Non-obstructive Azoospermia Patients Research Articles

Seminal Plasma ExLncRNA Pairs: Updating Perspectives in the Search for Testicular Spermatozoa Retrieval Biomarkers in Nonobstructive Azoospermia Patients with mTESE by WGCNA.

This study aims to find candidates for testicular spermatozoa retrieval biomarkers among the seminal plasma exLncRNA pairs. A set of exLncRNA pairs with the best potential biomarkers was selected and validated in 96 NOA samples. Weighted correlation network analysis (WGCNA) and Least Absolute Shrinkage and Selection Operator were used to identify possible biomarkers for these pairs (LASSO). These pairs' potential biomarkers were identified using receiver operating curves. Confusion matrices and sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), FP, false-negative rates (FNR), and F1 scores are calculated. Through F1 scores, we selected the best threshold value. The relative differential expression of each pair in testicular spermatozoa retrieval (+) and testicular spermatozoa retrieval (-) men were validated. The six pairs displayed the best biomarker potential. Among them, CCDC37.DT-LOCI00505685 pair and LOC440934- LOCI01929088 (XR_001745218.1) pair showed the most significant potential and stability for detecting testicular spermatozoa retrieval in the selected and validated cohort. CCDC37.DT-LOCI00505685 pair and LOC440934- LOCI01929088 (XR_001745218.1) pair have the potential to become new molecular biomarkers that could help to select clinical strategies for microdissection testicular sperm extraction.

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.

Factors associated with undergoing microdissection testicular sperm extraction among men with non-obstructive azoospermia following evaluation by a reproductive urologist.

Microdissection testicular sperm extraction (mTESE) is the gold standard treatment for men with non-obstructive azoospermia (NOA). However, many men do not elect to pursue this surgical intervention. We aimed to identify factors associated with NOA patients undergoing mTESE after initial evaluation by a reproductive urologist (RU) through a retrospective cohort study. We retrospectively reviewed NOA patient who underwent evaluation by a RU between 2002-2018. Demographic and clinical data were collected. Our primary outcome was electing to undergo mTESE. 44.4% (75/169) of NOA men underwent mTESE. These patients earned significantly higher median neighborhood income ($133,000 vs. $97,000, P<0.001), spent fewer years trying to conceive before seeking care {1.3 [interquartile range (IQR): 1-3] vs. 2.3 (IQR: 1-5), P=0.012}, and were more likely to be married (79.7% vs. 53.9%, P=0.001). On univariate analysis, married men [odds ratio (OR) 3.37, 95% confidence interval (CI): 1.67-6.79, P=0.001] and men with higher neighborhood income (OR 1.14, 95% CI: 1.06-1.21, P<0.001) were more likely to undergo mTESE, while couples attempting to conceive for a longer period of time prior to initial evaluation were less likely to undergo mTESE (OR 0.79, 95% CI: 0.68-0.92, P=0.003). On multivariable regression analysis, marital status and years attempting to conceive remained significantly associated with NOA patients undergoing mTESE (OR 4.61, 95% CI: 1.16-18.25, P=0.03; OR 0.67, 95% CI: 0.52-0.88, P=0.003, respectively). Higher neighborhood income and marital status were positively associated with patients undergoing mTESE, while couples who attempted to conceive for a longer period of time before seeking infertility care were less likely to undergo mTESE.

O-138 Factors influencing sperm retrieval rate of microdissection testicular sperm extraction in nonobstructive azoospermia patients with different etiologies: a retrospective study of 3104 patients

Abstract Study question What are predictors of successful sperm retrieval (SRR) with microdissection testicular sperm extraction (mTESE) in men with non-obstructive azoospermia (NOA) according to different etiologies? Summary answer Predictors for SRR were different in patients with various etiologies and effects of age on SRR in idiopathic NOA and KS patients were opposite. What is known already NOA patients due to spermatogenic dysfunction, accounting for about 60 percent of the total azoospermia cases, may have opportunities to obtain sperm by mTESE and overall SRR of mTESE in NOA patients is about 50%. There were some of predictive factors for SRR at mTESE including age, testis volume, serum follicle stimulating hormone (FSH), testosterone (T), inhibin B, anti-Mullerian hormone and testicular histopathology, while the predictive factors of SRR remains underappreciated, especially in accordance with different type of etiology. Most notably, there are some controversies about the relationship between SSR and age at mTESE surgery. Study design, size, duration This retrospective study involved 3104 NOA patients with different etiologies treated with the first mTESE at the Reproductive Medical Centre of Peking University Third Hospital from March 2012 to December 2022. Participants/materials, setting, methods 3104 NOA patients were classified into seven groups according to etiologies including 1530 males with idiopathic NOA (iNOA), 763 males with Klinefelter syndrome (KS), 345 males with microdeletion of the AZFc, 177 cases with the history of cryptorchidism, 131 with a history of mumps orchitis, 89 males with cryptozoospermia and 69 cases with other causes. The end-point was the presence of one or more sperm. Multi-variable logistic regression was used to analyze sperm retrieval outcome. Main results and the role of chance Overall SRR was 43.46%, and there were different SRR in NOA patients with various etiologies with highest SRR of 89.31% in patients with a history of mumps orchitis and lowest SRR of 29.35% in iNOA patients. For patients with a history of mumps orchitis, there was a negative relationship between T and SRR (0.805 [0.654, 0.991] p = 0.041). For cryptozoospermia patients, shorter infertility duration was predictive for successful SRR (0.837 [0.704, 0.995], p = 0.044). Males with bigger testes had more likelihood of positive SRR (1.077 [1.005; 1.153], p = 0.035) in patients with cryptorchidism. BMI was an independent factor of SRR in AZFc-deleted patients (1.084 [1.013, 1.161], p = 0.020). For KS patients, lower male age (0.938 [0.898, 0.979], p = 0.004) and bigger testes (1.214[1.099, 1.342], p＜0.001) were predictive for successful SRR. Lower levels of FSH (0.984[0.970, 0.998] p = 0.025) and higher male age(1.079[1.050, 1.110], p＜0.001) were predictive for successful sperm retrieval in iNOA patients. So, the optimum age range of iNOA male undergoing mTESE should be 30 to 35 years old with the SRR of 29.13% when considering low SRR of iNOA males aged＜30 years (23.96%) and the negative effect of female age on ICSI outcome. Limitations, reasons for caution Surgeon performing mTESE maybe another predictor for SRR which is a potential selection bias. Inhibin B and Anti-Müllerian hormone weren’t included due to limited information in our database. We couldn’t verify the conclusion in more population from multicenter despite the current study with the large sample size so far. Wider implications of the findings Our results provide valuable information for NOA patients who want to counsel surgeons about their treatments to help patients and surgeons to perform a shared decision-making for optimal therapy methods, especially about optimal age treated with mTESE in iNOA and KS patients. Trial registration number not applicable

