Male reproductive ageing is a complex process involving progressive and detrimental histological and physiological alterations to the testis and beyond. Age-related morbidities often confound reproductive function, making it difficult to disentangle systemic from reproductive male ageing. We have previously shown that healthy ageing is associated with full spermatogenesis, normal sperm production and hormonal secretion. However, the molecular mechanisms allowing the human testis to age without major loss of function remained elusive. In this study, we investigated the transcriptomic dynamics of the ageing human testis using bulk RNA sequencing of testicular samples with full spermatogenesis from young (24–31 years, n = 4), middle-aged (41–45 years, n = 3), and aged (54–75 years, n = 6) men. We found that, in healthy human testis, ageing is associated with widespread alternative splicing events, affecting genes involved metabolic pathways and DNA repair. Moreover, we identified significant transcriptional changes during ageing, particularly associated with inflammation and oxidative stress. Importantly, a subset of genes showing age-dependent expression patterns was involved in the formation of double-strand breaks (DSBs) and DNA repair and was expressed during early meiosis. Quantification of γH2AX, a marker of DSBs, in an independent validation cohort, did not show age-related abnormal accumulation of DSBs in the germline. These findings provide a comprehensive view of the transcriptional changes occurring during healthy ageing in the human testis, and we hypothesise that these reflect compensatory mechanisms that help preserve reproductive capacity over time in human males.

INTRODUCTION:Direct experimental investigation of Klinefelter syndrome (KS) in patients is limited because the syndrome manifests heterogeneously and affects multiple organ systems. Studying KS therefore requires a model that captures this complexity as accurately as possible while still permitting controlled experimental manipulation. METHODS:This review integrates findings from clinical studies and experimental research using male mice carrying a supernumerary X chromosome that replicate key features of the syndrome, with particular emphasis on their translational relevance, experimental utility, and inherent limitations. RESULTS:Over the past decades, these mouse models have demonstrated their value by enabling the successful translation of experimental findings into clinical applications. Using these models, researchers have investigated multiple aspects of KS, including neurocognitive function, social behavior, metabolism, bone health, brain structure and function, endocrine dysregulation, testicular degeneration, germ cell fate, Sertoli and Leydig cell function, vascular abnormalities, chromosomal imbalance, and X-chromosome dosage effects. CONCLUSION:Although these models have some limitations, their availability allows the investigation of developmental timing and tissue-specific mechanisms. They offer unique opportunities to dissect the genetic, hormonal, and systemic features of KS, thereby informing both basic research and clinical care. Given their demonstrated and ongoing value, we aim to encourage the scientific community to further adopt these models in future KS research.

Summary Sperm transfer genetic information from one generation to the next. These cells originate from gonocytes, which are specified during early embryonal development. To date however, there is limited information on the transcriptional gatekeepers governing the key events leading to sperm production: transition of gonocytes out of pluripotency around the time of birth, formation of the spermatogonial compartment prior to puberty, and initiation of germ cell differentiation at the time of puberty. We address this knowledge gap by employing the marmoset monkey ( Callithrix jacchus ) as a model for human postnatal testicular development. We analysed the transcriptional profiles of ∼48,000 neonatal, pre-pubertal, pubertal, and adult testicular cells and correlated these with histomorphometric measurements. We uncovered a transcriptional state linking gonocytes and spermatogonia characterized by CITED2 expression. Moreover, we propose NANOS2 and DPPA4 as regulators of spermatogonial plasticity from pre-puberty onwards and identify molecular gatekeepers of male germ cell differentiation.

