USP11 regulates proliferation and apoptosis of human spermatogonial stem cells via HOXC5-mediated canonical WNT/ß-catenin signaling pathway.

Spermatogonial stem cells (SSCs) are capable of transmitting genetic information to the next generations and they are the initial cells for spermatogenesis. Nevertheless, it remains largely unknown about key genes and signaling pathways that regulate fate determinations of human SSCs and male infertility. In this study, we explored the expression, function, and mechanism of USP11 in controlling the proliferation and apoptosis of human SSCs as well as the association between its abnormality and azoospermia. We found that USP11 was predominantly expressed in human SSCs as shown by database analysis and immunohistochemistry. USP11 silencing led to decreases in proliferation and DNA synthesis and an enhancement in apoptosis of human SSCs. RNA-sequencing identified HOXC5 as a target of USP11 in human SSCs. Double immunofluorescence, Co-immunoprecipitation (Co-IP), and molecular docking demonstrated an interaction between USP11 and HOXC5 in human SSCs. HOXC5 knockdown suppressed the growth of human SSCs and increased apoptosis via the classical WNT/ß-catenin pathway. In contrast, HOXC5 overexpression reversed the effect of proliferation and apoptosis induced by USP11 silencing. Significantly, lower levels of USP11 expression were observed in the testicular tissues of patients with spermatogenic disorders. Collectively, these results implicate that USP11 regulates the fate decisions of human SSCs through the HOXC5/WNT/ß-catenin pathway. This study thus provides novel insights into understanding molecular mechanisms underlying human spermatogenesis and the etiology of azoospermia and it offers new targets for gene therapy of male infertility.

Cyto- and Histopographic Assessment of CPA3-Positive Testicular Mast Cells in Obstructive and Non-Obstructive Azoospermia.

Infertility is an important personal and society disease, of which the male factor represents half of all causes. One of the aspects less studied in male infertility is the immunological testicular microenvironment. Mast cells (MCs), having high potential for regulating spermatogenesis due to fine-tuning the state of the integrative buffer metabolic environment, are one of the most crucial cellular subpopulations of the testicular interstitium. One important component of the MC secretome is proteases that can act as proinflammatory agents and in extracellular matrix (ECM) remodeling. In the testis, MCs are an important cell component of the testicular interstitial tissue (TIT). However, there are still no studies addressing the analysis of a specific MC protease-carboxypeptidase A3 (CPA3)-in cases with altered spermatogenesis. The cytological and histotopographic features of testicular CPA3+ MCs were examined in a study involving 34 men with azoospermia. As revealed, in cases with non-obstructive azoospermia, a higher content of CPA3+ MCs in the TIT and migration to the microvasculature and peritubular tissue of seminiferous tubules were observed when compared with cases with obstructive azoospermia. Additionally, a high frequency of CPA3+ MCs colocalization with fibroblasts, Leydig cells, and elastic fibers was detected in cases with NOA. Thus, CPA3 seems to be of crucial pathogenetic significance in the formation of a profibrogenic background of the tissue microenvironment, which may have direct and indirect effects on spermatogenesis.

Interaction between Chromodomain Y-like Protein and Androgen Receptor Signaling in Sertoli Cells Accounts for Spermatogenesis.

Spermatogenesis is a highly regulated process dependent on androgen receptor (AR) signaling in Sertoli cells. However, the pathogenic mechanisms of spermatogenic failure, by which loss of AR impairs downstream target genes to affect Sertoli cell function, remain incompletely understood. By using microarray analysis, we identified several AR-regulated genes involved in the maturation of spermatogenesis, including chromodomain Y-like protein (CDYL) and transition proteins 1 (TNP-1), that were significantly decreased in ARKO mouse testes. AR and CDYL were found to co-localize and interact in Sertoli cells. The AR-CDYL complex bound to the promoter regions of TNP1 and modulated their transcriptional activity. CDYL acts as a co-regulator of AR transactivation, and its expression is decreased in the Sertoli cells of human testes from patients with azoospermia. The androgen receptor-chromodomain Y-like protein axis plays a crucial role in regulating a network of genes essential for spermatogenesis in Sertoli cells. Disruption of this AR-CDYL regulatory axis may contribute to spermatogenic failure. These findings provide insights into novel molecular mechanisms targeting the AR-CDYL signaling pathway, which may have implications for developing new therapeutic strategies for male infertility.

Meiotic transcriptional reprogramming mediated by cell-cell communications in humans and mice revealed by scATAC-seq and scRNA-seq.

