Non-obstructive Azoospermia Group Research Articles

Testicular piRNA Analysis Identified Dysregulated piRNAs in Non-obstructive Azoospermia.

Male infertility has remained idiopathic in a remarkable proportion of all cases. Gonadal expression of PIWI-interacting RNAs (piRNAs) has been shown to be vital to normal spermatogenesis, as they are expressed in almost all types of testicular germ cells. These molecules and their related Piwi proteins strictly regulate transposable elements' activity and gene expression. We aimed to identify dysregulated piRNAs in idiopathic non-obstructive azoospermic (NOA) testis by global expression analysis. Testis tissue samples from 18 azoospermic patients (ten NOA and eight OA) were studied by small RNA sequencing. To validate high-throughput sequencing data, quantitative real-time polymerase chain reactions for two differentially altered piRNAs were performed. Bioinformatics analyses were undertaken to identify pathways affected by piRNA dysregulation. In the NOA group, 1328 piRNAs were identified to be differentially expressed, of which 1322 were downregulated and 6 were upregulated. Bioinformatics analysis corroborated the involvement of dysregulated piRNA in spermatogenesis. We also identified 64 clusters of differentially expressed piRNAs, of which 42 clusters had a minimum of ten absolute piRNA hits. Our study suggests that piRNAs show significant dysregulation in infertility. Their target genes play a role in their self-biogenesis, probably by regulating their own production through a feedback mechanism. The downregulated piRNAs may find value as biomarkers for the presence of spermatozoa in the testis of azoospermic individuals, while the upregulated piRNAs are great candidates for further investigation of their precise functions in spermatogenesis.

Correlation of serum kisspeptin and leptin with non-obstructive azoospermia: A cross-sectional study in a subset of Karachi population.

To determine the association of serum kisspeptin, leptin, and other hormonal profile with non-obstructive azoospermia (NOA) in infertile male subjects. This cross-sectional study was conducted at Australian Concept Infertility Medical Center, and Ziauddin University, Karachi from March 2018 to March 2020. The duration of the study was two years. Serum samples of 106 azoospermic participants were taken. Division of the subjects was done on a histological basis into obstructive azoospermia (OA) n=36, NOA n=70 which were further divided into spermatid maturation arrest (SMA), n=41, and sertoli cell-only syndrome (SCOS) n=29. Serum kisspeptin and leptin were measured by ELISA (Cloud-Clone Corp). The follicle-stimulating hormone (FSH) (p<0.01), luteinizing hormone (LH) (p<0.01), thyroid-stimulating hormone (TSH) (p<0.01), and estradiol (p<0.01) was significantly high in the NOA group. However, kisspeptin was significantly decreased (p<0.01) in the NOA group. In the multivariate analysis after adjusting for other variables, the results showed that with the decrease in kisspeptin, the chances of being NOA were increased. Moreover, with the increase in Leptin, FSH, LH, and TSH the chances of being NOA were significantly enhanced. Serum kisspeptin, leptin, FSH, LH, TSH, and estradiol can be a potential marker for NOA in terms of better diagnosis, targeted therapeutic management, and the decision to proceed with surgical intervention.

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

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

Proteomic Analysis of Testicular Interstitial Fluid in Men with Azoospermia

BackgroundThe primary microenvironment of the testis comprises testicular interstitial fluid (TIF) surrounding the seminiferous tubules and testicular interstitial tissue. The pathological alterations of germ and Sertoli cells could affect the TIF composition and might contain putative biomarkers for monitoring active spermatogenesis. ObjectiveWe identified differentially expressed proteins in the TIF of patients with obstructive (OA) or nonobstructive (NOA) azoospermia to elucidate the underlying etiology of defective spermatogenesis. Design, setting, and participantsWe prospectively enrolled nine patients, including three men with OA and six with NOA with (n = 3) and without (n = 3) successful sperm retrieval. Their TIF was collected during the testicular sperm extraction procedure. Outcome measurements and statistical analysisTIF was analyzed using liquid chromatography-tandem mass spectrometry to identify differentially expressed proteins specific to OA and NOA with or without successful sperm retrieval. The dysregulated protein was further validated using Western blotting. Results and limitationsAmong the 555 TIF proteins identified in NOA patients, 14 were downregulated relative to OA patients. These proteins participate in biological processes such as proteolysis, complement activation, and immune responses; complement and coagulation cascade pathways were also enriched. Furthermore, 68 proteins with significantly higher levels were identified in the TIF of NOA patients with successful sperm retrieval than in those with failed sperm retrieval; these are mainly implicated in oxidation-reduction processes. The expression of calreticulin, which can distinguish successful and failed testicular sperm retrieval in the NOA group, was validated by Western blotting. ConclusionsWe provide the first scientific evaluation of TIF protein composition in men with azoospermia. These findings will help identify the physiological and pathological roles of each protein in regulating sperm production. Thus, our study underscores the potential of TIF in sperm retrieval biomarker discovery and would serve as a foundation for further studies to improve treatment strategies against azoospermia. Patient summaryUsing a proteomic approach, we identified and analyzed the total protein content of testicular interstitial fluid in humans with defective spermatogenesis for the first time and discovered altered protein expression patterns in patients with nonobstructive azoospermia (NOA). Proteins related to oxidation-reduction processes were upregulated in NOA patients with successful sperm retrieval compared with those with failed sperm retrieval. This can aid the development of novel diagnostic tools for successful testicular sperm retrieval.