The analysis of individual cells is increasingly automated through deep learning techniques. This is particularly relevant for high-resolution whole slide images (WSIs), which can contain thousands of cells, making manual evaluation impractical. This increase in automation, however, requires higher levels of standardisation (with respect to the scanning hardware, settings and staining) and is further aggravated by the dynamics of the underlying cellular processes, rendering unique cell classifications difficult. To address these difficulties we investigated the entire processing pipeline (from imaging over annotation to model training) and study its underlying trade-offs. In particular, we created a new dataset comprising of more than 6, 300 labelled and 500, 000 unlabelled cells scanned using two different scan settings, resulting in fully registered image pairs with varying level of detail and quality. Using these alternative dataset versions we analysed the impact of inter- and intra-variability between three different annotators and addressed the challenge of limited labelled data by comparing the impact of different self-supervised pretraining strategies. Overall, our analyses provide new insights into the dependencies between imaging, annotation, self-supervision and deep learning-based classification, especially in the context of continuously developing cells and demonstrate the beneficial impact of these considerations on the overall classification accuracy. Code is available at https://zivgitlab.uni-muenster.de/cvmls/icdc and the data will be shared upon qualified request due to data privacy laws.

CONTEXT:Nonobstructive azoospermia (NOA) constitutes male infertility with complete absence of sperm in the ejaculate. NOA can originate in testicular malfunction or in endocrine dysregulation. Elevated follicle-stimulating hormone (FSH) levels are diagnostically valuable for NOA. OBJECTIVE:An azoospermic patient cohort comprising 79 men and exhibiting no obstruction but normal FSH levels was identified. Focusing on this normogonadotropic nonobstructive azoospermic (NNOA) group, the study aimed to characterize these patients in depth. METHODS:Whether the missing FSH upregulation in patients with NNOA is due to testicular or pituitary/hypothalamic malfunctions was examined by analyzing somatic, endocrine, and testicular parameters compared with 87 men with hypergonadotropic NOA and 88 normozoospermic men. RESULTS:Testicular phenotypes of patients with NNOA and NOA were compared in histologically stratified subgroups (most advanced germ cell type). Using flow cytometry, the samples were evaluated for testicular cell composition by ploidy analysis. Concerning the distinct histological classification (hypospermatogenesis, spermatogenic arrest, Sertoli cell only, tubular atrophy) NNOA men produced more elongated spermatids and showed higher sperm retrieval. Testicular tissue composition between patients with NNOA and patients with NOA only differed after meiosis. CONCLUSION:The missing FSH upregulation in NNOA might be due to a testicular malfunction, as both FSH and testosterone were normal and NNOA spermatogenesis differed only after meiosis. Two explanations are possible: NNOA represents a phenotype in which spermatogenesis fails-different from NOA-only at the postmeiotic level, leaving FSH regulation unaffected, or the same mechanism underlies both NNOA and NOA, but the groups are at different stages of progression of the same disorder.

Human chorionic gonadotropin (hCG) has structural similarities with thyroid-stimulating hormone (TSH) and may stimulate TSH receptors at higher concentrations. During pregnancy, placental hCG causes TSH suppression, contributing to hyperemesis. However, in males, clinical manifestations caused by excess hCG are rare. Herein, we describe complications of life-threatening thyroid storm caused by paraneoplastic hCG secretion from testicular germ cell tumours (GCTs) and aim to identify high-risk groups through retrospective analysis in n = 20 males (aged 17–55 years) with testicular hCG-positive GCTs. Seven hCG-positive testicular GCTs were classified as seminoma, and 13 were classified as non-seminomatous GCTs (NSGCTs). In 3/7 males with seminomas (43%), serum β-hCG concentrations were mildly elevated (median: 0.3 U/L; range: 0.3–82.1 U/L). In contrast, β-hCG was increased in 12/13 (92%) males with a NSGCT (median: 71.1 U/L; range: 0.3–1,600,000 U/L). In 10/13 males with NSGCT (77%), we detected components of embryonal cell carcinoma (EC), and in 7/13 (54%), we detected components of a choriocarcinoma (ChC). TSH was suppressed with high free thyroxine levels in two cases with NSGCT and excessively elevated β-hCG concentrations, but there was no TSH suppression in a further case with high β-hCG. One patient with NSGCT and high β-hCG levels presented with thyroid storm and imminent decompensation refractory to anti-thyroid treatment, requiring a total thyroidectomy. In the second patient, anti-thyroid treatment was initiated shortly after the diagnosis, successfully normalizing hyperthyroxinaemia. In conclusion, paraneoplastic β-hCG production, occurring in NSGCTs with components of ECs or ChCs, is a rare cause of thyrotoxicosis. Early recognition and treatment are critical to prevent a life-threatening thyroid storm.