Meiosis is a highly complex process significantly influenced by transcriptional regulation. However, studies on the mechanisms that govern transcriptomic changes during meiosis, especially in prophase I, are limited. Here, we performed single-cell ATAC-seq of human testis tissues and observed reprogramming during the transition from zygotene to pachytene spermatocytes. This event, conserved in mice, involved the deactivation of genes associated with meiosis after reprogramming and the activation of those related to spermatogenesis before their functional onset. Furthermore, we identified 282 transcriptional regulators (TRs) that underwent activation or deactivation subsequent to this process. Evidence suggested that physical contact signals from Sertoli cells may regulate these TRs in spermatocytes, while secreted ENHO signals may alter metabolic patterns in these cells. Our results further indicated that defective transcriptional reprogramming may be associated with non-obstructive azoospermia (NOA). This study revealed the importance of both physical contact and secreted signals between Sertoli cells and germ cells in meiotic progression.

Can we improve time to patency with vasoepididymostomy with an innovative epididymal occlusion stitch?

INTRODUCTION: Obstructive azoospermia occurs when there is a blockage in the male reproductive tract, leading to a complete absence of sperm in the ejaculate. It constitutes around 40% of all cases of azoospermia (1, 2). Blockages in the male reproductive tract can arise from either congenital or acquired factors, affecting various segments such as the epididymis, vas deferens, and ejaculatory ducts (3). Examples of congenital causes encompass conditions like congenital bilateral absence of the vas deferens and unexplained epididymal blockages (4). Acquired instances of obstructive azoospermia may result from factors like vasectomy, infections, trauma, or unintentional injuries caused by medical procedures (5). This complex condition affecting male fertility, presents two main treatment options: microsurgical reconstruction and surgical extraction of sperm followed by in vitro fertilization (IVF). Microsurgical reconstruction proves to be the most cost-effective option for treating obstructive azoospermia when compared with assisted reproductive techniques (6, 7). However, success rates of reconstruction defined by patency are as high as 99% for vasovasostomy (VV) but decline to around 65% if vasoepididymostomy (VE) is required (8, 9). Thus, continued refinement in technique is necessary in order to attempt to improve patency for patients undergoing VE. In this video, we show a comprehensive demonstration of microsurgical VE, highlighting the innovative epididymal occlusion stitch. The goal of this innovative surgical technique is to improve outcomes for VE. MATERIALS AND METHODS: The patient is a 39-year-old male diagnosed with obstructive azoospermia who presents for surgical reconstruction via VE. His partner is a 37-years-old female with regular menstrual cycles. The comprehensive clinical data encompasses a range of factors, including FSH levels, results from semen analysis, and outcomes from testicular sperm aspiration. This thorough exploration aims to provide a thorough understanding of our innovative surgical technique and its application in addressing complex cases of obstructive azoospermia. RESULTS: The procedure was started on the right, the vas deferens was identified and transected. The abdominal side of the vas was intubated and a vasogram performed, there was no obstruction. There was no fluid visible from the testicular side of the vas for analysis, thus we proceeded with VE. Upon inspection of the epididymis dilated tubules were identified. After selecting a tubule for VE, two 10-0 nylon sutures were placed, and it was incised. Upon inspection of the fluid motile sperm was identified. After VE, we performed a novel epididymal occlusion stitch technique. This was completed distal to the anastomosis by placing a 7-0 prolene through the tunica of the epididymis from the medial to lateral side. This stitch was then tightened down with the goal to largely occlude the epididymis so that sperm will preferentially travel through the anastomosis. The steps were then repeated on the left. At 3-month follow up, the patient had no change in testicular size as compared with preoperative size (18cc), he had no testicular or incisional discomfort, and on semen analysis he had presence of motile sperm. After 3 months post-surgery, the patient had motile sperm seen on semen analysis. DISCUSSION: The introduction of a novel epididymal occlusion stitch demonstrates a targeted strategy to enhance the success of microscopic VE. Encouragingly, a 3-month post-surgery follow-up reveals the presence of motile sperm, reinforcing the potential efficacy of our approach. This is promising given the historical lower patency, delayed time to patency, and higher delayed failure rates that patients who require VE experience (10). In total, 40% of all azoospermia cases can be attributed to obstruction. The conventional treatments for obstructive azoospermia involve microsurgical reconstruction and surgical sperm retrieval followed by IVF. While microsurgical reconstruction has proven to be economically viable, the quest for enhanced success rates has led to the exploration of innovative techniques. Historically, the evolution of VV and VE procedures, initially performed in the early 20th century, laid the foundation for contemporary microsurgical approaches (11). Notably, the microscopic VV demonstrated significant improvements in patency rates and natural pregnancy likelihood, as evidenced by the seminal Vasovastomy Study Group study in 1991 (8). In contemporary literature, success rates particularly for VE remain unchanged for the past three decades since the original published success rates by the Vasectomy Reversal Study Group (12). VE is associated with a longer time to patency as well with patients taking 2.8 to 6.6 months to have sperm return to ejaculate as compared to 1.7 to 4.3 months for those undergoing VV. Additionally, of those patients who successfully have sperm return to the ejaculate after VE up to 50% will have delayed failure compared to 12% for those undergoing VV who are patent. Finally, of those who experience delayed failure after undergoing VE it usually occurs earlier with studies reporting as early as 6 months post-operatively (10). Given the lack of improvement and significantly worsened outcomes with VE further surgical refinement is a constant goal for surgeons performing this procedure. CONCLUSION: In conclusion, this video is both a demonstration and a call to action for commitment to surgical innovation. We aim to raise the bar in VE success rates, ultimately bringing tangible benefits to patients and contributing to the ongoing evolution of reproductive medicine. The novel epididymal occlusion stitch emerges as a beacon of progress, promising not only enhanced safety but also potential reductions in patency time. Surgical excellence and methodological refinement, as exemplified in this video, lay the foundation for a future where male reproductive surgery continues to break new ground.

