Azoospermic Patients Research Articles

Alteration of the metabolite interconversion enzyme in sperm and Sertoli cell of non-obstructive azoospermia: a microarray data and in-silico analysis

Numerous variables that regulate the metabolism of Sertoli cells and sperm have been identified, one of which is sex steroid hormones. These hormones play a vital role in maintaining energy homeostasis, influencing the overall metabolic balance of the human body. The proper functioning of the reproductive system is closely linked to energy status, as the reproductive axis responds to metabolic signals. The aim of this study was to investigate the gene expression patterns of metabolite interconversion enzymes in testicular cells (Sertoli cells and spermatogonia) of non-obstructive azoospermia (NOA) patients, as compared to normal controls, to understand the molecular mechanisms contributing to NOA. We used microarray and bioinformatics techniques to analyze 2912 genes encoding metabolite interconversion enzymes, including methyltransferase, monooxygenase, transmembrane reductase, and phosphohydrolase, in both testicular cells and normal samples. In sperm, the upregulation of MOXD1, ACAD10, PCYT1A, ARG1, METTL6, GPLD1, MAOA, and CYP46A1 was observed, while ENTPD2, CPT1C, ADC, and CYB5B were downregulated. Similarly, in the Sertoli cells of three NOA patients, RPIA, PIK3C3, LYPLA2, CA11, MBOAT7, and HDHD2 were upregulated, while NAA25, MAN2A1, CYB561, PNPLA5, RRM2, and other genes were downregulated. Using STRING and Cytoscape, we predicted the functional and molecular interactions of these proteins and identified key hub genes. Pathway enrichment analysis highlighted significant roles for G1/S-specific transcription, pyruvate metabolism, and citric acid metabolism in sperm, and the p53 signaling pathway and folate metabolism in Sertoli cells. Additionally, Weighted Gene Co-expression Network Analysis (WGCNA) and single-cell RNA sequencing (scRNA-seq) were performed to validate these findings, revealing significant alterations in gene expression and cellular distribution in NOA patients. Together, these results provide new insights into the molecular mechanisms underlying NOA and identify potential therapeutic targets.

A New Sperm Concentration Threshold for Y Chromosome Microdeletion Analysis in Infertile Men: Could It Be Azoopermia?

We aimed to assess the frequency of Y-chromosome microdeletions (YCMs) in a non-multiethnic urban population in our region, define predictive factors, and determine a new clinical threshold for YCMs in infertile men. A total of 281 patients with a sperm concentration ≤5 million/mL were retrospectively evaluated. Oligozoospermic and/or azoospermic patients with a sperm concentration of ≤5 million/mL were screened for the YCM analysis. Y-chromosome microdeletion was detected in 9 (3.2%) of the 281 patients. All patients with YCM were azoospermic. The presence of azoospermia, a high folliclestimulating hormone level, and a high luteinizing hormone level were found to be important determinants for the identification of a microdeletion (P = .002, P = .002, and P=.021, respectively). If the presence of azoospermia and a sperm concentration threshold of <1 million/mL had been applied for the YCM test, the number of tests performed would have been reduced by 54.4% (153 tests) and 42.7% (120 tests), respectively, resulting in cost saving of approximately $11 474 and $9000, respectively. We recommend that the threshold for sperm concentration for YCM analysis be set at <1 million in individuals in developed countries and only in patients with azoospermia in developing countries, in order to reduce costs and save labor by excluding unnecessary tests. These proposed thresholds (azoospermia and sperm counts less than <1 million/mL) provide cost-effectiveness by significantly reducing the number of genetic tests ordered without affecting the diagnosis rate.

Pregnancy outcomes in patients with non-obstructive azoospermia undergoing micro-TESE: comparison of fresh vs. frozen-thawed testicular sperm.

The relative merits of fresh or frozen testicular sperm in ICSI remain a matter of contention. This study aims to compare the reproductive outcomes of non-obstructive azoospermia patients undergoing ICSI using fresh and frozen-thawed microdissection testicular sperm extraction (micro-TESE) sperm. A total of 223 men with non-obstructive azoospermia underwent micro-TESE to collect testicular spermatozoa. ICSI cycles were performed using fresh and frozen-thawed spermatozoa. The cleavage states and grading of embryos, fertilization, and pregnancy outcomes were compared between the groups to assess the impact of cryopreservation of testicular spermatozoa on embryo quality and ICSI outcomes. A total of 223 cases were evaluated, with fertilization observed in 208 cases and no fertilization observed in 15 cases. The number of day 3 total embryos and the number of cleavage embryos differed between the fresh and frozen micro-TESE groups, whereas the number of two-pronuclei oocytes, grading of embryos, fertilization, pregnancy, and live birth rates were found to be similar between the two groups. The cryopreservation of spermatozoa obtained by micro-TESE does not affect the fertilization rate or pregnancy outcome in cases of non-obstructive azoospermia. The present findings, when considered in conjunction with the extant evidence, may serve to alleviate concerns regarding the utilization of frozen-thawed micro-TESE sperm in patients with non-obstructive azoospermia.

Prediction of testicular histology in azoospermia patients through deep learning-enabled two-dimensional grayscale ultrasound.