Despite advances in computational pathology, manual tissue examinations remain the gold standard in diagnostics, resulting in thousands of whole slide image inspections in daily practice. Unfortunately, examination strategies and identified regions are often lost after inspection, preventing the use of manual efforts to train deep learning algorithms. To address this, we introduce MARTHA, a tool that combines passive eye tracking - which captures pathologists' gaze during tissue examinations - with deep learning-based image analysis. This approach simplifies and accelerates data interaction while improving the accuracy and efficiency of computer-assisted diagnostics by incorporating expert attention into neural networks. An intuitive graphical interface integrates traditional interaction methods, allowing seamless use across clinical routines and biomedical image domains. We evaluated MARTHA on human testicular tissues, generating the largest annotated dataset for the human testis, with over 83,000 cell nuclei from approximately 8000 tubules. Our results demonstrate strong performance in data interaction efficiency and semantic segmentation, offering valuable insights into testicular phenotypes and supporting pathologists in enhancing their analyses, which is a crucial step towards improving the diagnosis and treatment of infertile men with unfulfilled child-wish.

BACKGROUND:WFS1 spectrum disorder, also known as Wolfram syndrome (WS) is an ultra-rare (<1:500,000; ORPHA: 3463) monogenic (OMIM #222300) progressive neuroendocrine and neurodegenerative disorder, characterised by early-onset insulin-dependent diabetes, optic atrophy, central diabetes insipidus and sensi-neuronal deafness. It is caused predominantly by bi-allelic mutations in the WFS1 gene and exceptionally in the WFS2-gene. There is very limited published data on gonadal function in young people with WS. Expansion of the phenotype has previously included suggestions of abnormalities in puberty in adolescents with (WS) but with little detail.1-3 AIM: To assess testicular function and pubertal progression in a cohort of adolescent and young adult patients with classical WFS1 spectrum disorder (WS). METHODS:Retrospective case notes review of national patient cohorts comprising 21 males with WS aged 16-30 years. All patients were treated in two tertiary European health care centres: in Birmingham, UK and Münster, Germany. Hormonal parameters reflecting hypothalamic-pituitary-gonadal axis function and treatment with sex hormones were assessed. In addition, the presence or absence of erectile dysfunction was explored. In a subset of men, semen data were analysed. In one young man, testicular biopsies were examined histologically using light and electron microscopy. RESULTS:Severely delayed or arrested puberty was observed in 57% of male adolescents with WS, necessitating testosterone replacement for completion of pubertal development. Subclinical (compensated) hypergonadotropic hypogonadism with still adequate testosterone serum concentration for age, but elevated LH/FSH was observed in 28.6% (n = 6). In two males, aged 19 and 16 years (9.5%), inadequately low LH/FSH and testosterone levels indicated hypogonadotropic hypogonadism. In the subset of males with normal puberty and normal endocrine testicular function (43% of male patients), the oldest, aged 30 years had normal sperm count in semen. Another young man had oligozoospermia at age 20, but azoospermia at age 25 years. Histology of his testicular tissues evidenced structural alterations of Leydig and Sertoli cells and tubular atrophy with various stages of tubular degeneration and meiotic arrest of spermatogenesis. CONCLUSION:Endocrine testicular function and reproductive capacity are impaired in males with WS potentially due to premature degeneration of the testes, with 57% of adolescents developing hypogonadism with pubertal arrest.