Identification and validation of diagnostic signature genes in non-obstructive azoospermia by machine learning.

Non-obstructive azoospermia (NOA) is a common cause of male infertility, and no specific diagnostic indicators exist. In this study, we used human testis datasets GSE45885, GSE45887, and GSE108886 from GEO database as training datasets, and screened 6 signature genes (all lowly expressed in the NOA group) using Boruta algorithm and Lasso regression: C12orf54, TSSK6, OR2H1, FER1L5, C9orf153, XKR3. The diagnostic efficacy of the above genes was examined by constructing models with LightGBM algorithm: the AUC (Area Under Curve) of both ROC and Precision-Recall curves for internal validation was 1.0 (p < 0.05). For the external validation dataset GSE145467 (human testis), the AUC of its ROC curve was 0.9 and that of its Precision-Recall curve was 0.833 (p < 0.05). Next, we confirmed the cellular localization of the above genes using human testis single-cell RNA sequencing dataset GSE149512, which were all located in spermatid. Besides, the downstream regulatory mechanisms of the above genes in spermatid were inferred by GSEA algorithm: C12orf54 may be involved in the repression of E2F-related and MYC-related pathways, TSSK6 and C9orf153 may be involved in the repression of MYC-related pathways, while FER1L5 may be involved in the repression of spermatogenesis pathway. Finally, we constructed a NOA model in mice using X-ray irradiation, and quantitative Real-time PCR results showed that C12orf54, TSSK6, OR2H1, FER1L5, and C9orf153 were all lowly expressed in NOA group. In summary, we have identified novel signature genes of NOA using machine learning methods and complete experimental validation, which will be helpful for its early diagnosis.

MP01-07 A NEXT-GENERATION SEQUENCING ANALYSIS OF THE SEMEN MICROBIOME IN MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA

You have accessJournal of UrologyCME1 Apr 2023MP01-07 A NEXT-GENERATION SEQUENCING ANALYSIS OF THE SEMEN MICROBIOME IN MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA Katherine Campbell, Farah Rahman, Camila Suarez, Aaron Miller, Emad Ibrahim, Scott D. Lundy, and Ranjith Ramasamy Katherine CampbellKatherine Campbell More articles by this author , Farah RahmanFarah Rahman More articles by this author , Camila SuarezCamila Suarez More articles by this author , Aaron MillerAaron Miller More articles by this author , Emad IbrahimEmad Ibrahim More articles by this author , Scott D. LundyScott D. Lundy More articles by this author , and Ranjith RamasamyRanjith Ramasamy More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Detection of a microbiome profile coupled with advanced genetic sequencing has opened a new research frontier. We hypothesized that the semen microbiome profile in men with non-obstructive azoospermia (NOA) would differ from that of fertile controls (FC). METHODS: NOA was diagnosed using semen analysis and clinical phenotype (small testis volume, FSH >10 IU/mL). FC were identified as men undergoing vasectomy after fathering at least one child. Semen samples were collected and underwent Next Generation Sequencing (NGS) including quantitative PCR and 16s rRNA V1/V2 region comprehensive sequencing using Illumina MiSeq technology. Data was processes and analyzed in dada2. Alpha diversity was analyzed via Margalef. Beta diversity was analyzed via Bray-Curtis difference. Differential abundance was evaluated with ANCOMBC procedure. RESULTS: After rarefaction analysis, 33 samples were included (18 NOA and 15 FC) determined samples had sufficient sequencing depth to capture microbiome diversity. Alpha diversity was lower in the NOA men but did not reach statistical significance. Beta diversity showed a difference between the NOA and fertile groups primarily driven by differences in ethnicity. At the phylum level, NOA patients demonstrated lower levels of Proteobacteria and Firmicutes and higher levels of Actinobacteriota compared to the FC group consistent with prior published work. At the genus level, the known uropathogens Ureaplasma (1.3% vs 0.16%) and Eschrichia/Shigella (2.26% vs 0.42%) were higher in the NOA group compared to fertile controls. Other notable differences were Raoultella (39-fold higher in Fertile – 2% vs 0.09%) and Sneathia (9.5-fold higher in Fertile – 1% vs. 0.1%). Differential abundance analysis largely confirmed these findings. CONCLUSIONS: Compared to fertile controls, men with NOA have a less diverse seminal microbiome enriched for known pathogens and several understudied organisms. This work supports the presence of a testicular microbiome, which may become dysbiotic with infertility and could be leveraged as a biomarker to guide treatment decision making in the future. Source of Funding: MicrogenDX © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e4 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Katherine Campbell More articles by this author Farah Rahman More articles by this author Camila Suarez More articles by this author Aaron Miller More articles by this author Emad Ibrahim More articles by this author Scott D. Lundy More articles by this author Ranjith Ramasamy More articles by this author Expand All Advertisement PDF downloadLoading ...