Testicular histology based on testicular biopsy is an important factor for determining appropriate testicular sperm extraction surgery and predicting sperm retrieval outcomes in patients with azoospermia. Therefore, we developed a deep learning (DL) model to establish the associations between testicular grayscale ultrasound images and testicular histology. We retrospectively included two-dimensional testicular grayscale ultrasound from patients with azoospermia (353 men with 4357 images between July 2017 and December 2021 in The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China) to develop a DL model. We obtained testicular histology during conventional testicular sperm extraction. Our DL model was trained based on ultrasound images or fusion data (ultrasound images fused with the corresponding testicular volume) to distinguish spermatozoa presence in pathology (SPP) and spermatozoa absence in pathology (SAP) and to classify maturation arrest (MA) and Sertoli cell-only syndrome (SCOS) in patients with SAP. Areas under the receiver operating characteristic curve (AUCs), accuracy, sensitivity, and specificity were used to analyze model performance. DL based on images achieved an AUC of 0.922 (95% confidence interval [CI]: 0.908-0.935), a sensitivity of 80.9%, a specificity of 84.6%, and an accuracy of 83.5% in predicting SPP (including normal spermatogenesis and hypospermatogenesis) and SAP (including MA and SCOS). In the identification of SCOS and MA, DL on fusion data yielded better diagnostic performance with an AUC of 0.979 (95% CI: 0.969-0.989), a sensitivity of 89.7%, a specificity of 97.1%, and an accuracy of 92.1%. Our study provides a noninvasive method to predict testicular histology for patients with azoospermia, which would avoid unnecessary testicular biopsy.

A homozygous nonsense variant in HENMT1 causes male infertility in humans and mice.

HENMT1 encodes a small RNA methyltransferase that plays a crucial role in mouse spermatogenesis through the methylation of the 3' end of PIWI-interacting RNAs. Our study aims to elucidate the relationship between HENMT1 and male infertility in humans. A consanguineous family, having a single non-obstructive azoospermia patient was recruited for pathogenic variants screening. The research includes genetic analysis and experimental validation using mouse models. The patient was diagnosed with non-obstructive azoospermia. Whole-exome sequencing and subsequent bioinformatic analyses were performed to screen for candidate pathogenic variants. The pathogenicity of the identified variant was assessed and studied in vivo using a mouse model that mimicked the patient's mutation. Through whole-exome sequencing, we identified a homozygous nonsense variant (c.555G>A, p.Trp185*) in HENMT1 in the patient. The presence of the mutant HENMT1 mRNA was detected in the patient's blood, and the truncated HENMT1 protein was observed in transfected HEK293T cells. The mutant mice modeling this HENMT1 variant displayed an infertile phenotype similar to that of the patient, characterized by spermiogenesis arrest. Further analysis revealed a significant derepression of retrotransposon LINE1 in the testes of the Henmt1 mutant mice, and increased apoptosis of spermatids. Our findings provide the evidence of pathogenicity of the identified HENMT1 variant, thus shedding light on the indispensable role of HENMT1 in human spermatogenesis.

PTN from Leydig cells activates SDC2 and modulates human spermatogonial stem cell proliferation and survival via GFRA1

BackgroundSpermatogonial stem cells (SSCs) are essential for the maintenance and initiation of male spermatogenesis. Despite the advances in understanding SSC biology in mouse models, the mechanisms underlying human SSC development remain elusive.ResultsHere, we analyzed the signaling pathways involved in SSC regulation by testicular somatic cells using single-cell sequencing data (GEO datasets: GSE149512 and GSE112013) and identified that Leydig cells communicate with SSCs through pleiotrophin (PTN) and its receptor syndecan-2 (SDC2). Immunofluorescence, STRING prediction, and protein immunoprecipitation assays confirmed the interaction between PTN and SDC2 in spermatogonia, but their co-localization was observed only in approximately 50% of the cells. The knockdown of SDC2 in human SSC lines impaired cell proliferation, DNA synthesis, and the expression of PLZF, a key marker for SSC self-renewal. Transcriptome analysis revealed that SDC2 knockdown downregulated the expression of GFRA1, a crucial factor for SSC proliferation and self-renewal, and inhibited the HIF-1 signaling pathway. Exogenous PTN rescued the proliferation and GFRA1 expression in SDC2 knockdown SSC lines. In addition, we found downregulation of PTN and SDC2 as well as altered localization in non-obstructive azoospermia (NOA) patients, suggesting that downregulation of PTN and SDC2 may be associated with impaired spermatogenesis.ConclusionsOur results uncover a novel mechanism of human SSC regulation by the testicular microenvironment and suggest a potential therapeutic target for male infertility.