STUDY QUESTION: Are there subgroups among patients with cryptozoospermia pointing to distinct etiologies? SUMMARY ANSWER: We reveal two distinct subgroups of cryptozoospermic (Crypto) patients based on testicular tissue composition, testicular volume, and FSH levels. WHAT IS KNOWN ALREADY: Cryptozoospermic patients present with a sperm concentration below 0.1 million/ml. While the etiology of the severely impaired spermatogenesis remains largely unknown, alterations of the spermatogonial compartment have been reported including a reduction of the reserve stem cells in these patients. STUDY DESIGN, SIZE, DURATION: To assess whether there are distinct subgroups among cryptozoospermic patients, we applied the statistical method of cluster analysis. For this, we retrospectively selected 132 cryptozoospermic patients from a clinical database who underwent a testicular biopsy in the frame of fertility treatment at a university hospital. As controls (Control), we selected 160 patients with obstructive azoospermia and full spermatogenesis. All 292 patients underwent routine evaluation for endocrine, semen, and histological parameters (i.e. the percentage of tubules with elongated spermatids). Moreover, outcome of medically assisted reproduction (MAR) was assessed for cryptozoospermic (n = 73) and Control patients (n = 87), respectively. For in-depth immunohistochemical and histomorphometrical analyses, representative tissue samples from cryptozoospermic (n = 27) and Control patients (n = 12) were selected based on cluster analysis results and histological parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included two parts: firstly using clinical parameters of the entire cohort of 292 patients, we performed principal component analysis (PCA) followed by hierarchical clustering on principal components (i.e. considering hormonal values, ejaculate parameters, and histological information). Secondly, for histological analyses seminiferous tubules were categorized according to the most advanced germ cell type present in sections stained with Periodic acid Schif. On the selected cohort of 39 patients (12 Control, 27 cryptozoospermic), we performed immunohistochemistry for spermatogonial markers melanoma-associated antigen 4 (MAGEA4) and piwi like RNA-mediated gene silencing 4 (PIWIL4) followed by quantitative analyses. Moreover, the morphologically defined Adark spermatogonia, which are considered to be the reserve stem cells, were quantified. MAIN RESULTS AND THE ROLE OF CHANCE: The PCA and hierarchical clustering revealed three different clusters, one of them containing all Control samples. The main factors driving the sorting of patients to the clusters were the percentage of tubules with elongated spermatids (Cluster 1, all Control patients and two cryptozoospermic patients), the percentage of tubules with spermatocytes (Cluster 2, cryptozoospermic patients), and tubules showing a Sertoli cells only phenotype (Cluster 3, cryptozoospermic patients). Importantly, the percentage of tubules containing elongated spermatids was comparable between Clusters 2 and 3. Additional differences were higher FSH levels (P < 0.001) and lower testicular volumes (P < 0.001) in Cluster 3 compared to Cluster 2. In the spermatogonial compartment of both cryptozoospermic Clusters, we found lower numbers of MAGEA4+ and Adark spermatogonia but higher proportions of PIWIL4+ spermatogonia, which were significantly correlated with a lower percentage of tubules containing elongated spermatids. In line with this common alteration, the outcome of MAR was comparable between Controls as well as both cryptozoospermic Clusters. LIMITATIONS, REASONS FOR CAUTION: While we have uncovered the existence of subgroups within the cohort of cryptozoospermic patients, comprehensive genetic analyses remain to be performed to unravel potentially distinct etiologies. WIDER IMPLICATIONS OF THE FINDINGS: The novel insight that cryptozoospermic patients can be divided into two subgroups will facilitate the strategic search for underlying genetic etiologies. Moreover, the shared alterations of the spermatogonial stem cell compartment between the two cryptozoospermic subgroups could represent a general response mechanism to the reduced output of sperm, which may be associated with a progressive phenotype. This study therefore offers novel approaches towards the understanding of the etiology underlying the reduced sperm formation in cryptozoospermic patients. STUDY FUNDING/COMPETING INTEREST(S): German research foundation CRU 326 (grants to: SDP, NN). Moreover, we thank the Faculty of Medicine of the University of Munster for the financial support of Lena Charlotte Schulke through the MedK-program. We acknowledge support from the Open Access Publication Fund of the University of Munster. The authors have no potential conflicts of interest.