Microdissection testicular sperm extraction in non-obstructive azoospermic patients with solitary testis: a retrospective case-control study.

Obtaining sperm from the testis surgically and using these sperm with the intracytoplasmic sperm injection technique, has opened the way for the possibility of biological fathering in men with non-obstructive azoospermia (NOA). We aimed to evaluate our sperm retrieval rate (SRR) by microdissection testicular sperm extraction (micro-TESE) in NOA patients with solitary testis. In this retrospective case-control study, fortyfive patients with NOA who had a congenital or acquired solitary testis were included, between September 2003 and January 2022. These patients were randomly matched with patients with NOA who had bilateral testes, using a 1:3 matching ratio. We found that SRR by micro-TESE in patients with solitary testis was similar to NOA patients with bilateral testis (51.1% vs. 50.4%). Age, infertility period, ejaculate volume, serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone, history of varicocelectomy, history of orchiopexy, testicular stimulation therapy before micro-TESE, testicular volume, genetic status, TESE side, micro-TESE success, complications and histopathological evaluation results of both groups were evaluated, there was a statistically significant difference in only serum FSH and LH levels. There was no difference between the groups in terms of complications and hormonal effects in the early postoperative period. Micro-TESE in NOA patients with solitary testis has similar sperm retrieval and complication rates as NOA patients with bilateral testis.

Non-invasive biomarkers for sperm retrieval in non-obstructive patients: a comprehensive review.

Recent advancements in reproductive medicine have guided novel strategies for addressing male infertility, particularly in cases of non-obstructive azoospermia (NOA). Two prominent invasive interventions, namely testicular sperm extraction (TESE) and microdissection TESE (micro-TESE), have emerged as key techniques to retrieve gametes for assisted reproduction technologies (ART). Both heterogeneity and complexity of NOA pose a multifaceted challenge to clinicians, as the invasiveness of these procedures and their unpredictable success underscore the need for more precise guidance. Seminal plasma can be aptly regarded as a liquid biopsy of the male reproductive tract, encompassing secretions from the testes, epididymides, seminal vesicles, bulbourethral glands, and prostate. This fluid harbors a variety of cell-free nucleic acids, microvesicles, proteins, and metabolites intricately linked to gonadal activity. However, despite numerous investigations exploring potential biomarkers from seminal fluid, their widespread inclusion into the clinical practice remains limited. This could be partially due to the complex interplay of diverse clinical and genetic factors inherent to NOA that likely contributes to the absence of definitive biomarkers for residual spermatogenesis. It is conceivable that the integration of clinical data with biomarkers could increase the potential in predicting surgical procedure outcomes and their choice in NOA cases. This comprehensive review addresses the challenge of sperm retrieval in NOA through non-invasive biomarkers. Moreover, we delve into promising perspectives, elucidating innovative approaches grounded in multi-omics methodologies, including genomics, transcriptomics and proteomics. These cutting-edge techniques, combined with the clinical and genetics features of patients, could improve the use of biomarkers in personalized medical approaches, patient counseling, and the decision-making continuum. Finally, Artificial intelligence (AI) holds significant potential in the realm of combining biomarkers and clinical data, also in the context of identifying non-invasive biomarkers for sperm retrieval.

