Therapeutic strategies for uncomplicated cystitis in women.

Uncomplicated cystitis is affecting many women of all ages and has a great impact on the quality of life, especially in women suffering from recurrent, uncomplicated cystitis. By far the most frequent uropathogen, E. coli, may have acquired increasing resistance against a variety of oral antibiotics, which may differ between countries and regions. Therefore, local resistance data are important to be considered. On the other hand, non-antibiotic therapy has also become an option which should be discussed and offered to the patient. In patients suffering from recurrent uncomplicated cystitis, individual risk factors and possible behavioral changes should first be taken into account. Non-antimicrobial prophylactic strategies shown to be successful in well-designed clinical studies are the next options. Long term antibiotic prophylaxis, however, should only be considered as a last option. For some of those patients self-diagnosis and self-treatment may be suitable, e.g. by using a recognized questionnaire.

Impaired Semen Quality in Patients with Chronic Prostatitis.

Background/Objectives: Chronic prostatitis/chronic pelvic pain syndrome CP/CPPS is a rather common condition and in recent years many studies have shown contradictory results regarding its impact on semen quality. This prospective cohort study set out to investigate how CP/CPPS affected the parameters of semen in a prospective cohort of patients compared with the WHO 2021 reference group. Methods: From 2013 to 2022, a total of 1071 patients with suspicion of CP/CPPS received a comprehensive andrological examination. Complete semen analysis was carried out in compliance with WHO 2010 guidelines, comparing every study population semen variable to the WHO 2021 reference group (n~3500). Results: All evaluated semen parameters had median values that fell within a normal range. Nonetheless, approximately 25% of patients had values for each semen variable that were lower than the WHO reference group's fifth percentile. In particular, bacteriospermia was associated with a negative impact on semen volume. Conclusions: This is the largest study that compares all standard semen parameters in patients suffering from CP/CPPS to WHO 2021 reference values. It provides evidence of an impairment of conventional semen parameters.

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.

European Association of Urology Guidelines on Urological Infections: Summary of the 2024 Guidelines.

BACKGROUND AND OBJECTIVE: Urological infections significantly impact the wellbeing and quality of life of individuals owing to their widespread occurrence and diverse clinical manifestations. The objective of the guidelines panel was to provide evidence-based guidance on the diagnosis, treatment, and prevention of urinary tract infections (UTIs) and male accessory-gland infections, while addressing crucial public health aspects related to infection control and antimicrobial stewardship. METHODS: For the 2024 guidelines on urological infections, new and relevant evidence was identified, collated, and appraised via a structured assessment of the literature. Databases searched included Medline, EMBASE, and the Cochrane Libraries. Recommendations within the guidelines were developed by the panel to prioritise clinically important care decisions. The strength of each recommendation was determined according to a balance between desirable and undesirable consequences of alternative management strategies, the quality of the evidence (including the certainty of estimates), and the nature and variability of patient values and preferences. KEY FINDINGS AND LIMITATIONS: Key recommendations emphasise the importance of a thorough medical history and physical examination for patients with urological infections. The guidelines stress the role of antimicrobial stewardship to combat the rising threat of antimicrobial resistance, providing recommendations for antibiotic selection, dosing, and duration on the basis of the latest evidence. CONCLUSIONS AND CLINICAL IMPLICATIONS: This overview of the 2024 EAU guidelines offers valuable insights into managing urological infections and are designed for effective integration into clinical practice. PATIENT SUMMARY: The European Association of Urology has issued an updated guideline on urological infections. The guidelines provide recommendations for diagnosis, treatment, and prevention, with a particular focus on minimising antibiotic use because of the increasing global threat of antimicrobial resistance.

T cells in testicular germ cell tumors: new evidence of fundamental contributions by rare subsets.

BACKGROUND: Immune cell infiltration is heterogeneous but common in testicular germ cell tumors (TGCT) and pre-invasive germ cell neoplasia in situ (GCNIS). Tumor-infiltrating T cells including regulatory T (Treg) and follicular helper T (Tfh) cells are found in other cancer entities, but their contributions to TGCT are unknown. METHODS: Human testis specimens from independent patient cohorts were analyzed using immunohistochemistry, flow cytometry and single-cell RNA sequencing (scRNA-seq) with special emphasis on delineating T cell subtypes. RESULTS: Profound changes in immune cell composition within TGCT, shifting from macrophages in normal testes to T cells plus B and dendritic cells in TGCT, were documented. In most samples (96%), the CD4+ T cell frequency exceeded that of CD8+ cells, with decreasing numbers from central to peripheral tumor areas, and to tumor-free, contralateral testes. T cells including Treg and Tfh were most abundant in seminoma compared to mixed tumors and embryonal carcinoma. CONCLUSION: Despite considerable heterogeneity between patients, T cell subtypes form a key part of the TGCT microenvironment. The novel finding of rare Treg and Tfh cells in human testis suggests their involvement in TGCT pathobiology, with implications for understanding tumor progression, to assess patients' prognosis, and as putative targets for personalized immunotherapy.