O-135 Application of AMH determination in preoperative evaluation of micro-TESE in NOA patients

Abstract Study question To investigate the value of anti-Müllerian hormone(AMH) determination for estimating the sperm retrieval rate(SRR) of microdissection testicular sperm extraction(micro-TESE) in non-obstructive azoospermia(NOA) patients. Summary answer NOA with low AMH would have more opportunity to present heterogeneous seminiferous tubules when micro-TESE was performed and had higher SRR, especially in idiopathic cases. What is known already For infertile patients with NOA, micro-TESE is considered to have higher SRR than traditional surgery methods. However, serum inhibin B, follicle-stimulating hormone (FSH) and various clinical parameters are not reliable predictors for the presence of focal spermatogenesis and SRR. In male, AMH is a glycoprotein secreted by Sertoli cells and facilitate the regression of Müllerian structures in the developing foetus. It is still controversial that whether AMH level has value to predict the SRR of micro-TESE. Study design, size, duration This was a retrospective case-control study. From September 2014 to May 2022, 502 NOA patients treated with micro-TESE were divided into different groups according to their surgery outcome and seminiferous tubules appearance. Age, testis volume, serum AMH, FSH and testosterone level were compared between the different groups. The differences of SRR and AMH level in NOA patients with different etiologies were also compared. Participants/materials, setting, methods Micro-TESE was performed at x10 to x20 magnification. An attempt was made to identify seminiferous tubules that were larger and more opaque than others. The procedure was terminated when sperm were retrieved. If all tubules were seen to have an identical appearance, at least three samples (upper, middle, and lower) were extracted. Venous blood samples were drawn from each patient (7–10 AM) after an overnight fast. FSH and AMH were measured by electrochemiluminescence immunoassay. Main results and the role of chance Testicular sperms were successfully retrieved in 270 cases (SRR=53.8%). There were no statistical differences in age, testicular volume, FSH and testosterone levels between the patients who succeeded and failed to obtain sperm (all P&amp;gt;0. 05). The patients who obtained sperms had lower serum AMH level than those without sperm [0.81(0.16, 3.26)μg/L vs.1.37(0.21, 4.84)μg/L, P&amp;lt;0.05]. Patients with orchitis or AZFc deletion, cryptorchidism, KS, idiopathic azoospermia would have different AMH level [0.15(0.01, 0.41)μg/L, 5.71(3.57, 8.26)μg/L, 2.29(1.36, 3.81)μg/L, 0.15(0.05, 0.39)μg/L, 2.46(0.75, 5.49)μg/L, P&amp;lt;0.05]. Idiopathic azoospermia patients who obtained sperms had higher age but lower testosterone and AMH level than those without sperm [(35.2±8.9) years vs. (32.5±5.5) years, P&amp;lt;0.05, (3.1±1.4)μg/L vs. (3.7±2.1)μg/L, P&amp;lt;0.05; 1.63(0.35, 3.84)μg/L vs.3.00(1.20, 6.68)μg/L, P&amp;lt;0.05], there were no statistical differences in testicular volume and FSH level between the two groups (P&amp;gt;0.05). Receiver operating characteristic (ROC) curve showed that cut-off of serum AMH for successful sperm retrieval of idiopathic azoospermia patients was determined to be 2.96, with a sensitivity of 0.710 and specificity of 0.523, area under the curve (AUC) was 0.649. In the cases presenting heterogeneous seminiferous tubules during micro-TESE had lower AMH level and higher SRR than those presenting homogeneous seminiferous tubules [0.55(0.12, 2.05)μg/L vs.2.99(0.76, 6.11)μg/L, 75.9%(236/311)vs. 17.8%(34/191), all P&amp;lt;0.05]. Limitations, reasons for caution Pathology analysis should be involved in the following study. Randomized controlled trial comparing micro-TESE and traditional TESE would demonstrate that whether idiopathic azoospermia patients with higher serum AMH level would have less benefit by microsurgery than patients with lower AMH level. Wider implications of the findings Recently, in our pathologic research, NOA patients with extremely lower serum AMH level were observed to have more opportunity to present severe hyalinization in their seminiferous tubules. Tubules with severe hyalinization have less Sertoli cells and seem very thin. Therefore, tubules with spermatogenesis would be easy to identify during micro-TESE. Trial registration number not applicable

P-021 A novel SNP in HUWE1 promoter confers increased risk of NOA by affecting the RA/RARα pathway in Chinese individuals

Abstract Study question The present study aimed to investigate the role of HUWE1 in non-obstructive azoospermia (NOA) etiology and germ cell differentiation. Summary answer An SNP mutation in HUWE1 promoter region downregulates HUWE1 expression in NOA patients, the genetic polymorphisms of HUWE1 are related to the pathogenesis of NOA. What is known already HUWE1, a ubiquitin ligase, is crucial to the development of male reproductive system and embryonic development. However, the specific role of HUWE1 in regulating germ cell differentiation is unclear, and clinical evidence for the relationship between HUWE1 and the pathogenesis of male infertility is lacking. Study design, size, duration 190 NOA Chinese patients were recruited and analyzed single nucleotide polymorphisms (SNPs) in HUWE1 gene. Participants/materials, setting, methods Regulation of HUWE1 gene expression by RARα factor was evaluated by performing ChIP assay, EMSA, and siRNA-mediated RARα knockdown. We used C18-4 cells to determine whether HUWE1 participated in RA-mediated RARα signaling and performed luciferase assay, CCK-8 assay, immunofluorescence staining, RT-qPCR, and western blot analysis. The expression level of HUWE1 and RARa was evaluated in clinical samples obtained from NOA or obstructive azoospermia (OA) patients using RT-qPCR. Main results and the role of chance We explored the mechanism by which polymorphism of the HUWE1 promoter leads to spermatogenesis disorders, and focused on the identification of mutations in the SNP site of the HUWE1 regulatory sequence among patients with NOA, which may influence HUWE1 expression. The results of in vitro experiments showed that RARα binds to the HUWE1 promoter region and regulates its expression. However, the regulatory effect of RARα weakened following introduction of the mutation into the HUWE1 regulatory sequence. The expression of spermatogonial differentiation-related gene STRA8 was impaired, and γH2AX foci accumulated following inhibition of HUWE1 gene expression using siRARα. Finally, to determine whether HUWE1 expression was important in patients with NOA, its expression was verified using testicular puncture samples collected from patients with NOA and the results revealed that HUWE1 expression was abnormal (P &amp;lt; 0.01). In addition, RARα expression was significantly downregulated (P &amp;lt; 0.001). Overall, our result support the hypothesis that HUWE1 expression is induced by the RA/RARα signaling pathway and it inhibit γH2AX foci accumulation, thereby considerably promoting sperm differentiation and meiotic prophase. Limitations, reasons for caution The effect of SNPs in HUWE1 on male infertility is further studied in a wider population. Wider implications of the findings Our study of HUWE1 and RA/RARα signaling pathway provides new information on the regulatory mechanism of spermiogenesis, suggesting that SNPs in HUWE1 promoter are etiological factors in patients with NOA and providing a rationale for further drug development to target HUWE1 in these patients. Trial registration number the National Key Research and Development Program of China (2018YFC1003603)