Vas deferens to rete testis anastomosis for obstructive azoospermia.

PURPOSE: This video aims to present an in-depth, step-by-step tutorial on microsurgical reconstruction for obstructive azoospermia, featuring a distinctive case involving anastomosis from vas deferens to rete testis. The primary aim of this endeavor is to offer thorough and practical insights for healthcare professionals and researchers within the realm of reproductive medicine. The video endeavors to disseminate expertise, methodologies, and perspectives that can be advantageous to individuals grappling with obstructive azoospermia, providing a significant contribution to the progress of reproductive medicine and the augmentation of existing treatment alternatives. MATERIALS AND METHODS: Surgical footage was recorded using the ORBEYE 4K 3D Orbital Camera System by Olympus America, with patient consent acquired for research purposes. Additionally, a retrospective examination of patient records was undertaken to compile relevant medical histories. RESULTS: This video furnishes an exhaustive guide to microsurgical reconstruction for obstructive azoospermia, encompassing a distinctive instance of anastomosis from vas deferens to rete testis. State-of-the-art technology, such as the ORBEYE 4K 3D Orbital Camera, heightens procedural transparency, accentuating the significance of advanced instrumentation. The ethical underpinning is emphasized by obtaining patient consent for footage utilization, and a retrospective chart review augments the repository of valuable patient data. This comprehensive approach serves as an invaluable reservoir of knowledge for medical professionals and underscores excellence in clinical and ethical healthcare research. CONCLUSIONS: Anastomosis from vas deferens to rete testis emerges as a viable surgical reconstruction alternative for obstructive azoospermia, particularly when confronted with non-dilated tubules within the epididymis.

Examination of ejaculate fructose levels on male infertility patients at various times and centrifugation using semiautomatic method.

OBJECTIVE: Various factors, such as obstructive azoospermia, cause infertility in men. Biochemical examination of ejaculate, especially measurement of fructose, can be an additional investigation that can be used for this diagnosis in reproductive health. Examination of fructose is carried out after routine ejaculate analysis, resulting in prolonging the examination time so that it will affect the measurement of fructose level in the ejaculate and the accuracy of the diagnosis. This study aims to determine the best timing and procedure for measurement of fructose using a semiautomatic method. METHODS: This research is an analytic observational study conducted at Dr. Soetomo General Hospital, Surabaya. A total of 13 ejaculate samples from infertile male patients who met the inclusion criteria were evaluated. Each ejaculate was divided into eight aliquots that were examined for fructose using a semiautomated method after different intervals of time and centrifugation modalities. RESULTS: This study showed a significant difference in fructose levels when aliquots were centrifuged and examined immediately or after different interval of time (p=0.036). In addition, aliquots left standing for more than 60 minutes (p=0.012) and 120 minutes (p<0.001) before centrifugation, showed significantly lower levels compared to aliquots that were centrifuged and then immediately examined. CONCLUSIONS: We suggest that measuring fructose immediately after centrifugation is more reliable than measuring fructose left standing before or after centrifugation. Leaving the ejaculate standing will reduce the fructose level so that it does not resemble its real level.

NAD+ precursors promote the restoration of spermatogenesis in busulfan-treated mice through inhibiting Sirt2-regulated ferroptosis.