Abstract Study question What are the specific changes under gender affirming hormone therapy (GAHT) in the spermatogonial cell compartment in testicular tissues of transwomen? Summary answer GAHT has a negative effect on the most undifferentiated PIWIL4+ spermatogonia potentially leading to reduced chances for fertility preservation in transwomen. What is known already Transwomen take GAHT to achieve transition between the gender assigned at birth and their gender identity. GAHT leads to a reduction of the most advanced germ cells and a reduction of MAGEA4 positive spermatogonia (pan spermatogonial marker). Single-cell RNA sequencing studies identified spermatogonial subpopulations based on the expression of the marker genes PIWIL4, FGFR3, NANOS3, GFRA1, KIT, UTF1. PIWIL4+ cells are considered the origin of germ cell differentiation (state 0). State 1 and 2 spermatogonia are characterized by expression of GFRA1 and KIT, respectively. Study design, size, duration On the day of gender affirming surgery (GAS) testicular tissues and blood samples were collected of 25 age matched trans women (mean 28.1 yr), who underwent comparable GAHT regimens (10 or 12.5 mg cyproterone acetate and estrogens) between 2013 and 2018. 8 adult cis men (mean 34.5 yr) with complete spermatogenesis served as controls. Age, start and type of GAHT were assessed using a questionnaire. This study was approved by the local ethics committee. Participants/materials, setting, methods We performed immunohistochemical stainings to evaluate the expression of spermatogonial markers (MAGEA4, UTF1, PIWIL4, FGFR3, NANOS3, GFRA1) in Bouin’s-fixed testicular tissue sections. For each marker the number of positive cells per round tubular cross-section was determined in 25 round tubules. The spermatogonial proliferation rate was evaluated using immunofluorescence co-stainings for GFRA1/MIKI67/MAGEA4 and KIT/MKI67/MAGEA4. We scored 400 MAGEA4+ cells per individual and determined the proportion of GFRA1+/KIT+. MKI67+ cells among the GFRA1+/KIT+ were evaluated. Main results and the role of chance The number of MAGEA4+ spermatogonia and the proportion of PIWIL4+ spermatogonia within the MAGEA4+ spermatogonia were reduced in transwomen compared to controls (p < 0.001). In contrast, the relative proportion of UTF1+, FGFR3+ and NANOS3+ spermatogonia was comparable. The number of spermatogonia per tubule correlated negatively with the age at start of GAHT (p < 0.01) and the age on the day of GAS (p < 0.001). The treatment duration correlated negatively with the proportion of PIWIL4+ (p < 0.01) and NANOS3+ spermatogonia (p < 0.05). The immunofluorescence stainings in transwomen revealed a similar ratio of proliferating KI67+ spermatogonia, both within the GFRA1+ and KIT+ cells, compared to controls. These results show the negative effects of the GAHT itself on the population of spermatogonia as well as the individual susceptibility depending on age-related factors. These results have the potential to better define the chances for fertility preservation under GAHT. Limitations, reasons for caution Transwomen are a very heterogeneous patient group varying in age, different GAHT, different treatment durations, intake of co-medications and possible pre-existing medical conditions. Hence, testicular size and histological appearance of transwomen’s testes are heterogeneous. Wider implications of the findings Transwomen, who wish to have own biological children, have to be counseled with regard to fertility preservation, best before initiation of GAHT, in order to avoid adverse effects on spermatogonia. Individual counselling has to take place with regard to fertility preservation. Trial registration number 2012-555-f-S

Minipuberty is a transient activity period of the hypothalamic-pituitary-gonadal (HPG) axis in the postnatal and infant period in humans and non-human primates. Hallmarks of this period are surging serum concentrations of reproductive hormones. While in females, the role of minipuberty seems to be dispensable for future fertility, in males, it is significantly associated with reproductive function in later life. In males, this activity period promotes further masculinization, including testicular and penile growth, as well as completion of testicular descent if not already achieved at birth. At the testicular level, both, somatic and germ cells undergo proliferation and partial maturation during this period. Minipuberty is thought to prime male gonadal tissue for subsequent growth and maturation. Notably, perturbed or absent minipuberty is associated with reduced male reproductive function in adulthood. While the sustained HPG axis activity during adulthood is known to control reproductive function, minipuberty appears to be a prerequisite for obtaining full male reproductive function in later life, thereby determining future fertility potential, i.e. the ability to father a child. This review maps the role of male minipuberty for reproductive function and presents suitable animal models to study minipuberty. Also, it describes the development and maturation of testicular cell types, discusses short- and long-term effects of minipuberty and highlights future research perspectives.