The impact of semen parameters on ICSI and pregnancy outcomes in egg recipient cycles with PGT-A.

The egg donation model offers an opportunity to isolate the male factor and evaluate its impact on IVF-intracytoplasmic sperm injection and pregnancy outcomes. To study the effect of non-obstructive azoospermia on intracytoplasmic sperm injection and pregnancy outcomes compared with severe oligozoospermia and mild-to-moderate oligozoospermia in egg recipient cycles. This is a retrospective longitudinal cohort study, including 1594 patients who underwent intracytoplasmic sperm injection in egg recipient cycles with preimplantation genetic testing for aneuploidies. The cohort was divided into three groups: couples with non-obstructive azoospermia accounting for 479 patients (30%); couples with severe oligozoospermia (sperm number <5×106 /mL), accounting for 442 patients (27.8%); couples with mild-to-moderate oligozoospermia, with sperm number >5×106 and <15×106 /mL, accounting for 673 patients (42.2%). The fertilisation rate was significantly reduced in the non-obstructive azoospermia group as compared with the severe oligozoospermia and the mild-to-moderate oligozoospermia group: 30.3% versus 63% and 77.3% (p<0.05). Logistic regression analysis adjusted for confounders highlighted non-obstructive azoospermia as a negative predictor of obtaining a euploid blastocyst both per injected oocyte and per obtained blastocyst. The miscarriage rate in the non-obstructive azoospermia group was 11.8%; higher than the severe oligozoospermia and mild-to-moderate oligozoospermia groups (7% and 2.7%) (p<0.05). The live birth rate per embryo transfer (ET) was significantly lower in the non-obstructive azoospermia group compared with the severe oligozoospermia and the mild-to-moderate oligozoospermia group (20.4% vs. 30.3% and 35.4%, p<0.05). The risk of preterm labour was significantly higher in the non-obstructive azoospermia group, compared with the severe oligozoospermia and mild-to-moderate oligozoospermia group (55.1% vs. 46.8% and 16.1%, p<0.001), and this difference was observed in both singleton and twin pregnancies. In our retrospective comparative study, non-obstructive azoospermia significantly affects early embryonic potential and live birth rates per cycle and per embryo transfer. It is also associated with higher risk of preterm birth. Future prospective multi-centre studies are needed to highlight the effect of sperm quality on ART and pregnancy outcomes.

Diffusion-weighted magnetic resonance imaging and magnetic resonance spectroscopy for non-invasive characterization of azoospermia: A prospective comparative single-center study.

Azoospermia affects about 15% of childless males. The differential diagnosis between subtypes of azoospermia is the initial step in its management. To investigate the role of diffusion-weighted magnetic resonance imaging and proton magnetic resonance spectroscopy in distinguishing obstructive azoospermia from non-obstructive azoospermia and predicting sperm retrieval together with histological alterations in men with non-obstructive azoospermia. This prospective comparative study involved 60 men with obstructive azoospermia (group A) and 60 men with non-obstructive azoospermia (group B). Scrotal proton magnetic resonance spectroscopy and diffusion-weighted magnetic resonance imaging were conducted for all participants to respectively evaluate testicular metabolites and normalized apparent diffusion coefficient 1 week before sperm retrieval. Apparent diffusion coefficient was significantly higher in group B as compared to group A (0.47±0.11vs. 0.29±0.05; and 0.46±0.14vs. 0.28±0.02) for the right and left testis, respectively. Conversely, testicular choline and lipids were significantly higher in group A as compared to group B. Normalized apparent diffusion coefficient, choline, and lipids at cut-off levels of 0.353, 0.31, and 0.725 could differentiate between obstructive azoospermia and non-obstructive azoospermia (area under the curve=0.963; confidence interval=0.939-0.986, area under the curve=0.985; confidence interval=0.974-0.997, and area under the curve=0.970; confidence interval=0.940-0.999, respectively). Regarding the prediction of sperm retrieval in the non-obstructive azoospermia group, choline levels had the highest area under the curve (0.923), and its cut-off level was 0.195. The normalized apparent diffusion coefficient was significantly lower in men with positive sperm retrieval as compared to men with unsuccessful retrieval. Finally, it was revealed that all magnetic resonance imaging parameters except creatine could independently predict testicular histology in men with non-obstructive azoospermia. The highest prediction was 95% in normal spermatogenesis, and the least prediction was 40% in spermatid arrest. Regression analysis was used to detect final predictors and extrapolate an equation that could be used to predict testicular pathology CONCLUSIONS: Normalized apparent diffusion coefficient and proton magnetic resonance spectroscopy are helpful in differentiating obstructive azoospermia from non-obstructive azoospermia and predicting sperm retrieval and related histological alterations in men with non-obstructive azoospermia.