Rationale: In recent years, nicotinamide adenine dinucleotide (NAD+) precursors (Npre) have been widely employed to ameliorate female reproductive problems in both humans and animal models. However, whether and how Npre plays a role in the male reproductive disorder has not been fully clarified. Methods: In the present study, a busulfan-induced non-obstructive azoospermic mouse model was used, and Npre was administered for five weeks following the drug injection, with the objective of reinstating spermatogenesis and fertility. Initially, we assessed the NAD+ level, germ cell types, semen parameters and sperm fertilization capability. Subsequently, testis tissues were examined through RNA sequencing analysis, ELISA, H&E, immunofluorescence, quantitative real-time PCR, and Western blotting techniques. Results: The results indicated that Npre restored normal level of NAD+ in blood and significantly alleviated the deleterious effects of busulfan (BU) on spermatogenesis, thereby partially reestablishing fertilization capacity. Transcriptome analysis, along with recovery of testicular Fe2+, GSH, NADPH, and MDA levels, impaired by BU, and the fact that Fer-1, an inhibitor of ferroptosis, restored spermatogenesis and semen parameters close to CTRL values, supported such possibility. Interestingly, the reduction in SIRT2 protein level by the specific inhibitor AGK2 attenuated the beneficial effects of Npre on spermatogenesis and ferroptosis by affecting PGC-1α and ACLY protein levels, thus suggesting how these compounds might confer spermatogenesis protection. Conclusion: Collectively, these findings indicate that NAD+ protects spermatogenesis against ferroptosis, probably through SIRT2 dependent mechanisms. This underscores the considerable potential of Npre supplementation as a feasible strategy for preserving or restoring spermatogenesis in specific conditions of male infertility and as adjuvant therapy to preserve male fertility in cancer patients receiving sterilizing treatments.

Proteomic biomarkers in seminal plasma as predictors of reproductive potential in azoospermic men.

Introduction: Azoospermia, characterized by an absence of sperm in the ejaculate, represents the most severe form of male infertility. While surgical sperm retrieval in obstructive azoospermia (OA) is successful in the majority of cases, patients with non-obstructive azoospermia (NOA) show retrieval rates of only about 50% and thus frequently have unnecessary surgery. Surgical intervention could be avoided if patients without preserved spermatogenesis are identified preoperatively. This prospective study aimed to discover biomarkers in seminal plasma that could be employed for a non-invasive differential diagnosis of OA/NOA in order to rationalize surgery recommendations and improve success rates. Methods: All patients signed written informed consent, underwent comprehensive andrological evaluation, received human genetics to exclude relevant pathologies, and patients with azoospermia underwent surgical sperm retrieval. Using label-free LC-MS/MS, we compared the proteomes of seminal plasma samples from fertile men (healthy controls (HC), n=8) and infertile men diagnosed with 1) OA (n=7), 2) NOA with successful sperm retrieval (mixed testicular atrophy (MTA), n=8), and 3) NOA without sperm retrieval (Sertoli cell-only phenotype (SCO), n=7). Relative abundance changes of two candidate markers of sperm retrieval, HSPA2 and LDHC, were confirmed by Western Blot. Results: We found the protein expression levels of 42 proteins to be significantly down-regulated (p ≤ 0.05) in seminal plasma from SCO NOA patients relative to HC whereas only one protein was down-regulated in seminal plasma from MTA patients. Analysis of tissue and cell expression suggested that the testis-specific proteins LDHC, PGK2, DPEP3, and germ-cell enriched heat-shock proteins HSPA2 and HSPA4L are promising biomarkers of spermatogenic function. Western blotting revealed a significantly lower abundance of LDHC and HSPA2 in the seminal plasma of men with NOA (SCO and MTA) compared to controls. Discussion: The results indicate that certain testis-specific proteins when measured in seminal plasma, could serve as indicators of the presence of sperm in the testis and predict the success of sperm retrieval. Used in conjunction with conventional clinical assessments, these proteomic biomarkers may assist in the non-invasive diagnosis of idiopathic male infertility.

[Frequency and characteristics of Y chromosome microdeletions and karyotypic abnormalities among 4 278 infertile male patients from southwest China].

OBJECTIVE: To determine the frequency and characteristics of AZF microdeletions of Y chromosome and karyotypic abnormalities among infertile male patients from southwest China. METHODS: 4 278 infertile male patients treated at West China Second University Hospital of Sichuan University from September 2018 to July 2023 were selected as the study subjects. Results of Y chromosome microdeletion detection and G-banded karyotyping analysis were retrospectively reviewed. RESULTS: Clinical data of the patients were collected, which have included 2 048 patients with azoospermia, 1 536 patients with oligozoospermia, 310 patients with mild to moderate oligozoospermia, and 384 patients with infertility but normal sperm concentration. An abnormal karyotype was found in 213 (8.80%) of 2 421 patients who had undergone karyotyping analysis. The frequency of Y chromosome microdeletions was 9.86% (422/4 278), which had occurred in 10.4%, 13.28%, 0.97% and 0.52% of the cases with azoospermia, severe oligozoospermia, mild to moderate oligozoospermia, and infertility with normal sperm concentration, respectively. CONCLUSION: Y chromosome microdeletion detection and karyotyping analysis are crucial for assessing the cause of male infertility. Early diagnosis can facilitate the selection of reproductive methods.