EEF1B2 regulates the proliferation and apoptosis of human spermatogonial stem cell lines through TAF4B

BackgroundSpermatogonial stem cells (SSCs) are essential for male fertility, maintaining sperm production throughout life. While mouse SSCs have been studied extensively, the mechanisms regulating human SSCs are less understood. ObjectivesTo investigate the role of EEF1B2 in regulating human SSC proliferation and apoptosis. Material and methodsSingle cell RNA sequencing (scRNA-seq) analysis was utilized to investigate the differentially expressed genes of SSC. The distribution of EEF1B2 in the human testis was examined using immunofluorescence and immunohistochemistry techniques. Cell proliferation, DNA replication, and self-renewal were analyzed using CCK8, EdU, Western blot, and flow cytometry. RNA sequencing was employed to analyze the downstream target molecules and signaling pathways of EEF1B2. ResultsIn this study, we analyzed single-cell sequencing data from human testicular samples and identified EEF1B2 as a protein highly expressed in SSCs, with expression decreasing during development. Immunohistochemistry and immunofluorescence confirmed this pattern and showed co-localization with the proliferation marker KI67. Knockdown of EEF1B2 in human SSC lines impaired proliferation and viability, reducing self-renewal proteins like PLZF and CCNE1. RNA sequencing revealed decreased TAF4B following EEF1B2 knockdown, which could be rescued by replenishing TAF4B. Testicular SSCs from non-obstructive azoospermia (NOA) patients also showed reduced EEF1B2. Discussion and conclusionOur findings reveal a novel regulatory mechanism involving EEF1B2 and TAF4B in human SSCs, suggesting EEF1B2 deficiency may contribute to male infertility.

A novel homozygous nonsense variant of STX2 underlies non-obstructive azoospermia in a consanguineous Chinese family.

Male infertility is a widespread population health concern, causing various degrees of adverse fertility outcomes. We determined the genetic cause of an infertile male from a consanguineous family, expanding the mutant spectrum of male infertility. A non-obstructive azoospermia (NOA) patient was recruited, and histological type of human testicular tissue of the patient categorized as maturation arrest. We identified a novel loss-of-function variant of syntaxin 2 (STX2) (c.142C>T:p.Gln48*) by performing Whole-exome sequencing (WES) on the NOA patient from a consanguineous Chinese family. Sanger sequencing confirmed the p.Gln48* variant was maternally and paternally inherited. The variant was predicted to be deleterious and resulted in aberrant changes to structure and function of STX2 by In silico analysis. In summary, we reported for the first time that a nonsense variant occurred in the exon region of STX2 in an infertile male presenting with NOA, which was beneficial for diagnosis and therapies of NOA.

The effects of testicular stromal stem cells on surgically injured testicular tissue in rats.

This study investigated the effects of transplanted testicular stromal stem cells (tSSCs) on surgically damaged testis tissue. Ten-week-old male Wistar albino rats were divided into three groups: control (n = 6), damage (DG) (n = 6) and testicular stromal stem cell (TSSC) (n = 6) groups. Surgically induced damage was inflicted on the left testes of both the DG and TSSC groups, with no intervention on the right testes. In the TSSC group, damaged testes were treated with transplanted tSSCs, followed by orchiectomy after 15 days. Testes tissues were stained with haematoxylin-eosin (H&E), and recovery rates of functional structures were assessed by modified Johnsen scoring. The effects of tSSCs on testicular tissue were demonstrated by immunohistochemistry using BAX, BCL-2 and caspase 3. Serum testosterone levels were analysed using the enzyme-linked immunosorbent assay (ELISA) method. Surgical damage caused germ cell degeneration in some seminiferous tubules and a decrease in interstitial areas. With tSSC treatment, improvements in testicular architecture were identified through spermatogenesis in the seminiferous tubules and normal histological structures in the interstitial areas. Correspondingly, in the modified Johnsen score, the DG group showed a significant difference compared to the other groups (p = 0.001). High expressions of BAX, BCL-2 and caspase-3 in the DG group revealed prominent features of apoptosis. With the injection of tSSCs, these expressions significantly normalized according to H score analysis (all p = 0.004). Although serum testosterone levels in the tSSC group were higher compared to the control and DG groups, this difference was not statistically significant (p = 0.119). This study suggests transplanting tSSCs could accelerate tissue healing after testicular sperm extraction (TESE) surgery for azoospermia patients, potentially paving the way for a new and important clinical treatment.

Comparison of clinical characteristics and surgical outcomes in non-vasectomized epididymal obstructive azoospermia patients with or without concurrent vas-deferens obstruction.