P-080 Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval and cumulative live birth rates

Abstract Study question What is the added value of enzymatic processing of testicular biopsies on testicular sperm retrieval rates in patients with non-obstructive azoospermia (NOA)? Summary answer In addition to mechanical mincing, enzymatic digestion increased sperm retrieval rates in testicular biopsies of NOA patients with 48.9%. What is known already Many studies focus on the surgical approach to optimize recovery of testicular sperm in NOA, and in spite of that, there is still controversy whether the type of surgery makes any difference as long as multiple biopsies are taken. Few studies, however, focus on the role of the IVF laboratory and the benefit of additional lab procedures, like enzymatic digestion, in order to optimize sperm retrieval rates and CLB rate per TESE. Study design, size, duration A retrospective single-center cohort study including all patients who underwent their first TESE by open multiple-biopsy method from January 2004 till July 2022. Only patients with a normal karyotype, absence of Y-q deletions and a strict diagnosis of NOA based on histology were included. Primary outcome was sperm retrieval after mincing or enzymes for intracytoplasmic sperm injection (ICSI). Secondary outcome was CLB after ICSI with fresh TESE, and subsequent ICSI cycles with frozen TESE. Participants/materials, setting, methods Multiple biopsies were obtained from the testis, unilateral or bilateral, on the day of oocyte retrieval. Upon mechanical mincing, dishes were searched for 30 min; when no or insufficient numbers of spermatozoa were observed, enzymatic treatment was performed using collagenase type IV. Multivariable regression analysis was performed to predict CLB by adjusting for the following confounding factors: male age, male FSH level, cryptorchidism, enzymatic digestion, number of oocytes and female age. Main results and the role of chance Hundred-eighteen patients were included of which 61.0% had successful sperm retrieval. Spermatozoa were retrieved after mechanical mincing (23.7%; 28/118) or after additional enzymatic digestion of the remaining 90 patients (48.9%; 44/90). Mean male characteristics were not different between patients with sperm retrieval after mincing or enzymes: age (34.5 vs 34.5 y), testis volume (10.2 vs 10.6 ml), FSH (17.8 vs 16.9 IU/l), cryptorchidism (21.4 vs 34.1%) and histological diagnosis (Sertoli Cell Only 53.6 vs 47.7%, maturation arrest 21.4 vs 38.6%, sclerosis/atrophy 25.0 vs 13.6%), respectively. Of the 72 patients with sperm available for ICSI, 23/72 (31.9%) obtained a LB after the injection with fresh testicular sperm (fresh and frozen embryo transfers). Forty-nine patients remained without LB, of which 34 had supernumerary testicular sperm frozen. Of these, 9/47 (47.4%) had a LB after ICSI with frozen testicular sperm, giving rise to a total CLB per TESE of 32/118 (27.1%) or 32/72 (44.4%) CLB per TESE with sperm. Of the female characteristics (couples with sperm available), only female age (30.3 vs 32.7 y – p = 0,042) was significantly lower in the group with a live birth. Multivariable logistic regression analysis showed that enzymatic digestion was associated with significant decrease of CLB per TESE. Limitations, reasons for caution Limitations of the study are related to the retrospective design. The selection of only NOA patients with specific characteristics (normal karyotype and absence Y-q deletion) and having their first TESE ever strengthens our findings. Whether enzymatic digestion after a failed TESE without digestion may improve retrieval rate remains undecided. Wider implications of the findings Enzymatic processing increases the sperm retrieval rate from testicular biopsies of NOA patients compared to mechanical mincing, demonstrating the importance of an appropriate laboratory protocol. NOA patients should be counselled that if sperm has been found after enzymatic digestion, their chances to father a genetically own child will be lower. Trial registration number not applicable

O-147 Factors influencing ICSI outcome after sperm retrieval in nonobstructive azoospermia patients with different types of etiologies: a retrospective study of 1157 patients