[Tumor necrosis factor in the ejaculate as an indicator of reduced fertility].

INTRODUCTION: Pro-inflammatory cytokine - tumor necrosis factor-alpha (TNF) is one of the components of the seminal plasma proteome; its meaning has not been definitively revealed. A comparative analysis of the concentration of this protein in the blood serum and in the ejaculate and changes in its level in the semen of men with infertility is f scientific interest. THE PURPOSE OF THE STUDY: determination of TNF- level in the blood serum and seminal plasma of healthy men and patients with reduced fertility. MATERIALS AND METHODS: 70 men of reproductive age with azoospermia (main group, n=18), with oligoastenozoospermia (comparison group, n=18) and with normal spermogram parameters (control group, n=34) were examined. The ejaculate was examined using an SQA-V semen analyzer (MES, Israel). In seminal plasma samples, the concentration of TNF was determined using the alpha-TNF-ELISA-BEST test system (A-8756, Vector-Best LL, Russia). RESULTS: The concentration of TNF- in blood serum had a significant variation (CV=85.31%) and amounted to 2.75+/-2.18 pg/ml, which is 2.55 times lower than the same indicator in seminal plasma (7.01+/-5.98 pg/ml, CV=126.15%, p<0.00001). When comparing the content of TNF- in seminal plasma, significant differences were found in the examined patients (Kruskal-Wallis test H=24.75991; p<0.00001). Pairwise comparison revealed a statistically significant difference in the level of TNF- in seminal plasma between the comparison and control groups (p2-3=0.000023), as well as between the main group and the comparison group (p1-2=0.000043); there were no significant differences between the main and control groups (p>0.05). When determining the content of TNF- in the blood serum, there was no statistically significant difference between the groups (p>0.05). There were no correlations between the concentration of TNF- in blood serum and in seminal plasma (R=0.295374), and the total number of spermatozoa in the ejaculate (R=-0.027945); and the concentration of spermatozoa in the ejaculate (R=-0.042902). DISCUSSION: It is unlikely that TNF crosses into seminal plasma from serum against a concentration gradient. It is most likely that TNF is produced locally in the organs of the reproductive system by resident immune cells or cells involved in spermatogenesis. An increased content of TNF- in seminal plasma in patients of the comparison group may indicate the presence of an inflammatory process in the reproductive system and a reduced fertility of the ejaculate. CONCLUSION: The physiological role of TNF in sperm, its sources in the organs of the male reproductive system, and the pathogenetic mechanisms of the participation of the TNF in pathological processes in male reproductive system still remain unclear. All this justifies the need for further study of the TNF level in seminal plasma in normal conditions and in diseases of the urogenital tract in men.

Establishment and validation of a 5-factor diagnostic model for obstructive and non-obstructive azoospermia based on routine clinical parameters.

Azoospermia is a serious leading male-factor cause of infertility in couples of childbearing age. The two main azoospermia types, obstructive (OA) and non-obstructive (NOA) azoospermia, differ in their treatment approaches. Therefore, their clinical diagnosis is extremely important, requiring an accurate, efficient, and easy-to-use diagnostic model. This retrospective observational study included 707 patients with azoospermia treated between 2017 and 2021, 498 with OA, and 209 with NOA. Hematological and seminal plasma parameters, hormone levels, and testicular volume were used in logistic regression analysis to evaluate and compare their diagnostic performance, results showed that the optimal diagnostic model is constructed by five variables including semen volume, semen pH, seminal plasma neutral α-glucosidase activity, follicle-stimulating hormone in the serum, and testicular volume, compared with follicle-stimulating hormone-based and testicular volume-based models. The 5-factor diagnostic model had an accuracy of 90.4%, sensitivity of 96.4%, positive predictive value of 90.6%, negative predictive value of 89.8%, and area under the curve of 0.931, all higher than in the other two models. However, its specificity (76.1%) was slightly lower than in the other models. Meantime, the internal 5-fold cross-validation results indicated that the 5-factor diagnostic model had a good clinical application value. This study established an accurate, efficient, and relatively accessible 5-factor diagnostic model for OA and NOA, providing a reference for clinical decision-making when selecting an appropriate treatment.

Toward clinical exomes in diagnostics and management of male infertility.

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

Evaluation of human bone marrow mesenchymal stem cells in the treatment of non obstructive azoospermia.