The oxytocin antagonist cligosiban reduces human prostate contractility: Implications for the treatment of benign prostatic hyperplasia.

BACKGROUND AND PURPOSE: With increasing life expectancy, benign prostatic hyperplasia (BPH) consequently affects more ageing men, illustrating the urgent need for advancements in BPH therapy. One emerging possibility may be the use of oxytocin antagonists to relax smooth muscle cells in the prostate, similar to the currently used (although often associated with side effects) α1-adrenoceptor blockers. EXPERIMENTAL APPROACH: For the first time we used live-imaging, combined with a novel image analysis method, to investigate the multidirectional contractions of the human prostate and determine their changes in response to oxytocin and the oxytocin antagonists atosiban and cligosiban. Human prostate samples were obtained and compared from patients undergoing prostatectomy due to prostate cancer as well as from patients with transurethral resection of prostate tissue due to severe BPH. KEY RESULTS: The two cohorts of tissue samples showed spontaneous multidirectional contractions, which significantly increased after the addition of oxytocin. Different to atosiban, which showed ambiguous effects of short duration, only long-acting cligosiban reliably prevented, as well as counteracted, any contractile oxytocin effect. Furthermore, cligosiban visibly reduced not only oxytocin-induced contractions, but also showed intrinsic activity to relax prostatic tissue. CONCLUSION AND IMPLICATIONS: Thus, the oxytocin antagonist cligosiban could be an interesting candidate in the search for novel BPH treatment options.

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.

Genitourinary Tuberculosis: A Brief Manual for Urologists on Diagnosis and Treatment from the European Association of Urology Urological Infections Panel.

Although tuberculosis (TB) ranks among the most frequent infectious diseases worldwide, one of its extrapulmonary (EP) manifestations, genitourinary (GU) TB, is often underestimated by urologists, particularly in areas such as Europe where TB is not endemic. The aim of this review is to give urologists a concise overview of GUTB as a supplement to the more comprehensive European Association of Urology 2023 update on urological infections guidelines. EPTB can develop in 16% of TB cases. GUTB accounts for 4.6% of EPTB and is often asymptomatic or nonspecific, so it can be confused with other urogenital diseases. GUTB can be highly destructive, leading to failure of urogenital organs. Diagnosis is via microbiological, molecular, and histological testing for urine, genital secretions, or genitourinary tissue, supported by imaging. A 6-mo combinational medical regimen is the first-line treatment for GUTB. However, surgical interventions are also frequently required for the treatment of GUTB complications. Therefore, it is important to keep GUTB in mind for differential diagnosis. PATIENT SUMMARY: We reviewed scientific studies on the occurrence, diagnosis, and treatment of tuberculosis in the genitourinary tract. Our aim is to raise awareness among urologists from countries where this disease does not occur frequently, as urogenital tuberculosis can occur without any symptoms or with unspecific symptoms that can be confused with other diseases.

Effects of a Sertoli cell-specific knockout of Connexin43 on maturation and proliferation of postnatal Sertoli cells.

Adult male Sertoli cell-specific Connexin43 knockout mice (SCCx43KO) exhibit higher Sertoli cell (SC) numbers per seminiferous tubule compared to their wild type (WT) littermates. Thus, deletion of this testicular gap junction protein seems to affect the proliferative potential and differentiation of "younger" SC. Although SC have so far mostly been characterised as postmitotic cells that cease to divide and become an adult, terminally differentiated cell population at around puberty, there is rising evidence that there exist exceptions from this for a very long time accepted paradigm. Aim of this study was to investigate postnatal SC development and to figure out underlying causes for observed higher SC numbers in adult KO mice. Therefore, the amount of SC mitotic figures was compared, resulting in slightly more and prolonged detection of SC mitotic figures in KO mice compared to WT. SC counting per tubular cross section revealed significantly different time curves, and comparing proliferation rates using Bromodesoxyuridine and Sox9 showed higher proliferation rates in 8-day old KO mice. SC proliferation was further investigated by Ki67 immunohistochemistry. SC in KO mice displayed a delayed initiation of cell-cycle-inhibitor p27Kip1 synthesis and prolonged synthesis of the phosphorylated tumour suppressor pRb and proliferation marker Ki67. Thus, the higher SC numbers in adult male SCCx43KO mice may arise due to two different reasons: Firstly, in prepubertal KO mice, the proliferation rate of SC was higher. Secondly, there were differences in their ability to cease proliferation as shown by the delayed initiation of p27Kip1 synthesis and the prolonged production of phosphorylated pRb and Ki67. Immunohistochemical results indicating a prolonged period of SC proliferation in SCCx43KO were confirmed by detection of proliferating SC in 17-days-old KO mice. In conclusion, deletion of the testicular gap junction protein Cx43 might prevent normal SC maturation and might even alter also the proliferation potential of adult SC.