Abstract Study question What are predictors of fertilization, clinical pregnancy, miscarriage and livebirth delivery outcomes after microdissection testicular sperm extraction (mTESE)-ICSI in NOA patients with different etiologies? Summary answer Predictors for ICSI outcome were different in patients with various etiologies and type of NOA are associated with ICSI outcome in these patients. What is known already NOA patients due to spermatogenic dysfunction, accounting for about 60 percent of the total azoospermia cases, may have opportunities to be fathers with their own biological children by mTESE combined with ICSI. For them, there were some of predictive factors for ICSI outcome including sperm motility, sperm morphology, testis volume and levels of reproductive hormone including follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T), while the predictive factors of ICSI outcome remains underappreciated, especially about the relationship between ICSI outcome and male factors after reviewing the large scale clinical data. Study design, size, duration This retrospective study involved 1157 NOA patients with treated 1534 complete ICSI cycles at the Reproductive Medical Centre of Peking University Third Hospital from March 2012 to October 2021. Participants/materials, setting, methods 1534 ICSI cycles included 558, 359, 243, 155, 128, 56 and 35 cycles performed in males with idiopathic NOA (iNOA), Klinefelter syndrome (KS), AZFc microdeletions, cryptorchidism, a history of mumps orchitis, cryptozoospermia and with other causes separately. Primary outcome was livebirth delivery per ICSI cycle (LBR) and secondary outcomes were clinical pregnancy rate per ICSI cycle (PR) and fertilization rate (FR). Multi-variable logistic and linear regression was used to analyze ICSI outcome. Main results and the role of chance Overall LBR, PR and FR were 37.35%, 44.98%, 49.34% separately. Patients with mumps orchitis showed the highest LBR, PR and FR of 53.13%, 60.94% and 59.52% respectively, and AZFc-deleted patients displayed the lowest LBR, PR and FR of 25.93%, 30.04% and 32.87% separately. Lower female age (0.947 [0.904;0.993], p = 0.024), more transferred embryos (1.345 [1.225;1.477], p＜0.001), higher AFC (1.033[1.011;1.056], p = 0.003) were related with higher LBR, and higher male BMI (0.974[0.950; 0.999], p = 0.046) and lower AFC (1.057[1.034; 1.080], p＜0.001) were related with lower PR. Comparing outcome of mumps orchitis group, patients with cryptorchidism were associate with lower LBR(0.467[0.281; 0.778], p = 0.003), PR (0.440[0.268;0.721], p = 0.001) and FR(-0.169 [-0.226; -0.11], p＜0.001) separately. AZFc-deleted patients had a relationship with lower LBR (0.426[0.260; 0.697], p = 0.001), (0.273[0.170; 0.437], p＜0.001) and FR (-0.282 [-0.336; -0.228], p＜0.001) respectively. KS patients were related with lower LBR (0.455[0.288; 0.720], p = 0.001), PR (0.477 [0.306; 0.745], p = 0.001) and FR (-0.073 [-0.126; -0.020], p = 0.007) separately. iNOA patients were associated with lower PR (0.616 [0.409; 0.928], p = 0.020) and FR (-0.076 [-0.124; -0.029], p = 0.002) respectively. Furthermore, patients with cryptozoospermia were related with lower FR (-0.141 [-0.220; -0.061], p = 0.001). Limitations, reasons for caution Sperm characteristics maybe predictors for ICSI outcome which is a potential selection bias. Pituitary prolactin and Anti-Müllerian hormone weren’t included as covariates due to limited information in our database. We couldn’t verify the conclusion in more population from multicenter despite the current study with the large sample size so far. Wider implications of the findings Our results provide valuable information for NOA patients who want to counsel surgeons about their ICSI outcome to help patients and surgeons to perform a shared decision-making for optimal therapy methods, especially for AZFc-deleted patients with worse ICSI outcome who may consider donor semen to get better ICSI outcome. Trial registration number not applicable

O-137 Mapping microRNA profiling and dysregulated pathways in non-obstructive azoospermia cases with maturation arrest: A systematic review and in-silico analysis of the affected pathways

Abstract Study question Could microRNA profiling be a useful tool towards better understanding the pathophysiological mechanisms leading to non-obstructive azoospermia (NOA) due to maturation arrest? Summary answer MicroRNA profiling may be an efficient method towards unveiling maturation arrest pathophysiology as microRNAs constitute master posttranscriptional regulators of cell-cycle control during spermatogenesis. What is known already Non-obstructive azoospermia constitutes the most severe form of male factor infertility, affecting approximately 10% of infertile men. According to the histological subtype, NOA is classified into three main categories namely hypospermatogenesis, maturation arrest, and Sertoli-cell only syndrome. Maturation arrest is one of the most complex forms of NOA characterized by failure of spermatogenesis. Despite advances, the pathophysiology of maturation arrest remains a conundrum rendering management complex. Recent studies indicate that alterations on microRNA expression patterns could lead to maturation arrest. Mapping microRNA profiling and highlighting dysregulated pathways of maturation arrest cases may lead the way forward. Study design, size, duration A systematic review was performed in PubMed/Medline and Embase up to April 2022. Only full-length original studies in humans were included. Strict inclusion-exclusion criteria were applied aiming to isolate studies comparing microRNA profiling between maturation arrest cases versus normal fertile men, and/or men with obstructive azoospermia (OA). Following study selection, data on altered microRNA expression patterns were analyzed to underline differences between the abovementioned groups. Subsequently, in-silico analysis was performed to compare affected gene pathways. Participants/materials, setting, methods The studied population consisted of maturation arrest cases. Control groups consisted of men with normal spermatogenesis and/or men with OA. Predicted microRNA–target pairs were retrieved from microT-CDS, while a 0.8 cutoff threshold was applied. The GTEx repository was used to identify microRNA-targeted genes in the testis. Annotations derived from Ensembl and miRbase. Gene-set enrichment analysis was performed employing the KEGG-database. Fisher’s exact test was performed in R package limma, setting a 0.01 p-value threshold. Main results and the role of chance Ten studies reported altered microRNA expression patterns in maturation arrest versus normal spermatogenesis cases and six studies in maturation arrest versus OA cases. Considering the maturation arrest-normal spermatogenesis arm, analysis revealed that eight microRNAs, which were upregulated in maturation arrest cases, namely hsa-miR-449a, hsa-miR-370-3p, hsa-miR-10b-3p, hsa-miR-539-5p, hsa-miR-22-5p, hsa-miR-605-5p, hsa-miR-491-3p and hsa-miR-302d-3p, affected 106 statistically significant gene-targets in the testis. The three most significantly affected pathways included the “microRNAs in cancer” pathway (55 affected genes, p-value &amp;lt;0.001), the “EGFR tyrosine kinase inhibitor resistance pathway” (29 affected genes, p-value = 0.007), and the “Hedgehog signaling pathway” (22 affected genes, p-value=0.008). Regarding the second arm of maturation arrest-OA, functional analysis indicated that four microRNAs, presenting to be downregulated in maturation arrest cases, namely hsa-miR-181a-5p, hsa-miR-449a, hsa-miR-182-5p and hsa-miR-138-5p, affected 421 statistically significant gene-targets in testis. The three most significantly affected pathways included the “pathways in cancer” pathway (86 affected genes, p-value =0.009), the “microRNAs in cancer” pathway (48 affected genes, p-value &amp;lt; 0.001), and the “ cGMP-PKG signaling” pathway (36 affected genes, p-value=0.008). In summary, microRNAs affect the expression of master genes regulating cell-cycle and maturation processes during spermatogenesis, leading to maturation arrest as well as to other associated pathologies, including cancer. Limitations, reasons for caution The limited number of the included studies, as well as the small size population characterizing the great majority of them, constitute the main limitations of this study. Another reason for caution is the great heterogeneity observed among the studies regarding the molecular methods employed for microRNA profiling. Wider implications of the findings Data presented herein indicate that microRNA functional analysis may be a significant tool towards better understanding the pathophysiological basis of spermatogenesis maturation arrest, indicating future personalized diagnostic and therapeutic targets. Moreover, microRNA analysis may also constitute an efficient method of evaluating predisposition of NOA patients to other pathologies including cancer. Trial registration number Not applicable