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

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

Contribution of TEX15 genetic variants to the risk of developing severe non-obstructive oligozoospermia.

Background: Severe spermatogenic failure (SPGF) represents one of the most relevant causes of male infertility. This pathological condition can lead to extreme abnormalities in the seminal sperm count, such as severe oligozoospermia (SO) or non-obstructive azoospermia (NOA). Most cases of SPGF have an unknown aetiology, and it is known that this idiopathic form of male infertility represents a complex condition. In this study, we aimed to evaluate whether common genetic variation in TEX15, which encodes a key player in spermatogenesis, is involved in the susceptibility to idiopathic SPGF. Materials and Methods: We designed a genetic association study comprising a total of 727 SPGF cases (including 527 NOA and 200 SO) and 1,058 unaffected men from the Iberian Peninsula. Following a tagging strategy, three tag single-nucleotide polymorphisms (SNPs) of TEX15 (rs1362912, rs323342, and rs323346) were selected for genotyping using TaqMan probes. Case-control association tests were then performed by logistic regression models. In silico analyses were also carried out to shed light into the putative functional implications of the studied variants. Results: A significant increase in TEX15-rs1362912 minor allele frequency (MAF) was observed in the group of SO patients (MAF = 0.0842) compared to either the control cohort (MAF = 0.0468, OR = 1.90, p = 7.47E-03) or the NOA group (MAF = 0.0472, OR = 1.83, p = 1.23E-02). The genotype distribution of the SO population was also different from those of both control (p = 1.14E-02) and NOA groups (p = 4.33-02). The analysis of functional annotations of the human genome suggested that the effect of the SO-associated TEX15 variants is likely exerted by alteration of the binding affinity of crucial transcription factors for spermatogenesis. Conclusion: Our results suggest that common variation in TEX15 is involved in the genetic predisposition to SO, thus supporting the notion of idiopathic SPGF as a complex trait.

"Seminal testosterone", rising viewpoint of local spermatogenesis in nonobstructive azoospermia: One center long-term bidirectional cohort study.

Reproductive hormones are a traditional good method to evaluate spermatogenesis but might not accurately represent local spermatogenesis. To find a more accurate method, seminal reproductive hormones were studied. A bidirectional cohort study was performed. A total of 126 infertile men from 2018 to 2019 were retrospectively analyzed. They were divided into nonobstructive azoospermia (NOA), oligozoospermia (OLZ) and normal (NOR) groups. A prospective study was conducted on patients in the NOA and OLZ groups for 2 years. Microscopic testicular sperm extraction was performed for NOA patients, who were divided into a focal spermatogenesis group (FS) and an idiopathic azoospermia group (IA). Drug treatment was for OLZ patients, who were divided into a valid group (VA) and an invalid group (IN). The differences in sperm parameters and reproductive hormones were compared. ANOSIM analysis was used between and within groups. Pearson correlation analysis, CO inertia analysis and Proctor's analysis were for relationships. ROC curve for the specificity and sensitivity. Time series analysis was for the trends between hormones and time. The b-FSH, b-LH, s-T and ΔT in the NOA group were significantly higher than those in the OLZ and NOR groups. However, the s-FSH, s-E2, s-P, ΔFSH, ΔLH, ΔP and ΔE2 were lower. Thirty-one NOA patients underwent MTSE, of whom 12 had sperm (FS) and 19 had no sperm (IA). The s-FSH and s-E2 of the FS group were higher than those of the IA group. Twenty-six OLZ patients completed 30 days of treatment, of which 11 had an improved sperm count (VA) and 15 had no (IN). The ΔT of the VA group was higher than that of the IN group. After follow-up for 2 years, 18 patients' results showed that b-FSH, b-LH and s-T were different over time, with delays of 19, 3 and -19 days. SC is closely related to pH, s-FSH, s-LH, s-E2, s-P, s-T, b-FSH, b-LH, ΔFSH, ΔLH, ΔP, ΔE2 and ΔT. There were complex common trends and relationships between different kinds of hormones. s-FSH, s-LH, s-E2, s-P, s-T, b-FSH and b-LH were useful to judge spermatogenesis, of which s-T, b-FSH and b-LH were more sensitive. If s-T, b-FSH and b-LH reached 64.4, 9.4 and 4.7, respectively, their prediction performance was the strongest. Seminal testosterone is sensitive for judging local spermatogenesis in nonobstructive azoospermia patients, which may be the direction of local spermatogenesis in nonobstructive azoospermia. http://www.chictr.org.cn/index.aspx, identifier ChiCTR2200060463.