Microsurgical vasoepididymostomy is an effective surgical method for treating epididymal obstructive azoospermia but the surgical outcomes can be affected in some non-vasectomized epididymal obstructive azoospermia patients with concurrent vas-deferens obstruction. This study aimed to explore the clinical characteristics and surgical outcomes in non-vasectomized epididymal obstructive azoospermia patients with versus without concurrent vas-deferens obstruction. Retrospective study. To explore the clinical characteristics and surgical outcomes in non-vasectomized epididymal obstructive azoospermia patients with versus without concurrent vas-deferens obstruction, aiming to identify predictive factors for concurrent vas-deferens obstruction and evaluate the efficacy of microsurgical vasoepididymostomy in patients with epididymal obstructive azoospermia and concurrent short-segment vas-deferens obstruction. A retrospective analysis of 225 epididymal obstructive azoospermia cases was conducted at the First Affiliated Hospital of Fujian Medical University from November 2016 to March 2023. All patients underwent a comprehensive preoperative evaluation. During surgery, the vas deferens were assessed to determine the presence and extent of obstruction. Depending on the obstruction length, either a standard microsurgical vasoepididymostomy was performed, or the obstructed segment was resected followed by microsurgical vasoepididymostomy. If the remaining length post-resection was insufficient for anastomosis, the procedure was discontinued. Data on patient clinical characteristics, operative findings, and outcomes were collected and analyzed. Logistic regression was used to identify predictive factors for concurrent vas-deferens obstruction, and comparative analysis assessed patency and pregnancy rates between patients with and without concurrent vas-deferens obstruction. Of the 225 patients in the study, 77 (34.22%) presented with epididymal obstructive azoospermia and concurrent vas-deferens obstruction. Logistic regression analysis revealed that "the history of epididymitis" was a significant predictive factor for epididymal obstructive azoospermia patients with concurrent vas-deferens obstruction (odds ratio=9.06, p<0.001). The average length of vas deferens obstruction amenable to microsurgical vasoepididymostomy post-resection was 1.31±0.54cm (range from 0.50 to 2.50cm). In contrast, cases unsuitable for microsurgical vasoepididymostomy presented an average obstruction length of 15.26±5.79cm (p<0.001). The patency rates were 82.17% in epididymal obstructive azoospermia patients without concurrent vas-deferens obstruction and 74.14% in those with concurrent vas-deferens obstruction. The pregnancy rates followed a similar trend, at 34.11% and 34.48%, respectively. These differences were not statistically significant (p>0.05 for both). However, epididymal obstructive azoospermia patients with vas-deferens obstruction exhibited a decreased likelihood of bilateral microsurgical vasoepididymostomy (p<0.001). Our study identifies a noticeable occurrence of concurrent vas-deferens obstruction in non-vasectomized epididymal obstructive azoospermia patients, with approximately one-third of the cases (34.22%) exhibiting vas-deferens obstruction during surgical interventions. Notably, a small fraction (6.67%) of these individuals chose not to proceed with any microsurgical vasoepididymostomy, even on one side, due to the extensive length of the obstruction. Through logistic analysis, we have demonstrated that "the history of epididymitis" is a critical predictive factor for the presence of vas-deferens obstruction, underscoring its significance in preoperative evaluations.Furthermore, our research confirms that microsurgical vasoepididymostomy is still an effective treatment for epididymal obstructive azoospermia patients with concurrent short-segment vas-deferens obstruction, achieving significant patency and favorable pregnancy rates compared to those patients without vas-deferens obstruction. These insights are pivotal for enhancing surgical strategies and improving fertility outcomes in this patient cohort.

How exome sequencing improves the diagnostics and management of men with non-syndromic infertility.

Male infertility affects approximately 17% of all men and represents a complex disorder in which not only semen parameters such as sperm motility, morphology, and number of sperm are highly variable, but also testicular phenotypes range from normal spermatogenesis to complete absence of germ cells. Genetic factors significantly contribute to the disease but chromosomal aberrations, mostly Klinefelter syndrome, and microdeletions of the Y-chromosome have remained the only diagnostically and clinically considered genetic causes. Monogenic causes remain understudied and, thus, often unidentified, leaving the majority of the male factor couple infertility pathomechanistically unexplained. This has been changing mostly because of the introduction of exome sequencing that allows the analysis of multiple genes in large patient cohorts. As a result, pathogenic variants in single genes have been associated with non-syndromic forms of all aetiologic sub-categories in the last decade. This review highlights the contribution of exome sequencing to the identification of novel disease genes for isolated (non-syndromic) male infertility by presenting the results of a comprehensive literature search. Both, reduced sperm count in azoospermic and oligozoospermic patients, and impaired sperm motility and/or morphology, in asthenozoospermic and/or teratozoospermic patients are highly heterogeneous diseases with well over 100 different candidate genes described for each entity. Applying the standardized evaluation criteria of the ClinGen gene curation working group, 70 genes with at least moderate evidence to contribute to the disease are highlighted. The implementation of these valid disease genes in clinical exome sequencing is important to increase the diagnostic yield in male infertility and, thus, improve clinical decision-making and appropriate genetic counseling. Future advances in androgenetics will continue to depend on large-scale exome and genome sequencing studies of comprehensive international patient cohorts, which are the most promising approaches to identify additional disease genes and provide reliable data on the gene-disease relationship.

Evaluation of the Results of Intracytoplasmic Sperm Injection and Microdissection Testicular Sperm Extraction Treatments in Patients with Nonobstructive Azoospermia According to Etiological Factors: A Retrospective Analysis

Objective: This study aimed to retrospectively compare the results of microdissection testicular sperm extraction (microTESE) and Intracytoplasmic sperm injection (ICSI) treatments in nonobstructive azoospermia (NOA) patients with different aetiologies. Determinants (clinical characteristics) for microTESE outcomes were compared between patients with successful sperm retrieval (SSR) and sperm retrieval failure (SRF). Methods: A total of 510 NOA patients who underwent microTESE between January 2015 and January 2024 were included in this study. Patients were classified according to the cause of NOA and SSR, fertilisation rate, clinical pregnancy, and overall live birth rate were evaluated. Results: The SSR rate was 44.1% in the whole population. The idiopathic patient group had the lowest SSR rate (X2: 34.81; p&lt;0.01). There was no difference between the groups in terms of fertilisation rate, clinical pregnancy and overall live birth rate. There was a negative correlation between age and SSR rates in patients with idiopathic NOA (t:-0.27; p&lt;0.01). SSR rates were higher in patients with cryptorchidism (right: t:0.8; P:0.003; left: t:0.72; p:0.002) and mumps orchitis (right: t:0.76; P&lt;0.01; left: t:0.76; p=0.003). Conclusion: Etiology has a significant role in terms of SSR in patients with NOA. SSR was found to be significantly less in patients with idiopathic NOA compared to other causes. In addition, age and testicular volume were significant predictive factors for SSR in patients with idiopathic and acquired NOA.