Die erfolgreiche Befruchtung einer Eizelle ist das ultimative Ziel für ein reifes Spermium. Damit dieser Vorgang in vivo korrekt abläuft, folgt der natürliche Befruchtungsprozess einer zeit- und lokalisationsabhängigen Sequenz komplexer Schritte, beginnend bei der Produktion im Hoden, über die Reifung im Nebenhoden und letztendlich bei der Befruchtung der Eizelle. Spezifische Proteine und Oberflächenkomponenten in und an den Spermatozoen spielen dabei eine wichtige Rolle. Bei fertilen Männern erfolgt die natürliche Befruchtung der logisch bestimmten Reihenfolge der Ereignisse, bei infertilen Männern jedoch können diese Wege teilweise therapeutisch abgekürzt werden, um so letzendlich auch eine Vaterschaft herbeizuführen. So können nicht nur ejakulierte Spermatozoen, z. B. in vivo nach Insemination in den weiblichen Genitaltrakt, sondern auch Spermatozoen aus dem Hoden und dem Nebenhoden nach Injektion in die Eizelle in vitro vorherbestimmte Befruchtungsschritte ausführen und so zu einer intakten Schwangerschaft führen. Dieses Buchkapitel folgt dem Weg der Spermien vom Ort der Produktion bis zur befruchtungsfähigen Eizelle und beschreibt wichtige Prozesse, die Spermatozoen durchlaufen, um letztendlich ihr Ziel zu erreichen.

The spermatogonial compartment maintains spermatogenesis throughout the reproductive lifespan. Single-cell RNA sequencing (scRNA-seq) has revealed the presence of several spermatogonial clusters characterized by specific molecular signatures. However, it is unknown whether the presence of such clusters can be confirmed in terms of protein expression and whether protein expression in the subsets overlaps. To investigate this, we analyzed the expression profile of spermatogonial markers during the seminiferous epithelial cycle in cynomolgus monkeys and compared the results with human data. We found that in cynomolgus monkeys, as in humans, undifferentiated spermatogonia are largely quiescent, and the few engaged in the cell cycle were immunoreactive to GFRA1 antibodies. Moreover, we showed that PIWIL4+ spermatogonia, considered the most primitive undifferentiated spermatogonia in scRNA-seq studies, are quiescent in primates. We also described a novel subset of early differentiating spermatogonia, detectable from stage III to stage VII of the seminiferous epithelial cycle, that were transitioning from undifferentiated to differentiating spermatogonia, suggesting that the first generation of differentiating spermatogonia arises early during the epithelial cycle. Our study makes key advances in the current understanding of male germline premeiotic expansion in primates.

Objective Germ cells of transwomen are affected by gender-affirming hormone therapy (GAHT). Fertility will be lost after surgical intervention; thereby, fertility preservation becomes an increasingly imortant topic. This study investigated if the absolute number of spermatogonia in transwomen is comparable at the time of gender-affirming surgery (GAS) to that in pre-pubertal boys. Methods We carried out a retrospective study of testicular tissues from 25 selected subjects, which had undergone a comparable sex hormone therapy regimen using cyproterone acetate (10 or 12.5 mg) and estrogens. As controls, testicular biopsies of five cisgender adult men (aged 35–48 years) and five pre-/pubertal boys (5–14 years) were included. Testicular tissues were immunohistochemically stained for MAGE A4-positive cells, the most advanced germ cell type. The number of spermatogonia per area was assessed. Clinical values and serum hormone values for FSH, LH, testosterone, free testosterone, estradiol and prolactin were determined on the day of GAS for correlation analyses. Results Round spermatids were the most advanced germ cell type in 3 subjects, 5 had an arrest at spermatocyte stage, while 17 showed a spermatogonial arrest. On average, testicular tissues of transwomen contained 25.15 spermatogonia/mm 3 , a number that was significantly reduced compared to the two control groups ( P < 0.01, adult 80.65 spermatogonia/mm 3 and pre-/pubertal boys 78.55 spermatogonia/mm 3 ). Linear regression analysis revealed that testes with higher weight and high LH contained more spermatogonia. Conclusion Irrespective of treatment dose or duration, spermatogenesis was impaired. Spermatogonial numbers were significantly reduced in transwomen compared to the control groups. Lay summary When transwomen go through treatment to confirm their gender, their germ cells are affected. They lose their fertility after surgery, so fertility preservation becomes an important topic. We carried out a study looking at tissue from testes of 25 people who had been through the same sex hormone therapy until surgery. Blood samples were also taken. As controls, samples were taken from the testes of cisgender boys and adult men. On average, the samples from the testes of transwomen contained a smaller number of early sperm cells compared to the two control groups. Regardless of the dose or length of hormone treatment, the fertility of transwomen was significantly reduced so that counseling about fertility preservation should be offered before hormone therapy.