BACKGROUND: Non-obstructive azoospermia (NOA) represents an infertility problem that is usually difficult to treat. Such patients usually have testicular biopsy of germ cell aplasia or spermatogenic arrest. In recent decades, mesenchymal stem cells (MSCs) had been studied thoroughly and proved safe and effective regarding their capability for trans-differentiation into different cell types. The aim of this study was to evaluate the effect of MSCs local intratesticular injection in induction of spermatogenesis. PATIENTS AND METHOD: The current study included 87 infertile non-obstructive azoospermic patients. Clinical assessment and repeated semen analysis with centrifugation were done to confirm azoospermia. Karyotyping and AZF study were done. Some of the patients had previous testicular biopsy proving a lack of sperm in the testes. Single intratesticular injection of purified MSCs suspension was done. RESULTS: 20.7% of patients showed sperm in their semen after variable period of time. Hormonal profile among treated patients showed significant improvement regardless success of treatment. Also most of the treated patients appreciated the improvement of their sexual function and libido. CONCLUSIONS: Bone marrow derived MSCs could be a new hope and therapeutic modality for treatment of refractory cases of NOA.

Systematic molecular analyses for 115 karyotypically normal men with isolated non-obstructive azoospermia.

STUDY QUESTION: Do copy-number variations (CNVs) in the azoospermia factor (AZF) regions and monogenic mutations play a major role in the development of isolated (non-syndromic) non-obstructive azoospermia (NOA) in Japanese men with a normal 46, XY karyotype? SUMMARY ANSWER: Deleterious CNVs in the AZF regions and damaging sequence variants in eight genes likely constitute at least 8% and approximately 8% of the genetic causes, respectively, while variants in other genes play only a minor role. WHAT IS KNOWN ALREADY: Sex chromosomal abnormalities, AZF-linked microdeletions, and monogenic mutations have been implicated in isolated NOA. More than 160 genes have been reported as causative/susceptibility/candidate genes for NOA. STUDY DESIGN, SIZE, DURATION: Systematic molecular analyses were conducted for 115 patients with isolated NOA and a normal 46, XY karyotype, who visited our hospital between 2017 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 115 unrelated Japanese patients. AZF-linked CNVs were examined using sequence-tagged PCR and multiplex ligation-dependent probe amplification, and nucleotide variants were screened using whole exome sequencing (WES). An optimized sequence kernel association test (SKAT-O), a gene-based association study using WES data, was performed to identify novel disease-associated genes in the genome. The results were compared to those of previous studies and our in-house control data. MAIN RESULTS AND THE ROLE OF CHANCE: Thirteen types of AZF-linked CNVs, including the hitherto unreported gr/gr triplication and partial AZFb deletion, were identified in 63 (54.8%) cases. When the gr/gr deletion, a common polymorphism in Japan, was excluded from data analyses, the total frequency of CNVs was 23/75 (30.7%). This frequency is higher than that of the reference data in Japan and China (11.1% and 14.7%, respectively). Known NOA-causative AZF-linked CNVs were found in nine (7.8%) cases. Rare damaging variants in known causative genes (DMRT1, PLK4, SYCP2, TEX11, and USP26) and hemizygous/multiple-heterozygous damaging variants in known spermatogenesis-associated genes (TAF7L, DNAH2, and DNAH17) were identified in nine cases (7.8% in total). Some patients carried rare damaging variants in multiple genes. SKAT-O detected no genes whose rare damaging variants were significantly accumulated in the patient group. LIMITATIONS, REASONS FOR CAUTION: The number of participants was relatively small, and the clinical information of each patient was fragmentary. Moreover, the pathogenicity of identified variants was assessed only by in silico analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that various AZF-linked CNVs are present in more than half of Japanese NOA patients. These results broadened the structural variations of AZF-linked CNVs, which should be considered for the molecular diagnosis of spermatogenic failure. Furthermore, the results of this study highlight the etiological heterogeneity and possible oligogenicity of isolated NOA. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grants from the Japan Society for the Promotion of Science (21K19283 and 21H0246), the Japan Agency for Medical Research and Development (22ek0109464h0003), the National Center for Child Health and Development, the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The results of this study were based on samples and patient data obtained from the International Center for Reproductive Medicine, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Achieving an optimal pregnancy outcome through the combined utilization of micro-TESE and ICSI in cryptorchidism associated with a non-canonical splicing variant in RXFP2.