Cryopreserved testicular spermatozoa among patients with azoospermia.

To investigate cryopreserved testicular spermatozoa among patients with azoospermia. In this retrospective study spanning from October 1993 to December 2021, we examined men diagnosed with azoospermia who underwent testicular spermatozoa cryopreservation. Data from medical records included utilization and disposal of sperm samples, age at initial cryopreservation. We analyzed the data over 20 years using Kaplan-Meier curves, compared age with the log-rank test, and assessed hazard ratios (HR) with 95% confidence intervals (CI) using Cox regression analysis. A total of 356 patients with a mean age of 32.1 ± 6 were included. Of these, 225 patients utilized thawed testicular sperm for fertility treatments, with 118 patients using all their frozen straws and 107 patients partially using their stored straws. Additionally, 29 patients opted for disposal (six patients partially used their testicular spermatozoa before disposal), resulting in 108 patients who neither used nor disposed of their straws. From a laboratory standpoint, nearly 90% of patients contributed a single testicular sample, which was subsequently divided and cryopreserved as straws, with a median of 4 straws per sample. Notably, in the older age group (> 35 years old), there were a significantly lower usage rate and a higher disposal rate compared to the younger age groups (p < 0.05 for both), corroborated by univariable Cox analysis. This extensive study unveils unique patterns in the preservation and disposal of testicular spermatozoa among azoospermic patients. Most patients utilize a significant portion of their stored samples, while older patients tend to use their testicular spermatozoa less frequently.

Interaction between microbiome and testicular tissue mastocytes in male infertility

Introduction. Male infertility is a complex condition with many potential causes, including hormonal imbalances, anatomical problems, genetic factors, lifestyle factors and more. But today, there is a fairly large group of infertile men with unknown causes of the disease.Objective. To analyze the taxonomic microbial diversity of testicular tissue and the urogenital tract of infertile men and to identify correlations between the microbiome and mastocytes in the testicular parenchyma.Materials &amp; methods. The study was performed on testicular tissue samples from infertile patients with azoospermia (n = 33). All patients were divided into two groups based on the form of azoospermia: group 1 — infertile patients with non-obstructive azoospermia (NOA) (n=21); and group 2 — infertile with obstructive azoospermia (OA) patients (n=12). The bacterial diversity of testicular tissue was studied by the method of high-performance new generation sequencing (NGS). Immunohistochemical staining with anti-MCT (Anti-Mast Cell Tryptase) was used to determine the IHC expression of mastocyte markers.Results. The microbiome of patients with NOA differs markedly from the microbiome of patients with OA (p &lt; 0.05). In group 1, representatives of the Enterobacteriaceae and Xanthomonadaceae families, the genera Finegoldia, Bifidobacterium, Porphiromonas, Prevotella, Peptoniphilus and Pseudomonas are significantly more often found. A distinctive feature of group 2 is the rare occurrence of the genus Prevotella. Histochemical analysis revealed mastocytosis in the in-between-canalicular stroma approximately in 83% of azoospermia cases. Mastocytes are found in tubule structures in 68% of cases and correlate with the microbiome of testicular tissue.Conclusions. Injuries caused by mastocytes in the stroma and tubular structures are interrelated with the taxonomic diversity of testicular tissue. Moreover, the testicles of NOA-patients have a qualitatively and quantitatively more diverse spectrum both at the level of families and genera, unlike OA-patients.

Comparative analysis of the testicular and uretral microbiota in azoospermia patients using next generation sequencing (NGS)

Aim. To determine the role of taxonomic bacterial diversity of the urethral and testicular microbiota in the development of male infertility using high-throughput next-generation sequencing (NGS).Material and methods. In the period between 2018 and 2022 we examined 53 patients. Three study groups were formed: Infertile patients with nonobstructive azoospermia and concomitant varicocele (n = 13) – group 1; Infertile patients with obstructive azoospermia (n = 29) – group 2; Fertile patients with confirmed paternity and testicular pathology requiring histological verification to exclude testicular oncopathology (n = 11) – group 3. Each patient underwent TESE with testicular both and urethral tissue sampling in order to compare the bacterial landscape and control the purity of the method. The obtained material was sequenced using a high-throughput method (NGS).Results.According to our findings, the testicular tissue of obstructive azoospermia patients had significantly depleted bacterial diversity in comparison to nonobstructive azoospermia and concomitant varicocele ones. Also, the fertile group turned out to be the most diverse in its taxonomic community. These results may suggest the bacterial microbiome’s influence on men’s reproductive health.Discussion.Our urogenital bacterial diversity analysis showed that human testicular tissue is not a microbiologically sterile environment and also presented new data associated with testicular tissue and its possible relations with male infertility.Conclusion. A focus on the qualitative and quantitative composition of the testicular microbial community may form the new basis in the diagnosis and treatment of male infertility associated with various types of azoospermia

