Non-obstructive Azoospermia Group Research Articles

A comparative cross-platform analysis of cuproptosis-related genes in human nonobstructive azoospermia: An observational study.

This study aimed to identify novel biomarkers associated with cuproptosis in human nonobstructive azoospermia (NOA). We obtained 4 NOA microarray datasets (GSE145467, GSE9210, GSE108886, and GSE45885) from the NCBI Gene Expression Omnibus database and merged them into training set. Another NOA dataset (GSE45887) was used as validation set. Differentially expressed cuproptosis-related genes were identified from training set. Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted. Least absolute shrinkage and selection operator regression and support vector machine-recursive feature elimination were used to identify hub cuproptosis-related genes. We calculated the expression of the hub cuproptosis-related genes in both validation set and patients with NOA. Gene set variation analysis was used to explore their potential biological functions. The risk prediction model was built by logistic regression analysis and was evaluated in the validation set. Finally, we constructed a competing endogenous RNA network. The training set included 29 patents in the control group and 92 in the NOA group, and 10 cuproptosis-related differentially expressed genes were identified. Subsequently, we screened 6 hub cuproptosis-related genes (DBT, GCSH, NFE2L2, NLRP3, PDHA1, and SLC31A1) by least absolute shrinkage and selection operator regression and support vector machine-recursive feature elimination. GCSH, NFE2L2, NLRP3, and SLC31A1 expressed higher in NOA group than in control group (P < .05) in the validation set (4 patients in control and 16 in NOA groups), while the expression levels of GCSH, NFE2L2, NLRP3, PDHA1, and SLC31A1 were higher in NOA group than in control group (P < .05) in our patients (3 patients in control and 4 in NOA groups). The model based on the 6-gene signature showed superior performance with an AUC value of 0.970 in training set, while 1.0 in validation set. Gene set variation analysis revealed a higher enrichment score of "homologous recombination" in the high expression groups of the 6 hub genes. Finally, we constructed a competing endogenous RNA network and found hsa-miR-335-3p and hsa-miR-1-3p were the most frequently related to the 6 hub genes. DBT, GCSH, NFE2L2, NLRP3, PDHA1, and SLC31A1 may serve as predictors of cuproptosis and play important roles in the NOA pathogenesis.

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-194 Cumulative live birth and neonatal outcomes according to different male factors in IVF/ICSI cycles

Abstract Study question What are the impacts of different male factors on cumulative live birth and neonatal outcomes of IVF/ICSI cycles? Summary answer Severe male infertility factors may be negatively associated with fertilization and egg utilization, embryo development and cumulative live birth rate, but not with neonatal outcomes. What is known already Several studies have explored the effect of semen quality on IVF/ICSI clinical outcomes and have drawn contradictory conclusions because the semen quality of male partners could be fluctuated by many factors and there are many confounding factors from women partner. However, an all-round and definitive conclusion has not been reached yet, and clinicians have not yet determined whether men with abnormal semen parameters should be treated to improve their semen parameters and IVF clinical outcomes. Study design, size, duration The retrospective cohort study involved 6,580 males with abnormal sperm parameters and 17,254 males with normozoospermia who underwent their first IVF/ICSI cycle between January 2018 and September 2022 in the reproductive medicine center at a university hospital. They were divided into five different study groups: normozoospermia (N), moderate male factor (MMF), severe oligoasthenoteratozoospermia (OAT-S), azoospermia-husband (azoospermia-H), and azoospermia-donor (azoospermia-D). We compared fertilization, embryo development, cumulative live birth and neonatal outcome. Participants/materials, setting, methods Reproductive and neonatal outcomes of men with abnormal sperm parameters (MMF, OAT-S, azoospermia-H, and azoospermia-D) were studied through four propensity score matching (PSM) comparisons along with corresponding control groups(N) (match factors: including female age, BMI, Gn time, controlled ovarian stimulation protocol, number of oocytes obtained, endometrial thickness and duration of infertility). Fertilization outcomes were also compared stratified by IVF or ICSI. Main results and the role of chance The median ages of the females in the azoospermia, OAT-S, MMF, and N groups were 29, 30, 32 and 32 years old, respectively, which were comparable among groups after PSM. The fertilization rates were significantly reduced in OAT-S and azoospermia-H compared with N in ICSI cycles (67.05% and 65.05% versus 71.50%, p &amp;lt; 0.001, respectively). The oocyte utilization rates were also significantly declined in OAT-S and azoospermia-H compared with N in IVF/ICSI cycles (28.39% and 29.64% versus 34.83%, p &amp;lt; 0.001, respectively). Azoospermia-H group was divided into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). The conservative cumulative live birth rate (CLBR) in the OAT-S group with poor spermatogenic function decreased (63.6% vs. 72.9%, P &amp;lt; 0.001), and was also declined in the NOA group with the poorest spermatogenic function (62.4% vs. 72.9%, P &amp;lt; 0.001), compared with that in the OA group with mostly normal spermatogenic function. After binary multifactorial logistic regression, we found that conservative CLBR was lower in the OAT-S (aOR 0.675, 95%CI 0.536-0.850, P = 0.001) and NOA (aOR 0.574, 95%CI 0.384-0.857, P = 0.007) groups than in the OA group. No impact of sperm factor on obstetrical/perinatal outcomes was observed. In IVF/ICSI cycles, reproductive and neonatal outcomes were similar between MMF, azoospermia-D and normal semen groups. Limitations, reasons for caution The main limitation of this study is that it was a single-center, observational and retrospective study. However, the large sample size, use of PSMs and use of a multivariate regression model for a wide array of possible confounding factors rendered the conclusion relatively reliable. Wider implications of the findings This is the first study involving long-term outcomes (obstetrical and neonatal outcomes, cumulative pregnancy and live rates) from IVF/ICSI cycles for different male factors, which can help clinicians identify paternal influence in an all-round way. 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