P-086 Prevalence and clinical factors associated with biochemical hypogonadism in infertile men with non-obstructive azoospermia: a single-center cohort study including 761 patients

Abstract Study question What is the prevalence, and which factors are associated with hypogonadism in infertile men with non-obstructive azoospermia (NOA)? Summary answer The prevalence of biochemical hypogonadism among NOA males is significant. The condition is associated with testicular volume, estradiol levels, paternal age, and testicular histopathology results. What is known already NOA is a severe and irreversible condition that accounts for approximately 60% of azoospermia cases, primarily associated with intrinsic testicular deficiencies of various causes. NOA men can present signs of hypogonadism, associated or not with elevated circulating FSH levels; however, the exact prevalence is ill-reported. There is also a lack of data on the clinical factors associated with hypogonadism. We, therefore, estimated the prevalence of biochemical hypogonadism using real-world data and investigated its relationship with clinical factors. Study design, size, duration An observational cohort study was conducted, including 767 consecutive NOA patients with primary spermatogenic failure seeking fertility treatment at a University-affiliated tertiary center for male reproductive health between January 2014 and September 2021. Biochemical hypogonadism was defined as total testosterone (T) levels &amp;lt;350 ng/dL, measured by chemiluminescence immunoassay from a venous sample, collected in the morning, from 8:00 to 10:00 am, and confirmed on a second analysis at least one week apart. Participants/materials, setting, methods All included patients (age range: 23-55 years) had complete clinical, hormonal, genetic, seminal, and histopathology data and were naïve concerning previous sperm retrieval attempts. Patients with NOA due to hypogonadotropic hypogonadism and those using hormonal therapy were excluded. Prevalence rates were computed by Goodman’s method. The relationship between clinical factors and hypogonadism was assessed by logistic regression analysis. We also assessed the frequency of patients with high and within-range FSH levels in the cohort. Main results and the role of chance NOA patients with biochemical hypogonadism represented 80.3% (95% confidence interval [CI] 77.3–43.7) of the studied population. The median (interquartile range [IQR]) of T and FSH levels in hypogonadal men were 276 ng/dL (233.0-303 ng/dL) and 12.3 IU/L (8.9-18.2). Among them, hypergonadotropic (FSH&amp;gt;12 IU/L) and normogonadotropic (within-range FSH levels 1.5-12.0 IU/L) patients were 52.1% (95% CI 48.1-56.0) and 47.8% (43.9-51.8) of studied individuals, respectively. Logistic regression analyses revealed that testicular volume (p &amp;lt; 0.0001) and estradiol levels (p = 0.0005) had a significant inverse relationship with hypogonadism, whereas paternal age (p = 0.02) showed a positive relationship. Moreover, testicular histopathology was a relevant predictor (p = 0.003), with an inverse relationship between hypogonadism and germ cell maturation arrest and a positive relationship between hypogonadism and hypospermatogenesis or Sertoli cell-only. Using these variables, a model constructed to predict hypogonadism had an area under the ROC curve of 0.72. Limitations, reasons for caution Limitations include possible unmeasured confounding factors and the intra- and inter-assay electrochemiluminescence method variability. Another limitation is that the study center is a referral facility for azoospermic patients; thus, hypogonadism prevalence rates reported herein might not represent that of the general population of NOA males attending general fertility centers. Wider implications of the findings Efforts are needed to provide adequate care for this population due to its association with poor quality of life. Moreover, as recent evidence has suggested that hormonal therapy to boost T production might increase sperm retrieval success rates, studies investigating hormonal therapy in such patients may be warranted. Trial registration number Not applicable

P-016 WHO 2021-based comprehensive appraisal of sperm factor parameters’ association with embryological and clinical outcomes. A single center study of 4013 PGT-A cycles