P-078 Comparison of embryological and clinical outcomes in mumps orchitis and non-obstructive azoospermia patients treated by Microdissection testicular sperm extraction combined with ICSI (microTESE-ICSI)

Abstract Study question The objective of this retrospective study was to examine the embryological and clinical outcomes in mumps orchitis and non-obstructive azoospermia (NOA) patients undergoing microTESE-ICSI treatment. Summary answer microTESE combined with ICSI is an effective treatment regimen that can help mumps orchitis and NOA patients to have their biological offsprings. What is known already Non-obstructive azoospermia can be caused by different etiologies, and one of factors is mumps orchitis. These patients cannot retrieve spermatozoa via ejaculation, so microTESE is an intervention indicated to retrieve their sperm. Recent studies showed that the combination of microTESE and ICSI increase the success of in-vitro fertilization (IVF) treatment for NOA patients. IVF effectiveness can also be influenced by maternal age; however, most of the previous studies on NOA group reported sperm retrieval rate and male factors. Few studies have focused on embryo development and clinical outcomes, especially, under the adjustment for female partner’s age. Study design, size, duration This retrospective study was conducted to analyze the embryo development and clinical outcomes in 212 patients who had spermatozoa retrieved from microTESE and treated by ICSI from 4/2022 to 10/2023. 212 microTESE-ICSI patients were divided into two groups including mumps orchitis group (n = 101) and NOA group by other etiologies (n = 111). These patients had spouses who were under 35-year-old. Participants/materials, setting, methods Effectiveness of microTESE-ICSI between mumps orchitis and NOA groups was evaluated by comparing the embryological development and clinical outcomes. The mean of fertilization rate, good cleavage embryo rate (following alpha consensus) and good blastocyst rate (embryo quality higher than 2BB at day 5) in two groups were compared to assess embryological outcomes. Pregnancy rate, miscarriage rate, ongoing pregnancy rate (≥ 30 weeks), and live-birth rate were examined to evaluate the clinical outcomes. Main results and the role of chance The mean of paternal age in mumps orchitis group and NOA group was similar (32.0±4.4 vs 32.0±4.9). There was no difference in the mean of maternal age between these two groups (28.7±3.4 vs 27.8±3.8, p &amp;gt; 0.05). The means of retrieved oocytes and mature oocytes in two patient groups were not significantly different (19.4±8.7 vs 20.1±11.3; 13.7±6.5 vs 14.1±8.3, respectively). The fertilization rate of mumps orchitis group was significantly higher than NOA group (66.1% vs 57.7%, p &amp;lt; 0.01). Rate of good embryo on day 3 and rate of blastocyst on day 5 in mumps orchitis group were 17.9% and 49.8% respectively. These rates in NOA group accounted for 13.3% and 43.8% respectively. The pregnancy rate (positive beta hCG-test) in mumps orchitis patients was 80.2% (77/96), and this rate in NOA group was 75.5% (77/102). The miscarriage rates in these two groups were not significantly different (0.03% vs 0.06%, p &amp;gt; 0.05). There were no significant differences in ongoing pregnancy rate between mumps orchitis group and NOA group (38.5% (37/96) vs 37.3% (38/102); p &amp;gt; 0.05, respectively). Live-birth rate of these groups was also similar (mumps orchitis vs NOA: 25% (24/96) vs 20.6 (21/102, p &amp;gt; 0.05). Limitations, reasons for caution This research is a single-center retrospective study performed in a brief time, so our sample size is small. Our study had a lack of information of body mass index and male hormonal factors because the database missed in several patients at the retrospective time. Wider implications of the findings These data suggested that microTESE is an optimal method for mumps orchitis and NOA patients who cannot ejaculate retrieve their own sperm. microTESE-ICSI might increase the success rate of fertility treatment in these patients. Trial registration number Not applicable

O-262 Karyotyping of single sperm from non-obstructive azoospermia patients and obstructive azoospermia patients using a combination of micromanipulation and next-generation sequencing