Micro-TESE surgery combined with ICSI regimen in the treatment of non-obstructive azoospermia patients and its effect analysis.

This study aimed to analyze the clinical effects of microdissection testicular sperm extraction (micro-TESE) surgery combined with an intracytoplasmic sperm injection (ICSI) regimen in the treatment of non-obstructive azoospermia (NOA) patients with different etiologies. In total, 128 NOA patients participated in this study, in which they received infertility treatment by micro-TESE surgery combined with an ICSI regimen, and all patients were divided into three groups [the Klinefelter syndrome (KS), the idiopathic and the secondary NOA groups]. In addition, the sperm retrieval rate (SRR), fertilization rate, embryo development status and clinical treatment effects were analyzed. Among the 128 NOA patients, the SRR of KS NOA patients was 48.65%, those of idiopathic and the secondary patients were 33.82% and 73.91%, respectively. Regardless of etiologies, there was no correlation with age, hormone value or testicular volume. Further analysis showed that the SRR of the KS group was positively related with testosterone (T) values, and the SRR of the secondary group had a positive relationship with follicle-stimulating hormone or luteinizing hormone values. In the subsequent clinical treatment, the retrieved sperm was subjected to ICSI and achieved good treatment effects, especially in the secondary group, and the implantation rate (55.56%) and clinical pregnancy rate (68.42%) were both higher than those of the idiopathic group (28.75% and 40.00%) and KS group (22.05% and 30.77%). Micro-TESE surgery combined with ICSI insemination is the most effective treatment regimen for NOA patients. The SRR of NOA patients with different etiologies are related to certain specific factors, and micro-TESE surgery seems to be the ideal and only way to have biological children.

O-249 The impact of semen parameters on ICSI and pregnancy outcomes in egg recipient cycles with PGT-A

Abstract Study question To assess the effect of male factor on ICSI and pregnancy outcomes in egg recipient cycles combined with preimplantation aneuploidy testing Summary answer In egg donor cycles where sperm was obtained from azoospermic men there was a significant effect on ART and clinical outcomes. What is known already The increased incidence of chromosomal problems in men with azoospermia and its correlation with chromosomally abnormal embryos and with adverse pregnancy outcomes has been shown by multiple studies. Nevertheless, few studies in the literature outline the impact of non-obstructive azoospermia in the subgroup of couples who opt to use donated eggs. The egg donation model offers an opportunity to isolate the male factor and evaluate its impact on IVF-ICSI and pregnancy outcomes. The aim of our study was to study the effect of non-obstructive azoospermia(NOA) on ICSI outcomes compared with oligoasthenozoospermia(OATS) and moderate male factor(MMF) infertility, from ART parameters to clinical outcomes. Study design, size, duration This is a retrospective longitudinal cohort study involving 1,594 ICSI cycles using donor eggs performed between January 2016 and May 2020. The cohort was divided into three groups according to the male partner’s sperm parameters: couples with NOA accounting for 479 cycles (30%); couples with OAT-S (sperm number &amp;lt;5 x 106/ml), accounting for 442 cycles (27.8%); couples with moderate male factor, with sperm number &amp;gt;_5 x 106/ml and &amp;lt;15 x 106/ml, accounting for 673 cycles (42.2%). Participants/materials, setting, methods Participants: Subfertile couples who opted to use donor eggs. Donor eggs were utilized, due to history of severe female infertility, including low ovarian reserve, poor response to ovarian stimulation, repeated IVF failures or premature ovarian failure. Setting: Private IVF Unit Intervention: ICSI with ejaculated/surgically retrieved sperm, blastocyst culture, PGT-A and frozen-thawed euploid embryo transfer. Outcomes: The primary outcomes were live birth, fertilization, blastocyst development and euploidy rates; the secondary outcomes were clinical pregnancy, miscarriage rates, preterm labour and SGA. Main results and the role of chance The fertilization rate was significantly reduced in the NOA group as compared to the OATS and the MMF group: 30.3% versus 63% and 77.3%. Logistic regression analysis adjusted for confounders highlighted NOA as a negative predictor of obtaining an euploid blastocyst per inseminated oocyte. When the analysis was performed per obtained blastocyst, no correlation between male factor and euploidy rate was observed. The clinical pregnancy rates were similar in the MMF and OATS group, but significantly lower in the NOA group (38.2% versus 36.3% and 29.5% respectively). The miscarriage rate in the NOA group was 11.8%; higher than the OATS and MMF group (7% and 2.7%). The live birth rate per ET was significantly lower in the NOA group compared to the OATS and the MMF group (20.4% vs 30.3% and 35.4%,p&amp;lt;0.05). The live birth rate was significantly lower in the OATS group compared to the MMF group(p &amp;lt; 0.05). The risk of preterm labour was significantly higher in the NOA group, compared to the OATS and MMF group (55.1% versus 46.8 and 16.1%,p&amp;lt;0.001) and a higher number of babies born with a birth weight &amp;lt;2.5kg came from the NOA group, compared to the OATS and MMF group (34% versus 19.8% and 12.2%, p &amp;lt; 0.001). Limitations, reasons for caution The present study is limited by its retrospective design and the fact that the confounding factors of advanced maternal age, endometrial ageing and uterine factors cannot be fully evaluated. However, the study was controlled for female and male partner's age. Neonatal outcomes were not available. Wider implications of the findings Non-obstructive azoospermia can impair early embryonic potential and reduce live birth rates. Our study also shows an association between NOA and preterm birth. Future research should focus on prospective collection of data and long-term follow up of babies born by fathers with non-obstructive azoospermia. Trial registration number Not applicable

