Dynamics of HSD17B3 expression in human fetal testis: implications for the role of Sertoli cells in fetal testosterone biosynthesis.

Introduction: Androgens play a pivotal role in shaping male sexual characteristics, with testosterone being an essential hormone in orchestrating various developmental processes. Testosterone biosynthesis involves a series of enzymatic reactions, among which the 17ß-hydroxysteroid dehydrogenase type 3 (HSD17B3) holds significance. While its role in adult Leydig cells is well established, its localization and importance during the fetal period remain less known, especially in humans. This study aims to delineate the dynamics of HSD17B3 expression in human fetal testes to clarify the contribution of specific cell types to testosterone biosynthesis. Methods: Using immunofluorescence staining, we investigated the expression pattern of HSD17B3 in human fetal and adult testicular tissues. Results and discussion: The findings of this study revealed a distinct temporal and cellular expression pattern of HSD17B3 protein in the fetal period. We detected its expression exclusively in Sertoli cells, the highest during the second trimester. This unique localization suggests the inclusion of fetal Sertoli cells in testosterone production during the critical masculinization-programming window. Furthermore, we demonstrated a shift in HSD17B3 expression from Sertoli cells to Leydig cells in adulthood, corroborating findings from rodent studies. This study sheds light on the intricate, still underexplored regulation of steroidogenesis during fetal development, whose disturbance might lead to testicular dysgenesis. Further research is warranted to elucidate the regulatory pathways governing the expression of HSD17B3 and its transition between Sertoli and Leydig cells, potentially paving the way for novel therapeutic interventions in disorders of sexual development.

FANCM Gene Variants in a Male Diagnosed with Sertoli Cell-Only Syndrome and Diffuse Astrocytoma.

Azoospermia is a form of male infertility characterized by a complete lack of spermatozoa in the ejaculate. Sertoli cell-only syndrome (SCOS) is the most severe form of azoospermia, where no germ cells are found in the tubules. Recently, FANCM gene variants were reported as novel genetic causes of spermatogenic failure. At the same time, FANCM variants are known to be associated with cancer predisposition. We performed whole-exome sequencing on a male patient diagnosed with SCOS and a healthy father. Two compound heterozygous missense mutations in the FANCM gene were found in the patient, both being inherited from his parents. After the infertility assessment, the patient was diagnosed with diffuse astrocytoma. Immunohistochemical analyses in the testicular and tumor tissues of the patient and adequate controls showed, for the first time, not only the existence of a cytoplasmic and not nuclear pattern of FANCM in astrocytoma but also in non-mitotic neurons. In the testicular tissue of the SCOS patient, cytoplasmic anti-FANCM staining intensity appeared lower than in the control. Our case report raises a novel possibility that the infertile carriers of FANCM gene missense variants could also be prone to cancer development.

Monocytes expressing activin A and CCR2 exacerbate chronic testicular inflammation by promoting immune cell infiltration.