Abstract Study question Is there a difference in the frequency of aberrant karyotype in sperm when the cause of infertility is non-obstructive azoospermia (NOA) versus obstructive azoospermia (OA)? Summary answer Aberrant karyotype was found in 7 of 46 sperm with NOA, none of 26 sperm with OA and none of 28 sperm with control. What is known already It is known that aberrant karyotype occurs more frequently in sperm from infertile men than sperm from general population, and that the severity of infertility is proportional to the frequency of sperm chromosomal abnormality. In fact, miscarriage rates are high in cases where TESE-ICSI is performed for azoospermia, and embryonic chromosomal aneuploidy derived from sperm is thought to be one of the causes of miscarriage. Azoospermia is classified into OA and NOA. However, it is unclear whether there is a difference in the frequency of sperm chromosomal abnormality between NOA and OA. Study design, size, duration Sperm were picked up individually by micromanipulation for the OA and NOA groups who underwent TESE in clinical practice. For comparison, ejaculated sperm from males who underwent IVF or ICSI for female infertility were picked up. As a quality control, ejaculated sperm from balanced reciprocal translocation (RcT) carriers were also picked up. Each sperm was karyotyped by next-generation sequencing (NGS) to compare the number of normal haploid sperm and aberrant sperm. Participants/materials, setting, methods Under the ethical review of Yokohama City University and informed consent with patients, we collected human sperm which were discarded after clinical use. Sperm samples were picked up individually by micromanipulation. Following whole-genome amplification (WGA), karyotyping was performed by NGS. For each sperm, if even one of all chromosomes had abnormality, that sperm was counted as aberrant sperm, and if all chromosomes were haploid, it was counted as haploid sperm. Main results and the role of chance We analyzed 10 sperm per patient. Karyotyping analysis was performed on sperm from 2 patients in the RcT group,4 patients in the control group, 4 patients in the OA group, and 5 patients in the NOA group. WGA was successful in 90% (18/20) of RcT carriers group, 95% (38/40) of the control group, 72.5% (29/40) of the OA group, and 92% (46/50) of the NOA group. Acquisition rate of karyotyping was 90% (18/20) of RcT carriers group, 70% (28/40) of the Control group, 65% (26/40) of the OA group, and 95% (46/50) of the NOA group. The results of karyotype analysis showed 28 haploid sperm and no aberrant sperm in the control group, 26 haploid sperm and no aberrant sperm in the OA group, and 39 haploid sperm and 7 aberrant sperm in the NOA group. Karyotyping of sperm from RcT carriers group showed sperm with unbalanced chromosomes derived from translocations of the carriers. The frequency of aberrant karyotype was significantly higher in NOA than control (P = 0.0297). It was also higher in NOA than OA(P = 0.0363). Limitations, reasons for caution In our research, micromanipulation was performed manually to isolate sperm, and the number of sperm that could be analyzed was small. Sperm with good morphology are selected using micromanipulation and analyzed by NGS, so the results do not reflect the results of all sperm actually collected by TESE. Wider implications of the findings In this study, sperm with good morphology were selected by micromanipulation and analyzed, so it is thought that the karyotype of sperm actually reflects the karyotype used in clinical practice. These results suggest that PGT-A is particularly effective in preventing miscarriages among patients whose azoospermia was caused by NOA. Trial registration number not applicable

O-263 The risk of hypogonadism after microdissection testicular sperm extraction (micro TESE) in non-obstructive azoospermia (NOA) patients with various etiologies

Abstract Study question What is the risk factor of hypogonadism after micro TESE in NOA patients? Summary answer Klinefelter syndrome (KS), past history of cryptorchidism, preoperative testicular volume &amp;lt;8 ml or testosterone &amp;lt;287 ng/dL are significant risk factors of hypogonadism after micro TESE. What is known already Micro TESE combined with intracytoplasmic sperm injection (ICSI) is currently standard procedure to treat infertility in cases of NOA. Although many studies have reported the effectiveness of the treatment concerning about sperm retrieval rates (SRR) and the live birth rates, data on safety, especially on the risk of hypogonadism, are limited. Most previous studies, including our past study, regarding the risk of hypogonadism after TESE have been limited to relatively few in number of patients and the investigations only of the changes of mean serum testosterone levels. Study design, size, duration This study retrospectively investigated 535 NOA patients including 336 cases of unexplained NOA, 91 KS, 16 other chromosomal abnormalities, 31 azoospermia factor (AZF)c deletions, 31 past history of cryptorchidism, and 30 post anticancer chemotherapy, who had undergone micro TESE at our institution from September 2013 to August 2018. All of them had been examined serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone levels before and at least once in 1-3 months after micro TESE. Participants/materials, setting, methods In this study, hypogonadism was defined as total testosterone &amp;lt;250 ng/dL, which is established by the Japanese Society of Men’s Health as diagnostic criteria for late onset hypogonadism syndrome, and/or starting testosterone replacement therapy (TRT). We assessed risk factors of hypogonadism after micro TESE using indicators such as age, preoperative testicular volume and testosterone levels, past medical history, unilateral or bilateral, first time or not micro TESE. Main results and the role of chance In total of 535 men, FSH and LH after micro TESE significantly increased vs baseline (26.2 vs 29.9 IU/L, 9.6 vs 12.8 IU/L, respectively). On the other hand, testosterone levels significantly decreased after surgery (351.2 vs 297.5 ng/dL). Although postoperative hypogonadism was found in 42% (227/535), many of whom were cases of KS or preoperative testosterone levels &amp;lt;250 ng/dL. Sixteen men with KS, 3 men with unexplained NOA, and 2 men with past history of cryptorchidism started TRT because they complained clinical symptoms after micro TESE. Of the 21 men who started TRT, only 5 had new-onset low testosterone levels after micro TESE. According to the assessment of any association of various indicators and low postoperative total testosterone levels using multiple logistic regression analysis, significant risk factors of hypogonadism early after micro TESE were cases of KS (OR 11.4, 95% CI 4.4-29.9), preoperative testosterone levels &amp;lt;287 ng/dL (OR 7.5, 95% CI 4.7-12.0), preoperative testicular volume &amp;lt;8 ml (OR 3.9, 95% CI 2.3-6.4), past history of cryptorchidism (OR 2.9, 95% CI 1.1-7.8). Limitations, reasons for caution Most examinations of postoperative hormone levels were performed in short-term period after micro TESE, so we could only assess the risk of postoperative hypogonadism in a short time course in this cohort study. Wider implications of the findings The strength of the current study is that the risk factors of hypogonadism after micro TESE was assessed in a large population of NOA men with various etiologies. The results of this study provide useful clinical information about the risk of postoperative hypogonadism for NOA patients considering micro TESE. Trial registration number not applicable

