6% of men with NOA,but SRRs varied considerably (18.4-70.8%) and were mainly related to the treated population characteristics. Concerning the general population of NOA patients who have not undergone previous sperm retrieval (naïve population), the SRR by micro-TESEwas 46.8% (1833 of 3914 patients; range 20-70.8%; 28 studies). In studies reporting SR by micro-TESE for men who had failed percutaneous testicular sperm aspiration or non-microsurgical testicular sperm extraction, the SRR was 39.1% (127 of 325 patients; range 18.4-57.1%; 4 studies). Dataon adverse events indicated that micro-TESE was associated with low (~ 3%) short-term postoperative complication rates. The fertilizing ability of testicular sperm retrieved by micro-TESE and usedfor ICSI was adequate (~ 57%), whereas clinical pregnancy and live birth were obtained in 39% and 24% of couples who had an embryo transfer, respectively. The health of the resulting children seems reassuring, but the evidence is limited. The procedure increases sperm retrieval success compared to non-microsurgical retrieval methods, particularly in men with Sertoli cell-only testicular histopathology. We concluded that micro-TESE is an effective and safe method to retrieve sperm from men with NOA-relatedinfertility, with potential advantages over non-microsurgical methods. Nevertheless, high-quality, head-to-head comparative randomized controlled trials by sperm retrievalmethod,focusing on SRR, live birth rate and assessing long-term adverse events and health of children conceived using testicular sperm from NOA patients are lacking. Therefore, further research is required to determine the full clinical implications of micro-TESE in male infertility treatment.

Evaluation of immune status in testis and macrophage polarization associated with testicular damage in patients with nonobstructive azoospermia.

Immune cells residing in the testicular interstitial space form the immunological microenvironment of the testis. They are assumed to play a role in maintaining testicular homeostasis and immune privilege. However, the immune status and related cell polarization in patients with nonobstructive azoospermia (NOA) remains poorly characterized. System evaluation of the testis immunological microenvironment in NOA patients may help to reveal the mechanisms of idiopathic azoospermia. The gene expression patterns of immune cells in normal human testes were systematically analyzed by single-cell RNA sequencing (scRNA-seq) and preliminarily verification by the human protein atlas (HPA) online database. The immune cell infiltration profiles and immune status of patients with NOA was analyzed by single-sample gene set enrichment analysis (ssGSEA) and gene set variation analysis (GSVA) based on four independent public microarray datasets (GSE45885, GSE45887, GSE9210, and GSE145467), obtained from Gene Expression Omnibus (GEO) online database. The relationship between immune cells and spermatogenesis score was further analyzed by Spearman correlation analysis. Finally, immunohistochemistry (IHC) staining was performed to identify the main immune cell types and their polarization status in patients with NOA. Both scRNA-seq and HPA analysis showed that testicular macrophages represent the largest pool of immune cells in the normal testis, and also exhibit an attenuated inflammatory response by expressing high levels of tolerance proteins (CD163, IL-10, TGF-β, and VEGF) and reduced expression of TLR signaling pathway-related genes. Correlation analysis revealed that the testicular immune score and macrophages including M1 and M2macrophages were significantly negatively correlated with spermatogenesis score in patients with NOA (GSE45885 and GSE45887). In addition, the number of M1 and M2macrophages was significantly higher in patients with NOA (GSE9210 and GSE145467) than in normal testis. GSVA analysis indicated that the immunological microenvironment in NOA tissues was manifested by activated immune system and pro-inflammatory status. IHC staining results showed that the number of M1 and M2macrophages was significantly higher in NOA tissues than in normal testis and negatively correlated with the Johnson score. Testicular macrophage polarization may play a vital role in NOA development and is a promising potential therapeutic target.