Abstract Study question What is the impact of sperm parameters and paternal age on all embryological and clinical outcomes during ICSI-cycles with aneuploidy testing? Summary answer Reduced basal/post swim-up motility and patients with concentration+morphology+motility &amp;lt;5th-percentile (based on WHO-2021) are associated with poorer embryological outcomes and cumulative-live-birth-rate per concluded PGT-A cycle. What is known already Previous studies reported an association between oligoasthenoteratozoospermia (OAT), obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) and poorer fertilization/blastulation rates, but no impact on euploidy rates at the blastocyst stage. Similarly, the clinical outcomes were mostly independent from sperm characteristics when euploid blastocysts are transferred. The data about paternal age effect are controversial, varying between absent and severe embryological/clinical impact. Moreover, little is known about the association between each semen analysis parameters and embryological/clinical outcomes. Lastly, most of the evidence to date was based on WHO 2010 criteria, recently updated by WHO 2021, and did not mention cumulative-live-birth-rates (CLBR) per cycle. Study design, size, duration Retrospective study at a single private center involving 4013 ICSI+PGT-A cycles with own-oocytes conducted by 3101 couples (2013-2021). The primary embryological outcome was euploid-blastocyst-rate (EBR) per cohort of inseminated oocytes. The primary clinical outcome was CLBR per concluded cycle (i.e., LB achieved or no transferable/all transferred blastocysts). Intermediate embryological/clinical outcomes were also tested. All parameters were analyzed as continuous variables. For some analyses we categorized paternal age (&amp;lt;38/38-44/&amp;gt;44 years) and sperm factor (WHO-2021 parameters&amp;lt;5th percentile). Participants/materials, setting, methods GnRH-antagonist ovarian-stimulation, ICSI, trophectoderm biopsy without zona-pellucida drilling in day3, qPCR/NGS-based analysis to report non-mosaic aneuploidies (segmental aneuploidies reported from 2017 onwards; N = 1991 cycles) and vitrified-warmed euploid single-blastocyst-transfers were conducted. Semen analysis (volume, concentration, total sperm per ejaculate, basal/post swim-up motility, morphology), sperm characteristics (ejaculated/FNA/TESE; fresh/frozen), DNA-fragmentation-index (DFI, outlined via Tunel-test; N = 188 cycles), paternal BMI and age were investigated for their association with embryological/clinical outcomes adjusted for confounders via linear/logistic regressions. Main results and the role of chance Maternal and paternal age were 38.9±3.2(range:23-45) and 41.9±5.7(25-72) years, 10±6.3(1-44) cumulus-oocyte-complexes were retrieved and 7.3±4.6(1-32) metaphase-II oocytes inseminated. Cycles showing all parameters &amp;gt;5th-percentile were 47.1%(N = 1890), concentration&amp;lt;5th-percentile were 3.7%(N = 149), motility&amp;lt;5th-percentile were 3.9%(N = 155), morphology&amp;lt;5th-percentile were 10.1%(N = 405), concentration+motility&amp;lt;5th-percentile were 1.7%(N = 70), concentration+morphology&amp;lt;5th-percentile were 6.2%(N = 248), motility+morphology&amp;lt;5th-percentile were 5.0%(N = 70), concentration+motility+morphology&amp;lt;5th-percentile were 19.9%(N = 797), OA and NOA were 1.4%(N = 56) and 1.0%(N = 41), respectively. The only confounder upon EBR per cohort of metaphase-II oocytes was maternal age. Only basal/post swim-up motility was associated with this outcome. When categorized, only concentration+morphology&amp;lt;5th-percentile (-2.7%,95%CI -5.1 to -0.3%, adjusted-p=0.03), concentration+morphology+motility&amp;lt;5th-percentile (-4.0%,95%CI -5.5 to -2.6%, adjusted-p&amp;lt;0.01), OA (-5.5%,95%CI -10.3 to -0.7%, adjusted-p=0.03) and NOA (-5.8%,95%CI -11.4 to -0.1%, adjusted-p=0.05) showed significantly poorer results. Maternal age and number of metaphase-II oocytes were the confounders upon the chance to obtain ≥1 euploid blastocyst and ≥1 LB among concluded cycles. Only basal/post swim-up motility was associated with this outcome. When categorized, only concentration+morphology+motility&amp;lt;5th-percentile showed significantly lower chances (multivariate-OR:0.74,95%CI 0.61-0.90, adjusted-p&amp;lt;0.01 and multivariate-OR:0.74,95%CI 0.59-0.92, adjusted-p&amp;lt;0.01). No association was reported between semen parameters/sperm factor categories and 1PN/³3PN rates, day5-7 blastocyst-rate, morphological quality, outcomes per euploid transfer. Paternal age categories were associated with day5-blastocyst (N = 1131/3308,34.2%, N = 1179/3644,32.4%, and N = 919/3127,29.4%) and AA-blastocyst rates (N = 1732/3308,52.4%, N = 1815/3644,49.8%, and N = 1387/3127,44.4%) in men &amp;lt;38/38-44/&amp;gt;44 years, respectively (p &amp;lt; 0.01). Limitations, reasons for caution Some confounders were unbalanced among the study groups but accounted for in linear/logistic regressions. 9% of the cycles were not concluded by the time of abstract drafting. The sample size in the groups of surgically-retrieved sperm is limited, in part because NOA patients due to genetic causes were not included. Wider implications of the findings By comprehensively accounting for sperm factor characteristics, this report provides reproductive professionals with useful figures to counsel infertile couples about their chance of success during IVF. These estimates are useful for the decision-making process regarding the most effective clinical strategies to apply. Further studies including more NOA/OA patients are warranted. Trial registration number not applicable

A Machine Learning Approach for the Prediction of Testicular Sperm Extraction in Nonobstructive Azoospermia: Algorithm Development and Validation Study.

Testicular sperm extraction (TESE) is an essential therapeutic tool for the management of male infertility. However, it is an invasive procedure with a success rate up to 50%. To date, no model based on clinical and laboratory parameters is sufficiently powerful to accurately predict the success of sperm retrieval in TESE. The aim of this study is to compare a wide range of predictive models under similar conditions for TESE outcomes in patients with nonobstructive azoospermia (NOA) to identify the correct mathematical approach to apply, most appropriate study size, and relevance of the input biomarkers. We analyzed 201 patients who underwent TESE at Tenon Hospital (Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris), distributed in a retrospective training cohort of 175 patients (January 2012 to April 2021) and a prospective testing cohort (May 2021 to December 2021) of 26 patients. Preoperative data (according to the French standard exploration of male infertility, 16 variables) including urogenital history, hormonal data, genetic data, and TESE outcomes (representing the target variable) were collected. A TESE was considered positive if we obtained sufficient spermatozoa for intracytoplasmic sperm injection. After preprocessing the raw data, 8 machine learning (ML) models were trained and optimized on the retrospective training cohort data set: The hyperparameter tuning was performed by random search. Finally, the prospective testing cohort data set was used for the model evaluation. The metrics used to evaluate and compare the models were the following: sensitivity, specificity, area under the receiver operating characteristic curve (AUC-ROC), and accuracy. The importance of each variable in the model was assessed using the permutation feature importance technique, and the optimal number of patients to include in the study was assessed using the learning curve. The ensemble models, based on decision trees, showed the best performance, especially the random forest model, which yielded the following results: AUC=0.90, sensitivity=100%, and specificity=69.2%. Furthermore, a study size of 120 patients seemed sufficient to properly exploit the preoperative data in the modeling process, since increasing the number of patients beyond 120 during model training did not bring any performance improvement. Furthermore, inhibin B and a history of varicoceles exhibited the highest predictive capacity. An ML algorithm based on an appropriate approach can predict successful sperm retrieval in men with NOA undergoing TESE, with promising performance. However, although this study is consistent with the first step of this process, a subsequent formal prospective multicentric validation study should be undertaken before any clinical applications. As future work, we consider the use of recent and clinically relevant data sets (including seminal plasma biomarkers, especially noncoding RNAs, as markers of residual spermatogenesis in NOA patients) to improve our results even more.

Isolated follicle-stimulating hormone elevation as a prognostic factor for negative microTESE outcome

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

A novel SNP in HUWE1 promoter confers increased risk of NOA by affecting the RA/RARα pathway in Chinese individuals.