P-022 Klinefelter syndrome couples are expected higher live birth rates than unexplained NOA couples after ICSI with testicular spermatozoa

Abstract Study question What are sperm retrieval rates (SRR) by microdissection TESE (micro TESE), fertilization rates, and pre- and post- implantation development in Klinefelter syndrome (KS) couples? Summary answer In KS couples, SRR, clinical pregnancy rates (CPR) per embryo transfer (ET), and live birth rates (LBR) per patient were higher than unexplained NOA couples. What is known already Micro TESE, in combination with ICSI, is presently used to treat infertility in cases of NOA including KS, which is the most common sex-chromosome disorder among infertile males, with a prevalence of 1 in 660 men and is a frequent cause of hypogonadism and infertility. There were few reports regarding ICSI outcomes in the couples of KS. The aim of this study is to assess the prevalence and the significance including SRR by micro TESE of ICSI outcomes with embryonic development in KS couples. Study design, size, duration A retrospective study was conducted in 212 patients with non-mosaic KS and 963 unexplained NOA patients with 46, XY without past history (unexplained NOA; not including after orchidopexy, KS, cryptozoospermia, post chemotherapy, mumps orchitis, etc) who underwent micro TESE and ICSI with fresh or cryopreserved testicular spermatozoa for their wives between September 2013 to November 2023. Participants/materials, setting, methods A total of 2204 azoospermic patients were examined for chromosomal analysis. We evaluated SRR of microdissection TESE, two pronuclei (2PN) rates, blastocyst development rates, good-quality blastocyst (Grade 3BB and above on day 5 by the Gardner scoring) rates, CPR per ET, and LBR per couple patient who underwent micro TESE. We did not undergo preoperative hormonal therapy for KS patients. Statistical analysis was performed using unpaired t-tests and chi-squared tests. Main results and the role of chance We identified 212 KS out of 2204 azoospermic patients (9.6%). SRR of the first attempt micro TESE in KS (91/175=52.0%) was significantly higher than unexplained NOA (166/768=21.6%) (p &amp;lt; 0.001). 2PN, blastocysts development, and good-quality blastocysts rates were 49.2%, 42.9%, 16.9% in KS and 50.8, 45.8%, 21.0% in unexplained NOA respectively. Only good-quality blastocysts rate of KS was significantly lower than that of unexplained NOA (p &amp;lt; 0.01). Taking into account deflection of wives age, each result was calculated at their age 38 or under, but they were the same. CPR and LBR per ET in KS (42.6%, 30.4%) were significantly higher than unexplained NOA (33.1%, 23.7%) (p &amp;lt; 0.01, p &amp;lt; 0.05). The couples were 26.4% (56/212) in KS and 12.0% (116/963) in unexplained NOA (p &amp;lt; 0.001) who have given birth or ongoing pregnancy up to now after underwent micro TESE. That is, KS couples were significantly easier to give birth than unexplained NOA. Rates of congenital disorders had no significant difference between KS and unexplained NOA. Limitations, reasons for caution We did not show the data using ejaculated sperm with KS. The natal outcomes and development of these children has not been fully investigated, long-term follow-up of babies of the KS couples is needed. Wider implications of the findings Micro TESE is particularly helpful for KS cases, but embryonic development depend on the quality of spermatozoa. However, if a blastocyst can be obtained, the expectation of having a child is considered to be high. These results were shown to be useful in informing azoospermic couples undergoing micro TESE. Trial registration number not applicable

P-020 Clinical outcomes of 583 treatment cycles with fresh and cryopreserved testicular sperm in cases without karyotype abnormalities and Y chromosome microdeletions needing testicular sperm extraction

Abstract Study question What are the embryological, clinical and newborn outcomes using fresh and cryopreserved testicular sperm in non-obstructive azoospermia patients? Are the differences between both techniques significant? Summary answer In this population there was a high live birth delivery rate with no significant differences observed in the outcomes using fresh and cryopreserved testicular sperm. What is known already The use of cryopreserved testicular sperm has been considered of lower quality due to freezing/thawing consequences (1). Nevertheless, cryopreserved testicular sperm presents the advantages of avoiding female stimulation in cases of failed testicular sperm retrieval, or multiple testicular biopsies (2). However, the majority of the studies evidenced no significant differences in the clinical outcomes between the use of cryopreserved and fresh sperm (3). As only a few studies are dedicated to compare both techniques (4) we expanded the number of cycles and provided full demographic, stimulation, embryological, clinical and newborn outcomes.