STUDY QUESTION: Does the chemokine/chemokine receptor axis, involved in immune cell trafficking, contribute to the pathology of testicular inflammation and how does activin A modulate this network? SUMMARY ANSWER: Testicular chemokines and their receptors (especially those essential for trafficking of monocytes) are elevated in orchitis, and activin A modulates the expression of the chemokine/chemokine receptor network to promote monocyte/macrophage and T cell infiltration into the testes, causing extensive tissue damage. WHAT IS KNOWN ALREADY: The levels of CC motif chemokine receptor (CCR)2 and its ligand CC motif chemokine ligand (CCL)2 are increased in experimental autoimmune orchitis (EAO) compared with healthy testes, and mice deficient in CCR2 are protected from EAO-induced tissue damage. Activin A induces CCR2 expression in macrophages, promoting their migration. Moreover, there is a positive correlation between testicular activin A concentration and the severity of autoimmune orchitis. Inhibition of activin A activity by overexpression of follistatin (FST) reduces EAO-induced testicular damage. STUDY DESIGN, SIZE, DURATION: EAO was induced in 10-12-week-old male C57BL/6J (wild-type; WT) and B6.129P2-Ccr2tm1Mae/tm1Mae (Ccr2-/-) mice (n = 6). Adjuvant (n = 6) and untreated (n = 6) age-matched control mice were also included. Testes were collected at 50 days after the first immunization with testicular homogenate in complete Freund's adjuvant. In another experimental setup, WT mice were injected with a non-replicative recombinant adeno-associated viral vector carrying a FST315-expressing gene cassette (rAAV-FST315; n = 7-9) or an empty control vector (n = 5) 30 days prior to EAO induction. Appropriate adjuvant (n = 4-5) and untreated (n = 4-6) controls were also examined. Furthermore, human testicular biopsies exhibiting focal leukocytic infiltration and impaired spermatogenesis (n = 17) were investigated. Biopsies showing intact spermatogenesis were included as controls (n = 9). Bone-marrow-derived macrophages (BMDMs) generated from WT mice were treated with activin A (50 ng/ml) for 6 days. Activin-A-treated or untreated BMDMs were then co-cultured with purified mouse splenic T cells for two days to assess chemokine and cytokine production. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of chemokines in total testicular RNA collected from mice. Immunofluorescence staining was used to detect activin A, F4/80, and CD3 expression in mouse testes. The expression of chemokine/chemokine-receptor-encoding genes was examined in human testicular biopsies by qRT-PCR. Correlations between chemokine expression levels and either the immune cell infiltration density or the mean spermatogenesis score were analyzed. Immunofluorescence staining was used to evaluate the expression of CD68 and CCR2 in human testicular biopsies. RNA isolated from murine BMDMs was used to characterize these cells in terms of their chemokine/chemokine receptor expression levels. Conditioned media from co-cultures of BMDMs and T cells were collected to determine chemokine levels and the production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN)-γ by T cells. MAIN RESULTS AND THE ROLE OF CHANCE: Induction of EAO in the testes of WT mice increased the expression of chemokine receptors such as Ccr1 (P < 0.001), Ccr2 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.0001), CXC motif chemokine receptor (Cxcr)3 (P < 0.01), and CX3C motif chemokine receptor (Cx3cr)1 (P < 0.001), as well as that of most of their ligands. Ccr2 deficiency reversed some of the changes associated with EAO by reducing the expression of Ccr1 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.01), Cxcr3 (P < 0.001), and Cx3cr1 (P < 0.0001). Importantly, the biopsies showing impaired spermatogenesis and concomitant focal leukocytic infiltration exhibited higher expression of CCL2 (P < 0.01), CCR1 (P < 0.05), CCR2 (P < 0.001), and CCR5 (P < 0.001) than control biopsies with no signs of inflammation and intact spermatogenesis. The gene expression of CCR2 and its ligand CCL2 correlated positively with the immune cell infiltration density (P < 0.05) and negatively with the mean spermatogenesis score (P < 0.001). Moreover, CD68+ macrophages expressing CCR2 were present in human testes with leukocytic infiltration with evidence of tubular damage. Treatment of BMDMs, as surrogates for testicular macrophages, with activin A increased their expression of Ccr1, Ccr2, and Ccr5 while reducing their expression of Ccl2, Ccl3, Ccl4, Ccl6, Ccl7 Ccl8, and Ccl12. These findings were validated in vivo, by showing that inhibiting activin A activity by overexpressing FST in EAO mice decreased the expression of Ccr2 (P < 0.05) and Ccr5 (P < 0.001) in the testes. Interestingly, co-culturing activin-A-treated BMDMs and T cells reduced the levels of CCL2 (P < 0.05), CCL3/4 (P < 0.01), and CCL12 (P < 0.05) in the medium and attenuated the production of TNF (P < 0.05) by T cells. The majority of cells secreting activin A in EAO testes were identified as macrophages. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: BMDMs were used as surrogates for testicular macrophages. Hence, results obtained from the in vitro experiments might not be fully representative of the situation in the testes in vivo. Moreover, since total RNA was extracted from the testicular tissue to examine chemokine expression, the contributions of individual cell types as producers of specific chemokines may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS: Our data indicate that macrophages are implicated in the development and progression of testicular inflammation by expressing CCR2 and activin A, which ultimately remodel the chemokine/chemokine receptor network and recruit other immune cells to the site of inflammation. Consequently, inhibition of CCR2 or activin A could serve as a potential therapeutic strategy for reducing testicular inflammation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the International Research Training Group in 'Molecular pathogenesis on male reproductive disorders', a collaboration between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK1871/1-2) funded by the Deutsche Forschungsgemeinschaft and Monash University, a National Health and Medical Research Council of Australia Ideas Grant (1184867), and the Victorian Government's Operational Infrastructure Support Programme. The authors declare no competing financial interests.