P-538 KATNAL1 polymorphisms confer susceptibility to severe phenotypes of male infertility in a large European cohort

Abstract Study question What is the contribution of the common variation of KATNAL1 to the development of severe spermatogenic failure (SPGF) in a phenotypically well-characterized cohort? Summary answer An allelic combination of KATNAL1 single-nucleotide polymorphisms (SNPs) increases the risk to develop SPGF, likely by altering the expression and splicing pattern of the gene. What is known already Spermatogenesis is a process that requires an exhaustive control of gene expression, and subtle alterations of its molecular regulatory network can lead to male infertility. The aetiology of most SPGF cases remains unknown, and increasing evidence clearly suggests that the idiopathic form of SPGF represents a complex trait, in which genetic susceptibility is conferred by the accumulation of risk alleles in genetically predisposed men. In this regard, previous studies reported that rare genetic mutations and polymorphisms in the KATNAL1 locus lead to male infertility through the disruption of microtubule remodelling and premature germ cell exfoliation. Study design, size, duration We designed a case-control genetic association study including three SNPs (rs2077011, rs7338931, and rs2149971) in the 3' and 5' regulatory regions of KATNAL1, which tagged the common genetic variability in the region. The allele frequencies in the study population, composed of 715 infertile men diagnosed with idiopathic SPGF, were compared to those observed in a control group comprising 1058 fertile men from Spain and Portugal. Participants/materials, setting, methods The SPGF group comprised 210 men with severe oligospermia (SO) and 505 with non-obstructive azoospermia (NOA). The latter were phenotypically characterised according to the histological examination of testis biopsies and its outcome (Sertoli cell-only syndrome, SCO; maturation arrest, MA; hypospermatogenesis, HS; and testicular sperm extraction, TESE). After genotyping, case-control association analyses by logistic regression were conducted. In silico functional characterization of risk variants was also carried out using public multiomic databases and bioinformatic tools. Main results and the role of chance Significant genetic associations with different SPGF patterns and/or TESE outcome were observed even after correction for multiple testing when independent SNP models were tested. However, in all cases, the haplotype model including the three risk alleles (rs2077011*C | rs7338931*T | rs2149971*A) was the most informative. This haplotype was specifically over-represented in the SPGF group (P = 3.45E-02, OR = 2.33), which includes all infertile men, and in the NOA group (P = 8.22E-03, OR = 2.97). In addition, subtype-specific associations were observed with the most severe subgroups, namely MA (P = 2.44E-02, OR = 5.00), SCO (P = 4.03E-03, OR = 5.16), and unsuccessful TESE outcome (P = 2.22E-04, OR = 6.13), which indicates the relevant role of KATNAL1 in spermatogenesis development. We prioritized the most likely causal variant/s based on in silico analyses addressing the possible functional implication of the tagged variants. We observed that an alteration of the KATNAL1 splicing pattern, by favouring the overrepresentation of a short non-functional transcript isoform in the testis, might be the cause behind the observed genetic association. The analysis of the testis transcriptome at the single cell level showed that KATNAL1 transcripts were mostly presented in spermatocytes and early spermatids at puberty, which correlates with its effect of premature exfoliation and loss of the germ cells. Limitations, reasons for caution Although a previous low-powered study reported suggestive associations between KATNAL1 and SPGF, additional genetic association studies in independent populations should be conducted to confirm our findings. Moreover, the statistical power for the overall analysis was appropriate, but the subphenotype analyses were performed with reduced power due to smaller study groups. Wider implications of the findings Our results suggest a relevant role of the common genetic variation of KATNAL1 in the susceptibility to develop the most severe histological phenotypes of NOA (i.e., SCO and MA). Studies like the one presented here may definitively help to develop future non-invasive molecular markers of TESE success. Trial registration number Not Applicable

Micro-testicular sperm extraction outcomes for non-obstructive azoospermia in a single large clinic in Victoria.