An uncontrolled reproduction of animals in human hands should be avoided. To meet this goal, animals are widely castrated, i.e., the gonads are completely removed. Since the gonads are the most important source of sex hormones, this is a serious intervention in the entire endocrine system of an organism. Sterilization is a much less invasive procedure. Thus, it could have advantages over castration. Therefore, the overall aim of this study was to analyze the effect of castration vs. sterilization on the release of glucocorticoids, i.e., an important indicator for welfare. Taking domestic guinea pigs as a model system, we studied baseline and response cortisol values (cortisol is the main glucocorticoid in guinea pigs) in castrated, sterilized, sham-operated and intact males and baseline values in their cohoused females. Whereas baseline values of males did not differ between the groups, castrated males showed significantly higher cortisol response levels than intact, sham-operated and sterilized males. Females housed with castrated, sterilized, sham-operated or intact males did not differ in their cortisol concentrations, neither shortly after being placed with the respective male or after being co-housed for several weeks. Overall, the results support the hypothesis that castrated males exhibited a higher cortisol responsiveness during acute challenge which could point to a generalized impaired welfare of castrated males in comparison to intact, sham-operated and sterilized males. Our results provide first evidence for a potential negative impact of castration on the animals' welfare, while at the same time pointing toward sterilization representing a less invasive, promising alternative. Therefore, the results may stimulate future research on this topic to further detect potential welfare-related side effects of castration.

Background: Crypto-and azoospermia (very few/no sperm in the semen) are main con-tributors to male factor infertility. Genetic causes for spermatogenic failure (SPGF) include Klinefelter syndrome and Y-chromosomal azoospermia factor microdeletions, and CFTR mutations for obstructive azoospermia (OA). However, the majority of cases remain unexplained because monogenic causes are not analysed. Objective: To elucidate the monogenic contribution to azoospermia by prospective exome sequencing and strict application of recent clinical guidelines. Design, setting, and participants: Since January 2017, we studied crypto-and azoospermic men without chromosomal aberrations and Y-chromosomal microdeletions attending the Centre of Reproductive Medicine and Andrology, Munster. Outcome measurements and statistical analysis: We performed exome sequencing in 647 men, analysed 60 genes having at least previous limited clinical validity, and strictly assessed variants according to clinical guidelines. Results and limitations: Overall, 55 patients (8.5%) with diagnostic genetic variants were identified. Of these patients, 20 (3.1%) carried mutations in CFTR or ADGRG2, and were diagnosed with OA. In 35 patients (5.4%) with SPGF, mutations in 20 different genes were identified. According to ClinGen criteria, 19 of the SPGF genes now reach at least mod-erate clinical validity. As limitations, only one transcript per gene was considered, and the list of genes is increasing rapidly so cannot be exhaustive. Conclusions: The number of diagnostic genes in crypto-/azoospermia was almost doubled to 21 using exome-based analyses and clinical guidelines. Application of this procedure in routine diagnostics will significantly improve the diagnostic yield and clinical workup as the results indicate the success rate of testicular sperm extraction. Patient summary: When no sperm are found in the semen, a man cannot conceive naturally. The causes are often unknown, but genetics play a major role. We searched for genetic variants in a large group of patients and found causal mutations for one in 12 men; these predict the chances for fatherhood. (c) 2022 European Association of Urology. Published by Elsevier B.V. All rights reserved.