Sertoli cell-enriched proteins in mouse and human testicular interstitial fluid.

Sertoli cells support the development of sperm and the function of various somatic cells in the interstitium between the tubules. Sertoli cells regulate the function of the testicular vasculature and the development and function of the Leydig cells that produce testosterone for fertility and virility. However, the Sertoli cell-derived factors that regulate these cells are largely unknown. To define potential mechanisms by which Sertoli cells could support testicular somatic cell function, we aimed to identify Sertoli cell-enriched proteins in the testicular interstitial fluid (TIF) between the tubules. We previously resolved the proteome of TIF in mice and humans and have shown it to be a rich source of seminiferous tubule-derived proteins. In the current study, we designed bioinformatic strategies to interrogate relevant proteomic and genomic datasets to identify Sertoli cell-enriched proteins in mouse and human TIF. We analysed proteins in mouse TIF that were significantly reduced after one week of acute Sertoli cell ablation in vivo and validated which of these are likely to arise primarily from Sertoli cells based on relevant mouse testis RNASeq datasets. We used a different, but complementary, approach to identify Sertoli cell-enriched proteins in human TIF, taking advantage of high-quality human testis genomic, proteomic and immunohistochemical datasets. We identified a total of 47 and 40 Sertoli cell-enriched proteins in mouse and human TIF, respectively, including 15 proteins that are conserved in both species. Proteins with potential roles in angiogenesis, the regulation of Leydig cells or steroidogenesis, and immune cell regulation were identified. The data suggests that some of these proteins are secreted, but that Sertoli cells also deposit specific proteins into TIF via the release of extracellular vesicles. In conclusion, we have identified novel Sertoli cell-enriched proteins in TIF that are candidates for regulating somatic cell-cell communication and testis function.

Linguistic and Clinical Validation of the Tajik Acute Cystitis Symptom Score for Diagnosis and Patient-Reported Outcome in Acute Uncomplicated Cystitis.

Background and Objectives: Acute Cystitis Symptom Score (ACSS) is a self-reporting questionnaire for clinical diagnosis and follow-up of acute uncomplicated cystitis (AC) in women. The ACSS, originally developed in Uzbek and Russian, both considered original languages, is now available in several other languages. This study aimed to translate and validate the ACSS in the Tajik language. Material and Methods: Linguistic validation was carried out according to the Linguistic Validation Manual for Patient-Reported Outcomes Instruments guidelines. Clinical validation was performed by enrolling fifty-four Tajik-speaking women. All women included in this study were first interviewed about the understandability of all questions and statements in the final Tajik ACSS and were asked to fill in form A at the first visit (diagnostics) and form B at any follow-up visit (patient-reported outcome). Results: Thirty-three women, median (range) age of 35 (18-77), were diagnosed with AC (patient group), while twenty-one women, median (range) age of 34 (20-61) (p = 0.109), were enrolled as the control group without any other urological disease. For the diagnostics of AC, a summary score of the six typical symptoms ("Typical" domain) showed the best balance between sensitivity (0.73) and specificity (0.71) at 5 and above. Cronbach's alpha [95% CI] and split-half reliability [95%] were 0.82 [0.76; 0.98] and 0.84 [0.77; 0.87], respectively. At the follow-up visit, the patients reported a significant reduction in the "Typical" domain and an improvement in the "Quality of Life" domain. Conclusion: The Tajik ACSS showed good reliability and diagnostic values and may be used as a reliable tool for the diagnosis and patient-reported outcome in women with AC in clinical and epidemiological studies and for daily practice.

Polish version of the Acute Cystitis Symptom Score for patients with acute uncomplicated cystitis.