P-069 The expression patterns of ALOX15 and GPX4 in the seminiferous epithelium of men with non-obstructive azoospermia (NOA) due to primary testicular failure

Abstract Study question Does the expression of ALOX15 and GPX4 differ in NOA men with a history of cryptozoospermia from NOA who never had sperm in their ejaculate? Summary answer There was no significant difference between these 2 groups of patients. What is known already Oxidative stress (OS) has been investigated as a prime suspect for many unexplained pathologies, including primary testicular failure (PTF). The fact that male germ cells are rich in polyunsaturated fatty acids (PUFAs) but have a limited amount of cytoplasmic scavenging enzymes, is the reason behind their extreme vulnerability to OS. The accumulation of lipid peroxidation markers leads to the inactivation of glutathione peroxidase 4 (GPX4), the lipid repair enzyme, and the exacerbation of arachidonate 15-lipoxygenase (ALOX15), a lipoxygenase enzyme that promotes lipid degradation. This study investigates the activity of these antagonizing enzymes in the testicular tissue of NOA patients. Study design, size, duration This observational study was conducted in the period from May 2020, following the Ethics Committee of the University of Split School of Medicine approval, to October 2023. The testicular tissue from 57 patients diagnosed with NOA (according to WHO criteria) and referred to their first microTESE as a part of fertility treatment was collected and processed. Participants/materials, setting, methods Samples were prepared for histological evaluation and immunodetection (fixation; embedding in paraffin; H&amp;E staining; and staining with anti-ALOX15 and anti-GPX4 antibodies). Two groups of NOA patients were observed: Crypto-NOA and NOA group. The main outcome measure was the number of ALOX15 positive germ cells per 1 mm² of the seminiferous tubule surface (N/ mm²) and the percentage (%) of GPX4 positive surface per surface of the tubule measured by the ImageJ program. Main results and the role of chance Spermatozoa were retrieved in 37 of 57 (64,91 %) men; samples of 4 patients had to be excluded due to poor fixation. There were 18 patients in the Crypto-NOA group, and 10 in the NOA group, while 5 patients were diagnosed with obstructive azoospermia (OA) and were used as controls. For comparison of the baseline data of Crypto-NOA and NOA patients, we used the Mann-Whitney test, and with statistical significance set at the level of P &amp;lt; 0.05, we found that there were significantly more patients with cryptorchism in the NOA group. As a tool for histological evaluation, we used the diagnostic code designed by McLachlan et al. and found similar spermatogenic phenotypes between Crypto NOA and NOA patients. The most represented phenotype was hypospermatogenesis with a moderate reduction in late spermatid number mixed with normal tubules (38,8 % in Crypto-NOA; 40% in NOA), following the phenotype where late spermatids are present in some tubules but mixed with tubules showing no progression past the spermatocyte stage. Using the Mann-Whitney test, we did not find a significant difference in the expression of ALOX15 and GPX4 in the seminiferous epithelium of Crypto-NOA and NOA patients. Limitations, reasons for caution The testicular tissue of NOA patients is characterized by focal or patchy spermatogenesis which led to the exclusion of several samples due to the fixation of tissue with a homogenous pattern of Sertoli cell-only or germ-cell arrest. Wider implications of the findings This study will provide additional information regarding the cause of impaired spermatogenesis and help to determine if the patient is a potential candidate for antioxidant therapy. Our next step is to associate the expression patterns of ALOX15 and GPX4 with the success rates of in vitro fertilization procedure outcomes. Trial registration number not applicable

Cтруктура микробиоты фертильных и инфертильных пациентов с различными видами азооспермии.