Enhancement of Vascularization and Ovarian Follicle Survival Using Stem Cells in Cryopreserved Ovarian Tissue Transplantation-A Systematic Review.

The increase in cancer survival rates has put a focus on ensuring fertility preservation procedures for cancer patients. Ovarian tissue cryopreservation presents the only option for prepubertal girls and patients who require immediate start of treatment and, therefore, cannot undergo controlled ovarian stimulation. We aimed to provide an assessment of stem cells' impact on cryopreserved ovarian tissue grafts in regard to the expression of growth factors, angiogenesis promotion, tissue oxygenation, ovarian follicle survival and restoration of endocrine function. For this systematic review, we searched the Scopus and PubMed databases and included reports of trials using murine and/or human cryopreserved ovarian tissue for transplantation or in vitro culture in combination with mesenchymal stem cell administration to the grafting site. Of the 1201 articles identified, 10 met the criteria. The application of stem cells to the grafting site has been proven to support vascular promotion and thereby shorten the period of tissue hypoxia, which is reflected in the increased number of remaining viable follicles and faster recovery of ovarian endocrine function. Further research is needed before implementing the use of stem cells in OT cryopreservation and transplantation procedures in clinical practice. Complex ethical dilemmas make this process more difficult.

Global Practice Patterns and Variations in the Medical and Surgical Management of Non-Obstructive Azoospermia: Results of a World-Wide Survey, Guidelines and Expert Recommendations.

PURPOSE: Non-obstructive azoospermia (NOA) is a common, but complex problem, with multiple therapeutic options and a lack of clear guidelines. Hence, there is considerable controversy and marked variation in the management of NOA. This survey evaluates contemporary global practices related to medical and surgical management for patients with NOA. MATERIALS AND METHODS: A 56-question online survey covering various aspects of the evaluation and management of NOA was sent to specialists around the globe. This paper analyzes the results of the second half of the survey dealing with the management of NOA. Results have been compared to current guidelines, and expert recommendations have been provided using a Delphi process. RESULTS: Participants from 49 countries submitted 336 valid responses. Hormonal therapy for 3 to 6 months was suggested before surgical sperm retrieval (SSR) by 29.6% and 23.6% of participants for normogonadotropic hypogonadism and hypergonadotropic hypogonadism respectively. The SSR rate was reported as 50.0% by 26.0% to 50.0% of participants. Interestingly, 46.0% reported successful SSR in <10% of men with Klinefelter syndrome and 41.3% routinely recommended preimplantation genetic testing. Varicocele repair prior to SSR is recommended by 57.7%. Half of the respondents (57.4%) reported using ultrasound to identify the most vascularized areas in the testis for SSR. One-third proceed directly to microdissection testicular sperm extraction (mTESE) in every case of NOA while others use a staged approach. After a failed conventional TESE, 23.8% wait for 3 months, while 33.1% wait for 6 months before proceeding to mTESE. The cut-off of follicle-stimulating hormone for positive SSR was reported to be 12-19 IU/mL by 22.5% of participants and 20-40 IU/mL by 27.8%, while 31.8% reported no upper limit. CONCLUSIONS: This is the largest survey to date on the real-world medical and surgical management of NOA by reproductive experts. It demonstrates a diverse practice pattern and highlights the need for evidence-based international consensus guidelines.