The ubiquitin ligase HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 is essential for the establishment and maintenance of spermatogonia. However, the role of HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 in regulating germ cell differentiation remains unclear, and clinical evidence linking HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 to male infertility pathogenesis is lacking. This study aims to investigate the role of HUWE1 in germ cell differentiation and the mechanism by which a HUWE1 single nucleotide polymorphism increases male infertility risk. We analyzed HUWE1 single nucleotide polymorphisms in 190 non-obstructive azoospermia patients of Han Chinese descent. We evaluated HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 regulation by retinoic acid receptor alpha using chromatin immunoprecipitation assays, electrophoretic mobility shift assays, and siRNA-mediated RARα knockdown. Using C18-4 spermatogonial cells, we determined whether HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 participated in retinoic acid-mediated retinoic acid receptor alpha signaling. We performed luciferase assays, cell counting kit-8 assays, immunofluorescence, quantitative real-time polymerase chain reaction, and western blotting. We quantified HUWE1 and retinoic acid receptor alpha in testicular biopsies from non-obstructive azoospermia and obstructive azoospermia patients using quantitative real-time polymerase chain reaction and immunofluorescence. Three HUWE1 single nucleotide polymorphisms were significantly associated with spermatogenic failure in 190 non-obstructive azoospermia patients; one (rs34492591) was in the HUWE1 promoter. Retinoic acid receptor alpha regulates HUWE1 gene expression by binding to its promoter. HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 participates in retinoic acid/retinoic acid receptor alpha signaling pathway and regulates the expression of germ cell differentiation genes STRA8 and SCP3 to inhibit cell proliferation and reduce γH2AX accumulation. Notably, significantly lower levels of HUWE1 and RARα were detected in testicular biopsy samples from non-obstructive azoospermia patients. An HUWE1 promoter single nucleotide polymorphism significantly downregulates its expression in non-obstructive azoospermia patients. Mechanistically, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 regulates germ cell differentiation during meiotic prophase through its participation in retinoic acid/retinoic acid receptor alpha signaling and subsequent modulation of γH2AX. Taken together, these results strongly suggest that the genetic polymorphisms of HUWE1 are closely related to spermatogenesis and non-obstructive azoospermia pathogenesis.

Single-cell multi-omics analysis reveals dysfunctional Wnt signaling of spermatogonia in non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is the most severe type that leads to 1% of male infertility. Wnt signaling governs normal sperm maturation. However, the role of Wnt signaling in spermatogonia in NOA has incompletely been uncovered, and upstream molecules regulating Wnt signaling remain unclear. Bulk RNA sequencing (RNA-seq) of NOA was used to identify the hub gene module in NOA utilizing weighted gene co-expression network analyses (WGCNAs). Single-cell RNA sequencing (scRNA-seq) of NOA was employed to explore dysfunctional signaling pathways in the specific cell type with gene sets of signaling pathways. Single-cell regulatory network inference and clustering (pySCENIC) for Python analysis was applied to speculate putative transcription factors in spermatogonia. Moreover, single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) determined the regulated genes of these transcription factors. Finally, spatial transcriptomic data were used to analyze cell type and Wnt signaling spatial distribution. The Wnt signaling pathway was demonstrated to be enriched in the hub gene module of NOA by bulk RNA-seq. Then, scRNA-seq data revealed the downregulated activity and dysfunction of Wnt signaling of spermatogonia in NOA samples. Conjoint analyses of the pySCENIC algorithm and scATAC-seq data indicated that three transcription factors (CTCF, AR, and ARNTL) were related to the activities of Wnt signaling in NOA. Eventually, spatial expression localization of Wnt signaling was identified to be in accordance with the distribution patterns of spermatogonia, Sertoli cells, and Leydig cells. In conclusion, we identified that downregulated Wnt signaling of spermatogonia in NOA and three transcription factors (CTCF, AR, and ARNTL) may be involved in this dysfunctional Wnt signaling. These findings provide new mechanisms for NOA and new therapeutic targets for NOA patients.

Predicting Micro-TESE among Heterogeneous Nonobstructive Azoospermic Patients: The Impact on Surgical Decision and ICSI

Background. Previously published predictive models for microdissection testicular sperm extraction (micro-TESE) were generally assumed patients with nonobstructive azoospermia (NOA) a homogenous population, i.e., the laboratory predictors were associated with sperm retrieval rate (SRR) in a similar way among different subpopulations. In addition, previous studies primarily regarded the success of sperm retrieval as the sole endpoint, although live birth is the ultimate goal for the couples. Objectives. The main objective is to develop and evaluate the clinical benefit of a model predicting the clinical outcome of micro-TESE in heterogeneous population with NOA. The outcome of pregnancy was taken into account via assessing the association between the predicted outcome of micro-TESE and pregnancy. Materials and Methods. A development cohort of 1,292 patients with NOA and an external validation cohort of 530 patients were included. Sperm retrieval was performed using micro-TESE. Clinical outcomes, including sperm retrieval, clinical pregnancy, and live birth, were collected. We developed a model using the machine learning method random forest and provided a web-based calculator. Results. The SRR was 38.1% (492/1,292) in the development cohort and 48.5% (257/530) in the validation cohort. The final model includes etiology, AMH, sperm retrieval surgical history, testicular volume, FSH, LH, and age as predictors (ordered by variable importance). The area under the curve of our model was 0.76 (0.74–0.79) in the development cohort and 0.75 (0.71–0.79) in the external validation cohort. The decision curve analysis showed that personalized model-based surgical decision provides additional clinical benefit. The clinical pregnancy rate (CPR) and cumulative live birth rate (CLBR) were 45.3% (405/895) and 57.6% (338/587), respectively, in the overall population. For patients of different SRR, the CPR and CLBR of whom had successful sperm retrieval were similar. Discussion and Conclusion. Our model predicting the SRR of micro-TESE was generalizable and easy to use. Predicted pregnancy outcomes like CPR and CLBR could also be derived from predicted SRR. A model-based surgical decision after personalized consultation would be beneficial to patients with NOA.

Predictors of successful salvage microdissection testicular sperm extraction (mTESE) after failed initial TESE in patients with non-obstructive azoospermia: A systematic review and meta-analysis.