Introduction. The study of the human microbiome has become of increasing scientific interest. A balanced and diverse microbiota of the male re- productive system is essential for optimal reproductive health. Today, there are no diagnostic tests to determine the effect of specific microorganisms on the quality of seminal fluid, however, the use of next-generation sequencing (NGS), using the 16S rRNA gene sequence as a molecular marker, provides us with the opportunity to understand the complexity of the relationship between the microbiome and its host. The aim of the study. To conduct a comparative analysis of the taxonomic structure of the microbiota between patients with obstructive and nonobstructive azoospermia and the fertility group (patients with a history of having children). Materials and methods. The study included (n=57) patients (infertile patients with obstructive azoospermia (OA), non-obstructive azoospermia (NOA) and a control group (FERT). All patients underwent percutaneous aspiration of sperm from the testicle (testicular sperm aspiration, MESA), with subsequent in vitro fertilization using ICSI technology, or cryopreservation of biological material. The amplicons of the bacterial 16S rRNA gene were analyzed using high-performance next-generation sequencing (NGS). For this research of bacterial composition of the urogenital tract and control the purity of the method, samples has been taken from the urethra of each patient who underwent testicular biopsy. The data was processed using the QUICKTIME program (version 1.9.1). Results. No significant differences were found in the comparison groups during the study of the urethral bacterial composition. The species diversity, as well as the total number of detected taxonomic units (OTE) in the NOA and OA groups are significantly lower than in the FERT group. The presentation of the phylum like Proteobacteria, Firmicutes, Bacteroidetes, Bifidobacterium adolescentis in the studied groups is different. Three microorganisms were identified with the same frequency in the urethra and testicular tissue (the exact Fisher criterion was used, adjusted for multiple comparison by the Benjamin-Hochberg method). The representation of the phylum Prevotella, Comamonadaceae, Veillonella dispa, Comamonadaceae in the studied groups differs. Conclusion. Testicular tissue is not sterile and has its own unique microbial landscape for each comparison group. Bacterial communities of testicular tissue in patients with azoospermia are characterized by a reduced diversity and a specific composition that differs from the urethral microbiota. These results may be useful in further to study the role of microbiota in the pathology of spermatogenesis and developing new approaches to the treatment and diagnosis of male infertility.

Intratesticular Testosterone and Its Precursors among Azoospermic Men: A Pilot Study.

The study aimed to comprehensively analyze testosterone and precursor concentrations in the testicular interstitial fluid (TIF) of men with azoospermia, exploring their significance in the testicular microenvironment and their correlation with testicular sperm retrieval outcomes. We analyzed 37 TIF samples, including 5 from men with obstructive azoospermia (OA) and 32 from men with non-obstructive azoospermia (NOA). Liquid chromatography with tandem mass spectrometry quantified testosterone and precursor levels. Comparative assessments of the outcomes of testicular sperm retrieval were performed between the OA and NOA groups as well as among men with NOA. Men with NOA who had not undergone hormone treatment exhibited significantly higher intratesticular concentrations of testosterone (median 1,528.1 vs. 207.5 ng/mL), androstenedione (median 10.6 vs. 1.9 ng/mL), and 17-OH progesterone (median 13.0 vs. 1.8 ng/mL) than men diagnosed with OA. Notably, in the subgroup of patients with NOA subjected to medical treatment, men with successful sperm retrieval had significantly reduced levels of androstenedione (median androstenedione 5.7 vs. 18.5 ng/mL, p=0.004). Upon a more detailed analysis of these men who underwent hormone manipulation treatment, the testosterone/androstenedione ratio (indicative of HSD17B3 enzyme activity) was markedly increased in men with successful sperm retrieval (median: 365.8 vs. 165.0, p=0.008) compared with individuals with NOA who had unsuccessful sperm recovery. Furthermore, within the subset of men with NOA who did not undergo medical treatment before microdissection testicular sperm extraction but achieved successful sperm retrieval, the ratio of 17-OH progesterone/progesterone (indicative of CYP17A1 activity) was substantially higher. The study suggests distinct testosterone biosynthesis pathways in men with compromised spermatogenesis and those with normal spermatogenesis. Among NOA men with successful retrieval after hormone optimization therapy, there was decreased androstenedione and increased HSD17B3 enzyme activity. These findings have diagnostic and therapeutic implications for the future.

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