Global Practice Patterns in the Evaluation of Non-Obstructive Azoospermia: Results of a World-Wide Survey and Expert Recommendations.

PURPOSE: Non-obstructive azoospermia (NOA) represents the persistent absence of sperm in ejaculate without obstruction, stemming from diverse disease processes. This survey explores global practices in NOA diagnosis, comparing them with guidelines and offering expert recommendations. MATERIALS AND METHODS: A 56-item questionnaire survey on NOA diagnosis and management was conducted globally from July to September 2022. This paper focuses on part 1, evaluating NOA diagnosis. Data from 367 participants across 49 countries were analyzed descriptively, with a Delphi process used for expert recommendations. RESULTS: Of 336 eligible responses, most participants were experienced attending physicians (70.93%). To diagnose azoospermia definitively, 81.7% requested two semen samples. Commonly ordered hormone tests included serum follicle-stimulating hormone (FSH) (97.0%), total testosterone (92.9%), and luteinizing hormone (86.9%). Genetic testing was requested by 66.6%, with karyotype analysis (86.2%) and Y chromosome microdeletions (88.3%) prevalent. Diagnostic testicular biopsy, distinguishing obstructive azoospermia (OA) from NOA, was not performed by 45.1%, while 34.6% did it selectively. Differentiation relied on physical examination (76.1%), serum hormone profiles (69.6%), and semen tests (68.1%). Expectations of finding sperm surgically were higher in men with normal FSH, larger testes, and a history of sperm in ejaculate. CONCLUSIONS: This expert survey, encompassing 367 participants from 49 countries, unveils congruence with recommended guidelines in NOA diagnosis. However, noteworthy disparities in practices suggest a need for evidence-based, international consensus guidelines to standardize NOA evaluation, addressing existing gaps in professional recommendations.

Pathohistological features of the aging human lacrimal gland.

AIM: To assess sex-related differences in the pathohistological features of the human lacrimal gland and to investigate age-related and sex-related differences in stereologically measured volume density of the secretory tissue, connective tissue, and fat. METHODS: We performed an observational analysis of acinar atrophy, periacinar fibrosis, periductal fibrosis, ductal dilation, ductal proliferation, fatty infiltration, and lymphocyte infiltration of hematoxylin and eosin-stained lacrimal gland samples from 81 cornea donors. Stereological analysis of the volume density of the secretory tissue, connective tissue, and fat was performed on samples from 66 donors. RESULTS: Up to 69% of all samples showed degenerative changes. Female samples had a higher frequency of all observed degenerative changes, except ductal dilation. While acinar atrophy was significantly more prevalent in women, ductal dilation was significantly more prevalent in men. Stereological analysis indicated lower portions of acini and higher portions of connective tissue and fat, as well as a more pronounced age-related progression of degenerative changes in female samples. CONCLUSION: Female lacrimal glands are more susceptible to degeneration, and this susceptibility could play an important role in the higher incidence of dry eye disease in older women. A further stereological analysis using more samples from younger age groups is needed to elucidate age-related and sex-related differences in the structure of the human lacrimal gland and their impact on dry eye disease.

Testicular Germ Cell Tumor Tissue Biomarker Analysis: A Comparison of Human Protein Atlas and Individual Testicular Germ Cell Tumor Component Immunohistochemistry.