There has been no systematic review and meta-analysis to analyze and summarize the predictive factors of successful sperm extraction in salvage microdissection testicular sperm extraction. We aimed to investigate the factors predicting the result of salvage microdissection testicular sperm extraction in patients with non-obstructive azoospermia who failed the initial microdissection testicular sperm extraction or conventional testicular sperm extraction. We conducted a systematic literature search in PubMed, Web of Science, EMBASE, and the Cochrane Library for literature that described the characteristics of patients with non-obstructive azoospermia who underwent salvage microdissection testicular sperm extraction after failing the initial microdissection testicular sperm extraction or conventional testicular sperm extraction published prior to June 2022. This meta-analysis included four retrospective studies with 332 patients with non-obstructive azoospermia who underwent a failed initial microdissection testicular sperm extraction and three retrospective studies with 177 non-obstructive azoospermia patients who underwent a failed conventional testicular sperm extraction. The results were as follows: among non-obstructive azoospermia patients whose first surgery was microdissection testicular sperm extraction, younger patients (standard mean difference: -0.28, 95% confidence interval [CI]: -0.55 to -0.01) and those with smaller bilateral testicular volume (standard mean difference: -0.55, 95% CI: -0.95 to -0.15), lower levels of follicle-stimulating hormone (standard mean difference: -0.86, 95% CI: -1.18 to -0.54) and luteinizing hormone (standard mean difference: -0.68, 95% CI: -1.16 to -0.19), and whose testicular histological type was hypospermatogenesis (odds ratio: 3.52, 95% CI: 1.30-9.53) were more likely to retrieve spermatozoa successfully, while patients with Sertoli-cell-only syndrome (odds ratio: 0.41, 95% CI: 0.24-0.73) were more likely to fail again in salvage microdissection testicular sperm extraction. Additionally, in patients who underwent salvage microdissection testicular sperm extraction after a failed initial conventional testicular sperm extraction, those with testicular histological type of hypospermatogenesis (odds ratio: 30.35, 95% CI: 8.27-111.34) were more likely to be successful, while those with maturation arrest (odds ratio: 0.39, 95% CI: 0.18-0.83) rarely benefited. We found that age, testicular volume, follicle-stimulating hormone, luteinizing hormone, hypospermatogenesis, Sertoli-cell-only syndrome, and maturation arrest were valuable predictors of salvage microdissection testicular sperm extraction, which will assist andrologists in clinical decision-making and minimize unnecessary injury to patients.

Biallelic mutations in RNA-binding protein ADAD2 cause spermiogenic failure and non-obstructive azoospermia in humans.

What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis? Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice. NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification. Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2Mut/Mut mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and Adad2Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2WT/Mut male mice and six Adad2WT/Mut female mice. A total of 120 Adad2Mut/Mut, Adad2WT/Mut, and WT mice were used in this study. The entire study was conducted over 3 years. Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages. Three recessive mutations were identified in ADAD2 (MT1: c.G829T, p.G277C; MT2: c.G1192A, p.D398N; MT3: c.917_918del, p.Q306Rfs*43) in patients from three unrelated Pakistani families. MT1 and MT2 dramatically reduced the testicular expression of ADAD2, likely causing spermiogenesis failure in the NOA patients. Immunofluorescence analysis of the Adad2Mut/Mut male mice with the corresponding MT3 mutation showed instability and premature degradation of the ADAD2 protein, resulting in the spermiogenesis deficiency phenotype. Through ROSI, the Adad2Mut/Mut mice could produce pups with comparable embryonic development (46.7% in Adad2Mut/Mut versus 50% in WT) and birth rates (21.45 ± 10.43% in Adad2Mut/Mut versus 27.5 ± 3.536% in WT, P = 0.5044) to WT mice. The Adad2WT/Mut progeny from ROSI (17 pups in total via three ROSI replicates) did not show overt developmental defects and had normal fertility. N/A. This is a preliminary report suggesting that ROSI can be an effective treatment for infertile Adad2Mut/Mut mice. Further assisted reproductive attempts need to be carefully examined in humans during clinical trials. Our work provides functional evidence that mutations in the ADAD2 gene are deleterious and cause consistent spermiogenic defects in both humans and mice. In addition, preliminary results show that ROSI can help Adad2Mut/Mut to produce biological progeny. These findings provide valuable clues for genetic counselling on the ADAD2 mutants-associated infertility in human males. This work was supported by the National Natural Science Foundation of China (32000587, U21A20204, and 32061143006), and the National Key Research and Developmental Program of China (2019YFA0802600 and 2021YFC2700202). This work was also supported by Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China. The authors declare no competing interests.

Global Analysis in Nonobstructive Azoospermic Testis Identifies miRNAs Critical to Spermatogenesis

Introduction. The etiology of male infertility characterized by non-obstructive azoospermia is largely unknown, especially at the molecular level. Identifying dysregulated microRNAs (miRNAs) in male infertility would be useful to achieve a more profound understanding of its pathogenesis. Methods. Small RNA sequencing was performed on the testicular tissues of 10 nonobstructive azoospermic patients with the Sertoli cell only syndrome (SCOS) and 8 obstructive azoospermic individuals with normal spermatogenesis. The expressions of two dysregulated miRNAs were validated by quantitative real-time polymerase chain reaction, confirming the results obtained by sequencing analysis. Bioinformatic analysis was undertaken to identify the main pathways impaired in complete spermatogenic failure. Results. A total of 136 miRNAs were detected to be differentially expressed in the Sertoli cell only syndrome group in comparison with the obstructive azoospermia group. Bioinformatic analysis suggested that the altered miRNAs were substantially involved in pathways related to spermatogenesis. Conclusions. Our study investigates the entire profile of miRNAs with emphasis on the crucial role of miRNAs in idiopathic Sertoli cell only syndrome, suggesting potential targets for employing molecular therapeutic strategies in the treatment of spermatogenic failure.

Germ Cell–Specific Proteins AKAP4 and ASPX Facilitate Identification of Rare Spermatozoa in Non-Obstructive Azoospermia

Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intra-cytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis- and germ cell-specific proteins would facilitate flow cytometry-assisted identification of rare spermatozoa in semen cell pellets of NOA patients, thus enabling non-invasive diagnostics prior to mTESE. Data mining, targeted proteomics, and immunofluorescent microscopy identified and verified a panel of highly testis-specific proteins expressed at the continuum of germ cell differentiation. Late germ cell-specific proteins AKAP4_HUMAN and ASPX_HUMAN (ACRV1 gene) revealed exclusive localization in spermatozoa tails and acrosomes, respectively. A multiplex imaging flow cytometry assay facilitated fast and unambiguous identification of rare but morphologically intact AKAP4+/ASPX+/Hoechst+ spermatozoa within debris-laden semen pellets of NOA patients. While the previously suggested markers for spermatozoa retrieval suffered from low diagnostic specificity, the multi-step gating strategy and visualization of AKAP4+/ASPX+/Hoechst+ cells with elongated tails and acrosome-capped nuclei facilitated fast and unambiguous identification of the mature intact spermatozoa. AKAP4+/ASPX+/Hoechst+ assay may emerge as a non-invasive test to predict retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of severe forms of male infertility.