PURPOSE: To identify the genetic cause of a cryptorchidism patient carrying a non-canonical splicing variant highlighted by SPCards platform in RXFP2 and to provide a comprehensive overview of RXFP2 variants with cryptorchidism correlation. METHODS: We identified a homozygous non-canonical splicing variant by whole-exome sequencing and Sanger sequencing in a case with cryptorchidism and non-obstructive azoospermia (NOA). As the pathogenicity of this non-canonical splicing variant remained unclear, we initially utilized the SPCards platform to predict its pathogenicity. Subsequently, we employed a minigene splicing assay to further evaluate the influence of the identified splicing variant. Microdissection testicular sperm extraction (micro-TESE) combined with intracytoplasmic sperm injection (ICSI) was performed. PubMed and Human Genome Variant Database (HGMD) were queried to search for RXFP2 variants. RESULTS: We identified a homozygous non-canonical splicing variant (NM_130806: c.1376-12A > G) in RXFP2, and confirmed this variant caused aberrant splicing of exons 15 and 16 of the RXFP2 gene: 11 bases were added in front of exon 16, leading to an abnormal transcript initiation and a frameshift. Fortunately, the patient successfully obtained his biological offspring through micro-TESE combined with ICSI. Four cryptorchidism-associated variants in RXFP2 from 90 patients with cryptorchidism were identified through a literature search in PubMed and HGMD, with different inheritance patterns. CONCLUSION: This is the first cryptorchidism case carrying a novel causative non-canonical splicing RXFP2 variant. The combined approach of micro-TESE and ICSI contributed to an optimal pregnancy outcome. Our literature review demonstrated that RXFP2 variants caused cryptorchidism in a recessive inheritance pattern, rather than a dominant pattern.

Sox9 downregulation in non-obstructive azoospermia by UTF1 and mediator role of POU5F1.

BACKGROUND: Spermatogenesis is the process of producing mature sperm from Spermatogonial stem cells (SSCs) and this process requires a complex cooperation of different types of somatic and germ cells. Undifferentiated spermatogonia initiate the spermatogenesis and Sertoli cells as the only somatic cells inside of the seminiferous tubule play a key role in providing chemical and physical requirements for normal spermatogenesis, here, we investigated the dysfunction of these cells in non-obstructive azoospermia. MATERIAL AND METHOD: In this study, we analyzed the expression of sox9 and UTF1 in the non-obstructive human testis by immunohistochemistry. Moreover, we used the KEGG pathway and bioinformatics analysis to reveal the connection between our object genes and protein. RESULTS: The immunohistochemistry analysis of the non-obstructive human seminiferous tubule showed low expression of Sox9 and UTF1 that was detected out of the main location of the responsible cells for these expressions. Our bioinformatics analysis clearly and strongly indicated the relation between UTF1 in undifferentiated spermatogonia and Sox9 in Sertoli cells mediated by POU5F1. CONCLUSION: Generally, this study showed the negative effect of POU5F1 as a mediator between Sertoli cells as the somatic cells within seminiferous tubules and undifferentiated spermatogonia as the spermatogenesis initiator germ cells in non-obstructive conditions.

Comparative evaluation of double- and single-armed two-suture longitudinal intussusception techniques in microsurgical vasoepididymostomy: An updated systematic review and meta-analysis.

BACKGROUND: This study aimed to compare the outcomes of double-armed two-suture longitudinal intussusception microsurgical vasoepididymostomy (LIVE) and single-armed two-suture LIVE techniques in patients with epididymal obstructive azoospermia (EOA). The main outcomes assessed were patency rates, patency time, semen quality and natural pregnancy rates. METHODS: Data from patients with EOA who underwent two-suture LIVE were obtained from databases including PubMed, EMBASE, and Web of Science. Weighted data were analyzed using a random-effects model, and weighted mean differences were reported. RESULTS: A total of 1574 patients with EOA from 24 studies were included. The overall patency rate was approximately 68% (95% confidence interval [CI]: 63-72%), with a patency time of approximately 4.63 months (95% CI: 4.15-5.12). The sperm concentration reached 26.90 million/ml and the sperm motility was 23.74%. The natural pregnancy rate was 38% (95% CI: 31-46%). The different definitions of patency do not seem to have any meaningful impact when comparing patency rates. There was no significant difference in patency rates, patency time, semen quality and natural pregnancy rates between the double-armed and single-armed LIVE techniques. CONCLUSION: The single-armed LIVE is a potential alternative surgical option when high quality double-needle sutures are not easily accessible.