The accurate management of testicular germ cell tumors (TGCTs) depends on identifying the individual histological tumor components. Currently available data on protein expression in TGCTs are limited. The human protein atlas (HPA) is a comprehensive resource presenting the expression and localization of proteins across tissue types and diseases. In this study, we have compared the data from the HPA with our in-house immunohistochemistry on core TGCT diagnostic genes to test reliability and potential biomarker genes. We have compared the protein expression of 15 genes in TGCT patients and non-neoplastic testicles with the data from the HPA. Protein expression was converted into diagnostic positivity. Our study discovered discrepancies in three of the six core TGCT diagnostic genes, POU5F1, KIT and SOX17 in HPA. DPPA3, CALCA and TDGF1 were presented as potential novel TGCT biomarkers. MGMT was confirmed while RASSF1 and PRSS21 were identified as biomarkers of healthy testicular tissue. Finally, SALL4, SOX17, RASSF1 and PRSS21 dysregulation in the surrounding testicular tissue with complete preserved spermatogenesis of TGCT patients was detected, a potential early sign of neoplastic transformation. We highlight the importance of a multidisciplinary collaborative approach to fully understand the protein landscape of human testis and its pathologies.

Circulating Cell-Free Tumor Deoxyribonucleic Acid Analysis as a Tool for the Diagnosis and Monitoring of Pediatric Solid Tumors.

The utility of cell-free tumor deoxyribonucleic acid analysis is currently being evaluated in a wide range of clinical studies. The validity of cell-free tumor deoxyribonucleic acid analysis methods used for screening and detecting malignant diseases, monitoring the effectiveness of treatment and disease progression, and identifying potential relapse is tested. Molecular technologies used for cell-free tumor deoxyribonucleic acid analysis include targeted polymerase chain reaction assays and next-generation sequencing approaches along with newly introduced epigenetic analysis methods such as methylation-specific polymerase chain reaction. The aim of this review was to compare the methods, pitfalls, and advantages of tests developed for the analysis of cell-free tumor deoxyribonucleic acid in the diagnosis and treatment of pediatric solid tumors. For this purpose, the PubMed database was searched for articles published in the last 10 years, in English, that investigated a human cohort aged 0 to 18 years. A total of 272 references were analyzed. A total of 33 studies were included in the review. Cell-free tumor deoxyribonucleic acid analysis has emerged as a promising novel approach that could potentially bring a significant improvement in the field of pediatric oncology, but the implementation of cell-free tumor deoxyribonucleic acid in clinical practice is largely hindered by the lack of standardized methods for processing and analysis.

Expression of Androgen and Estrogen Receptors in the Human Lacrimal Gland.

Lacrimal gland dysfunction causes dry eye disease (DED) due to decreased tear production. Aqueous-deficient DED is more prevalent in women, suggesting that sexual dimorphism of the human lacrimal gland could be a potential cause. Sex steroid hormones are a key factor in the development of sexual dimorphism. This study aimed to quantify estrogen receptor (ER) and androgen receptor (AR) expression in the human lacrimal gland and compare it between sexes. RNA was isolated from 35 human lacrimal gland tissue samples collected from 19 cornea donors. AR, ERα, and ERß mRNA was identified in all samples, and their expression was quantified using qPCR. Immunohistochemical staining was performed on selected samples to evaluate protein expression of the receptors. ERα mRNA expression was significantly higher than the expression of AR and ERß. No difference in sex steroid hormone (SSH) receptor mRNA expression was observed between sexes, and no correlation was observed with age. If ERα protein expression is found to be concordant with mRNA expression, it should be investigated further as a potential target for hormone therapy of DED. Further research is needed to elucidate the role of sex steroid hormone receptors in sex-related differences of lacrimal gland structure and disease.

Accuracy of Non-Invasive Prenatal Testing for Duchenne Muscular Dystrophy in Families at Risk: A Systematic Review.