Introduction: The Acute Cystitis Symptom Score (ACSS) is a self-reporting questionnaire to evaluate the symptoms and quality of life in women with uncomplicated acute cystitis (AC). The aim of the current study was the additional cognitive and clinical validation of the Polish version. Material and methods: Professional forward and backward translations from original Russian to Polish were performed by Mapi SAS. For cognitive assessment, women with different ages and educational levels were asked to comment on each item of the Polish ACSS to establish the final study version. The clinical validation was performed as a prospective, non-interventional cohort study. Women with AC (Patients) and those without (Controls) filled in the Polish ACSS during their visits to a physician's office and at a follow-up visit. Statistical analysis included ordinary descriptive values, calculation of reliability, validity, discriminative ability, responsiveness (sensitivity, specificity), and comparative analysis. Results: The cognitive assessment was performed in 60 women with a median (range) age of 44.5 (21-88) years and different educational levels: grade school (n = 8), high school (n = 25), college (n = 22), and postgraduate education (n = 5). Forty-three patients were recruited for the clinical validation study along with 34 controls. Statistical analyses resulted in excellent values of internal consistency, discriminative ability, and validity for diagnosis of AC. At a summary score of 6 and higher in the ´Typical´ domain, positive and negative predictive values were 97% and 79%, and sensitivity and specificity were 79% and 97%, respectively. Conclusions: The Polish version of the ACSS has demonstrated benefits for diagnosis and patient-reported outcome assessment. It is objective, fast, and cost-effective, and it may help to easily confirm the accurate diagnosis of AC. The Polish ASCSS can now be recommended for use in clinical and epidemiological studies, in clinical practice, or for self-diagnosis and patient-reported outcome in women with symptoms of AC.

Acute orchitis deciphered: Coxsackievirus B strains are the main etiology and their presence in semen is associated with acute inflammation and risk of persistent oligozoospermia.

Although various viruses are considered to be the clinical cause for acute orchitis, it is completely unclear to what extent and which viruses are etiologically involved in acute orchitis and what the clinic and course of these patients are like. Therefore, a prospective study was set up to decipher acute isolated orchitis. Between July 2007 and February 2023, a total of 26 patients with isolated orchitis were recruited and compared with 530 patients with acute epididymitis. We were able to show for isolated orchitis, that (1) orchitis is usually of viral origin (20/26, 77%) and enteroviruses with coxsackievirus B strains (16/26, 62%) are predominant, (2) virus isolates could be received from semen indicating the presence of replication-competent virus particles, (3) a polymerase chain reaction (PCR) for enteroviruses should be conducted using semen provided at the onset of disease, because the virus is not detectable in serum/urine, (4) there is a circannual occurrence with the maximum in summer, (5) orchitis is associated with a characteristic inflammatory cytokine panel in the semen and systemic inflammation, (6) orchitis is usually rapidly self-limiting, and (7) about 30% of patients (6/20) suffer ongoing oligozoospermia. These seven emerging aspects are likely to fundamentally change thinking and clinical practice regarding acute isolated orchitis.

Comprehensive evaluation of hematospermia in patients with acute epididymitis compared to patients with isolated hematospermia.

BACKGROUND: Among the most commonly known causes of hematospermia are infections in the genitourinary tract, but no study exists that has comprehensively investigated hematospermia in patients with acute epididymitis. OBJECTIVES: To assess the impact of hematospermia in patients with acute epididymitis and its association with clinical, microbiological, and semen parameters. MATERIALS AND METHODS: Since May 2007, a total of 324 sexually active patients with acute epididymitis were recruited in a prospective cohort study. Patients received a comprehensive medical and sexual history, and clinical, sonographic, laboratory, and microbiological diagnostics. Antibiotic therapy was given according to European Association of Urology guidelines. Semen analysis was offered 14 days after the first presentation and initiation of therapy. Since 2013, a separate control group of 56 patients presenting with isolated hematospermia (= no other urogenital symptoms) was prospectively recruited, and differences between the groups were statistically evaluated. RESULTS: Of 324 patients with acute epididymitis, 50 patients (15%) had self-reported hematospermia. This occurred with a median of 24 h before the onset of scrotal symptoms and was associated with significantly elevated prostate-specific antigen levels compared to 274 patients without hematospermia (3.1 vs. 1.8 ng/ml, p < 0.01). The two most common etiological pathogens were Escherichia coli and Chlamydia trachomatis, and the bacterial spectrum was comparable in both epididymitis subgroups (p = 0.859). Semen analysis at 14 days still showed hematospermia in 24% of patients associated with massive leukocytospermia. Compared to the hematospermia control group, the two epididymitis subgroups showed significantly increased inflammation markers (pH, leukocytes, and elastase), reduced sperm concentration, and reduced levels of alpha-glucosidase and zinc (always p < 0.01). DISCUSSION AND CONCLUSION: In sexually active patients who develop acute epididymitis, self-reported hematospermia is evident in 15% of patients as early as one day before the onset of scrotal symptoms. Conversely, none of the 56 patients presenting with isolated hematospermia developed epididymitis within the next 4 weeks.