To evaluate the results of microdissection testicular sperm extraction (micro-TESE) and intracytoplasmic sperm injection (ICSI) for treatment of non-obstructive azoospermia (NOA). We retrospectively analysed data of 88 consecutive patients with clinical NOA who were treated with micro-TESE by a single surgeon, between August 2014 and September 2020, in Melbourne, Victoria. Upon a successful sperm retrieval, sperm was either used fresh for ICSI, frozen for future use or both. The outcome measures were sperm retrieval rate (SRR), and in vitro fertilisation (IVF)/ICSI results. Furthermore, SRR was calculated for the predominant causes and histopathological patterns. The overall SRR was 61.2%. It was significantly higher in patients with a history of cryptorchidism and other childhood diseases (100%) than in the other NOA groups (P<0.05). Patients with Klinefelter syndrome had a 75% SRR. Among the different types of testicular histology, the highest SRR were noted in patients with complete hyalinisation (100%) and hypospermatogenesis (92.9%), and low with Sertoli cell-only syndrome (46.3%). The SRR has significantly increased from 33.3% in 2015-2016 to 73.6% in 2019-2020 (P=0.009). Of the 52 patients with SSR, 47 underwent IVF/ICSI. Fertilisation rate was 42.4%. Twenty-nine couples achieved at least one good-quality embryo and had embryo transfer. Nineteen achieved pregnancy (40.4%), and in three patients a miscarriage resulted. This is the first report from Australia showing that micro-TESE is an effective treatment for NOA with high SRR. The increasing success rates over several years indicate the importance of surgical skill and laboratory staff experience.

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.

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.

A magnetic resonance imaging study in etiology of nonobstructive azoospermia.

Testicular magnetic resonance imaging parameters, including apparent diffusion coefficient, fractional anisotropy, magnetization transfer ratio, and normalized metabolite concentrations represent useful noninvasive fingerprints of nonobstructive azoospermia. Nonobstructive azoospermia etiology might correlate with the spermatogenesis status. To assess the possible association between apparent diffusion coefficient, fractional anisotropy, magnetization transfer ratio, and normalised metabolite concentrations with nonobstructive azoospermia etiology. This retrospective study included 48 consecutive men with nonobstructive azoospermia and 18 age-matched controls. All participants underwent scrotal magnetic resonance imaging. The testicular apparent diffusion coefficient, fractional anisotropy, magnetization transfer ratio, and normalized metabolite concentrations were calculated. nonobstructive azoospermia men were classified into three groups, based on etiology: group 1, idiopathic; group 2, genetic causes; and group 3, non-genetic causes. Parametric and nonparametric statistical tests were used to evaluate differences in magnetic resonance imaging parameters between nonobstructive azoospermia groups and normal testes (group 4). Regression analysis was performed to assess the most predictive magnetic resonance imaging factor of nonobstructive azoospermia etiology. Differences in mean apparent diffusion coefficient (p<.001), fractional anisotropy (p<.001), magnetization transfer ratio (p<.001), and normalized concentrations of total choline (p=.005), glucose (p=.012), myo-inositol (p=.024), and lipids (p=.010) were observed among groups. Regression analysis failed to identify the most discriminating magnetic resonance imaging feature for nonobstructive azoospermia etiology. Apparent diffusion coefficient, fractional anisotropy, magnetization transfer ratio, and normalized concentrations of total choline, glucose, myo-inositol, and lipids are helpful in discriminating nonobstructive azoospermia etiology. Magnetic resonance imaging may provide useful, noninvasive information on the microstructural and biochemical milieu of nonobstructive azoospermia testes.