BACKGROUND: Methodological advancements, such as relative haplotype and relative mutation dosage analyses, have enabled non-invasive prenatal diagnosis of autosomal recessive and X-linked diseases. Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by progressive proximal muscular dystrophy and a high mortality rate before the age of twenty. We aimed to systematically present obtainable data regarding a non-invasive prenatal diagnosis of DMD and provide a comprehensive resume on the topic. The emphasis was given to the comparison of different available protocols and molecular methods used for fetal inheritance deduction, as well as their correlation with prognostic accuracy. METHODS: We searched the Scopus and PubMed databases on 11 November 2022 and included articles reporting a non-invasive prenatal diagnosis of DMD in families at risk using relative dosage analysis methods. RESULTS: Of the 342 articles identified, 7 met the criteria. The reported accuracy of NIPT for DMD was 100% in all of the studies except one, which demonstrated an accuracy of 86.67%. The combined accuracy for studies applying indirect RHDO, direct RHDO, and RMD approaches were 94.74%, 100%, and 100%, respectively. Confirmatory results by invasive testing were available in all the cases. Regardless of the technological complexity and low prevalence of the disease that reduces the opportunity for systematic research, the presented work demonstrates substantial accuracy of NIPT for DMD. CONCLUSIONS: Attempts for its implementation into everyday clinical practice raise many ethical and social concerns. It is essential to provide detailed guidelines and arrange genetic counseling in order to ensure the proper indications for testing and obtain informed parental consent.

Early development of the human embryonic testis.

Human gonadal development culminating in testicular differentiation is described through analysis of histologic sections derived from 33-day to 20-week human embryos/fetuses, focusing on early development (4-8 weeks of gestation). Our study updates the comprehensive studies of Felix (1912), van Wagenen and Simpson (1965), and Juric-Lekic et al. (2013), which were published in books and thus are unsearchable via PubMed. Human gonads develop from the germinal ridge, a thickening of coelomic epithelium on the medial side of the urogenital ridge. The bilateral urogenital ridges contain elements of the mesonephric kidney, namely the mesonephric duct, mesonephric tubules, and mesonephric glomeruli. The germinal ridge, into which primordial germ cells migrate, is initially recognized as a thickening of coelomic epithelium on the urogenital ridge late in the 4th week of gestation. Subsequently, in the 5th week of gestation, a dense mesenchyme develops sub-adjacent to the epithelium of the germinal ridge, and together these elements bulge into the coelomic cavity forming bilateral longitudinal ridges attached to the urogenital ridges. During development, primordial cells migrate into the germinal ridge and subsequently into testicular cords that form within the featureless dense mesenchyme of the germinal ridge at 6-8 weeks of gestation. The initial low density of testicular cords seen at 8 weeks remodels into a dense array of testicular cords surrounded by α-actin-positive myoid cells during the second trimester. Human testicular development shares many features with that of mice being derived from 4 elements: coelomic epithelium, sub-adjacent mesenchyme, primordial germ cells, and the mesonephros.

Analysis of 29 Targeted Genes for Non-Obstructive Azoospermia: The Relationship between Genetic Testing and Testicular Histology.

PURPOSE: To analyze the presence of potentially pathogenic variants of 29 candidate genes known to cause spermatogenic failure (SPGF) in patients with non-obstructive azoospermia (NOA) who underwent testicular histology. MATERIALS AND METHODS: Forty-eight patients with unexplained NOA referred to the Department of Transfusion Medicine and Transplantation Biology, University Hospital Center Zagreb, Zagreb, Croatia for testicular biopsy. They were divided into three groups: those who had cryptorchidism (n=9), those with varicocele (n=14), and those with idiopathic NOA (n=25). All included patients underwent blood withdrawal for next-generation sequencing (NGS) analysis and gene sequencing. RESULTS: We found a possible genetic cause in 4 patients with idiopathic NOA (16%) and in 2 with cryptorchidism (22%). No pathogenic or possibly pathogenic mutations were identified in patients with varicocele. Variants of undetermined significance (VUS) were found in 11 patients with idiopathic NOA (44%), 3 with cryptorchidism (33%), and 8 patients with varicocele (57%). VUSs of the USP9Y gene were the most frequently as they were found in 14 out of 48 patients (29%). In particular, the VUS USP9Y c.7434+14del was found in 11 patients. They showed varied histological pictures, including Sertoli cell-only syndrome, mixed atrophy, and hypospermatogenesis, regardless of cryptorchidism or varicocele. No direct correlation was found between the gene mutation/variant and the testicular histological picture. CONCLUSIONS: Different mutations of the same gene cause various testicular histological pictures. These results suggest that it is not the gene itself but the type of mutation/variation that determines the testicular histology picture. Based on the data presented above, it remains challenging to design a genetic panel with prognostic value for the outcome of testicular sperm extraction in patients with NOA.

Trends in the treatment of undescended testes: a pediatric tertiary care center experience from Croatia.

Objective: Undescended testes (UDT) is the most common anomaly of the male genitourinary tract. The guidelines suggest that orchidopexy in congenitally UDT should be performed between 6 months and 18 months of age, while in acquired UDT, orchidopexy should be performed before puberty. Delay in treatment increases the risk of cancer and infertility. The main aim of this study was to determine whether we meet international standards in the treatment of UDT. Methods: The present study included all boys who underwent orchidopexy either due to congenital or acquired UDT in 2019 (from January 1 to December 31). For each group, laterality, location, associated anomalies, premature birth and in how many cases ultrasound was applied were determined. Additionally, for each group, the types of surgery, the number of necessary reoperations, and in how many cases atrophy occurred were determined. Finally, ages of referral, of clinical examination, and of orchidopexy were determined. Results: During this period, 198 patients with 263 UDT underwent orchidopexy. The median time of orchidopexy for the congenital group was 30 months, while that for the acquired group was 99 months. In the congenital group up to 18 months of age, orchidopexy was performed in 16 (16%) boys, while in the acquired group up to 13 years of age, orchidopexy was performed in 95 (96.94%) boys. Conclusion: Given the well-known risks of late treatment of UDT, orchidopexy needs to be performed much earlier, especially in the congenital group.

Diverse monogenic subforms of human spermatogenic failure.

Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification.

Activin A and CCR2 regulate macrophage function in testicular fibrosis caused by experimental autoimmune orchitis.

Experimental autoimmune-orchitis (EAO), a rodent model of chronic testicular inflammation and fibrosis, replicates pathogenic changes seen in some cases of human spermatogenic disturbances. During EAO, increased levels of pro-inflammatory and pro-fibrotic mediators such as TNF, CCL2, and activin A are accompanied by infiltration of leukocytes into the testicular parenchyma. Activin A levels correlate with EAO severity, while elevated CCL2 acting through its receptor CCR2 mediates leukocyte trafficking and recruits macrophages. CCR2 + CXCR4 + macrophages producing extracellular matrix proteins contribute widely to fibrogenesis. Furthermore, testicular macrophages (TMs) play a critical role in organ homeostasis. Therefore, we aimed to investigate the role of the activin A/CCL2-CCR2/macrophage axis in the development of testicular fibrosis. Following EAO induction, we observed lower levels of organ damage, collagen deposition, and leukocyte infiltration (including fibronectin+, collagen I+ and CXCR4+ TMs) in Ccr2-/- mice than in WT mice. Furthermore, levels of Il-10, Ccl2, and the activin A subunit Inhba mRNAs were lower in Ccr2-/- EAO testes. Notably, fibronectin+ TMs were also present in biopsies from patients with impaired spermatogenesis and fibrotic alterations. Overexpression of the activin A antagonist follistatin reduced tissue damage and collagen I+ TM accumulation in WT EAO testes, while treating macrophages with activin A in vitro increased the expression of Ccr2, Fn1, Cxcr4, and Mmp2 and enhanced migration along a CCL2 gradient; these effects were abolished by follistatin. Taken together, our data indicate that CCR2 and activin A promote fibrosis during testicular inflammation by regulating macrophage function. Inhibition of CCR2 or activin A protects against damage progression, offering a promising avenue for therapeutic intervention.