MP21-03 INHIBIN B IS A PREDICTIVE FACTOR OF SPERM RECOVERY IN NON-OBSTRUCTIVE AZOOSPERMIA

You have accessJournal of UrologyInfertility: Epidemiology & Evaluation I (MP21)1 Sep 2021MP21-03 INHIBIN B IS A PREDICTIVE FACTOR OF SPERM RECOVERY IN NON-OBSTRUCTIVE AZOOSPERMIA Isadora Badalotti-Teloken, Mariangela Badalotti, Guilherme Santiago, Bruna Santos, Bruna Araujo, Ana Karoline Rosa, Alvaro Petracco, and Claudio Teloken Isadora Badalotti-TelokenIsadora Badalotti-Teloken More articles by this author , Mariangela BadalottiMariangela Badalotti More articles by this author , Guilherme SantiagoGuilherme Santiago More articles by this author , Bruna SantosBruna Santos More articles by this author , Bruna AraujoBruna Araujo More articles by this author , Ana Karoline RosaAna Karoline Rosa More articles by this author , Alvaro PetraccoAlvaro Petracco More articles by this author , and Claudio TelokenClaudio Teloken More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002006.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Inhibin B is a glycoprotein produced by seminiferous tubules, predominantly by Sertoli cells, and has been proposed as a non-invasive marker of spermatogenesis. This study aimed to assess the potential of inhibin B as a spermatogenesis marker compared with follicle-stimulating hormone (FSH) and in non-obstructive azoospermic men, its association with testicular cyto-histology, and its potential to predict the presence of sperm at testicular sperm extraction (TESE) procedure. METHODS: Clinical and laboratory data of men treated at a Brazilian human reproduction center were reviewed. To evaluate the correlation between inhibin B and FSH, the patients were divided in groups: non-obstructive azoospermia (NOA), severe oligospermia (SO, sperm concentration <5.106/ml), and mild to moderate oligospermia (MO, sperm concentration 5-14.9.106/ml); fertile normozoospermic patients composed the control group (CG). In the NOA group the correlation between inhibin B and testicular cyto-histology (CE–complete spermatogenesis; MA–maturation arrest at level of spermatid; SC–only Sertoli cells, spermatocyte or spermatogonia) was evaluated. Testicular specimens were obtained in 3 different areas of each testi. In patients who underwent TESE, the presence of sperm was evaluated. The statistical analysis was carried out using Kruskal-Wallis, Spearman, and Mann-Whitney U test, multiple regression and ROC curve, considering p<0.05 significant. RESULTS: A total of 246 cases was eligible; NOA n=135 (mean age 36.9 yo), SO n=89 (mean age 36.7 yo), MO n=10 (mean age 37.2 yo) and CG n=11. The median level of inhibin B (pg/ml) was 36.8 in NOA, 74.0 in SO, 109.0 in MO and 196.0 in CG (p<0.0001). The correlation between inhibin B and FSH was significant (r=0,701; p<0.0001). In the cyto-histological comparison, the median value of inhibin B was 87.6 in CE, 42.7 in MA and 22.0 in SC (p<0.0001). Patients with inhibin B ≥88,0 have a 73.3% chance of having spermatozoa or spermatid in cyto-histology, and those with inhibin B ≤22.0 have a 76.3% chance of not having these cells (p<0.0001). In the TESE results, the median level of inhibin B was 80.0 in the group with sperm and 27.6 in the group with no sperm. Inhibin B ≥88,0 also has an 85% positive predictive value for having spermatozoa in TESE (p=0.047). CONCLUSIONS: This study shows a cutoff point for inhibin B ≥ 88.0 for a good prognosis of finding sperm in azoospermic individuals for assisted reproduction through TESE. Furthermore, an inhibin B ≤22 points to a bad prognosis, suggesting that the patients look further into infertility treatment alternatives, such as sperm bank. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e346-e347 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Isadora Badalotti-Teloken More articles by this author Mariangela Badalotti More articles by this author Guilherme Santiago More articles by this author Bruna Santos More articles by this author Bruna Araujo More articles by this author Ana Karoline Rosa More articles by this author Alvaro Petracco More articles by this author Claudio Teloken More articles by this author Expand All Advertisement Loading ...

Immunohistochemical examination of androgen receptor and estrogen receptor alpha expressions in obstructive and non-obstructive azoospermia

ABSTRACT In this study, the expression of the androgen receptor (AR) and estrogen receptor alpha (ERα) in testicular tissue of male patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) were evaluated by immunohistochemistry. NOA (n = 23) and OA (n = 21) groups were created according to clinical and laboratory archival records. Testicular sperm extraction tissue sections were evaluated according to Johnsen’s tubular biopsy scoring (JTBS) method. ERα and AR immunostaining results were evaluated semiquantitatively. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and estradiol were analyzed. Serum FSH and LH concentrations were greater, and testosterone concentrations were lower than the normal values in the NOA group, whereas the OA group revealed normal hormonal values. Serum estradiol concentrations in groups were in the normal range. JTBSs were significantly lower in the NOA group. Decreased AR expression and increased ERα expression were observed in the NOA group compared to the OA group. This suggests that ERα and AR are expressed in Sertoli cells, Leydig cells, and myoid cells and are required for normal testicular function. Decreased expression of the AR and increased expression of ERα in the testis may negatively affect spermatogenesis. Abbreviations: AR: androgen receptor; ER: estrogen receptor; ERα: estrogen receptor alpha; FSH: follicle-stimulating hormone; JTBS: Johnsen’s tubular biopsy scoring; LH: luteinizing hormone; NOA: non-obstructive azoospermia; OA: obstructive azoospermia; TESE: testicular sperm extraction