Bi-allelic variants in INSL3 and RXFP2 cause bilateral cryptorchidism and male infertility.

STUDY QUESTION: What is the impact of variants in the genes INSL3 (Insulin Like 3) and RXFP2 (Relaxin Family Peptide Receptor 2), respectively, on cryptorchidism and male infertility? SUMMARY ANSWER: Bi-allelic loss-of-function (LoF) variants in INSL3 and RXFP2 result in bilateral cryptorchidism and male infertility, whereas heterozygous variant carriers are phenotypically unaffected. WHAT IS KNOWN ALREADY: The small heterodimeric peptide INSL3 and its G protein-coupled receptor RXFP2 play a major role in the first step of the biphasic descent of the testes, and variants in the INSL3 and RXFP2 genes have long been implicated in inherited cryptorchidism. However, only one single homozygous missense variant in RXFP2 has clearly been linked to familial bilateral cryptorchidism, so the effects of bi-allelic variants in INSL3 and heterozygous variants in both genes on cryptorchidism and male infertility remain unclear. STUDY DESIGN, SIZE, DURATION: Exome data of 2412 men from the MERGE (Male Reproductive Genomics) study cohort including 1902 infertile men with crypto-/azoospermia, of whom 450 men had a history of cryptorchidism, were screened for high-impact variants in INSL3 and RXFP2. PARTICIPANTS/MATERIALS, SETTING, METHODS: For patients with rare, high-impact variants in INSL3 and RXFP2, detailed clinical data were collected and the testicular phenotype was determined. Genotyping of family members was performed to analyse the co-segregation of candidate variants with the condition. Immunohistochemical staining for INSL3 in patient testicular tissue and measuring serum INSL3 concentration was performed to analyse the functional impact of a homozygous loss-of-function variant in INSL3. For a homozygous missense variant in RXFP2, its impact on the protein's cell surface expression and ability to respond to INSL3 in CRE reporter gene assay was determined. MAIN RESULTS AND THE ROLE OF CHANCE: This study presents homozygous high-impact variants in INSL3 and RXFP2 and clearly correlates these to bilateral cryptorchidism. Functional impact of the identified INSL3 variant was demonstrated by absence of INSL3-specific staining in patients' testicular Leydig cells as well as undetectable blood serum levels. The identified missense variant in RXFP2 was demonstrated to lead to reduced RXFP2 surface expression and INSL3 mediated receptor activation. LIMITATIONS, REASONS FOR CAUTION: Further investigations are needed to explore a potential direct impact of bi-allelic INSL3 and RXFP2 variants on spermatogenesis. With our data, we cannot determine whether the infertility observed in our patients is a direct consequence of the disruption of a possible function of these genes on spermatogenesis or whether it occurs secondarily due to cryptorchidism. WIDER IMPLICATIONS OF THE FINDINGS: In contrast to previous assumptions, this study supports an autosomal recessive inheritance of INSL3- and RXFP2-related bilateral cryptorchidism while heterozygous LoF variants in either gene can at most be regarded as a risk factor for developing cryptorchidism. Our findings have diagnostic value for patients with familial/bilateral cryptorchidism and additionally shed light on the importance of INSL3 and RXFP2 in testicular descent and fertility. STUDY FUNDING/COMPETING INTEREST(S): This study was carried out within the frame of the German Research Foundation (DFG) funded by Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG, CRU326). Research at the Florey was supported by an NHMRC grant (2001027) and the Victorian Government Operational Infrastructure Support Program. A.S.B. is funded by the DFG ('Emmy Noether Programme' project number 464240267). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

DDX3Y is likely the key spermatogenic factor in the AZFa region that contributes to human non-obstructive azoospermia.

Non-obstructive azoospermia, the absence of sperm in the ejaculate due to disturbed spermatogenesis, represents the most severe form of male infertility. De novo microdeletions of the Y-chromosomal AZFa region are one of few well-established genetic causes for NOA and are routinely analysed in the diagnostic workup of affected men. So far, it is unclear which of the three genes located in the AZFa chromosomal region is indispensible for germ cell maturation. Here we present four different likely pathogenic loss-of-function variants in the AZFa gene DDX3Y identified by analysing exome sequencing data of more than 1,600 infertile men. Three of the patients underwent testicular sperm extraction and revealed the typical AZFa testicular Sertoli cell-only phenotype. One of the variants was proven to be de novo. Consequently, DDX3Y represents the AZFa key spermatogenic factor and screening for variants in DDX3Y should be included in the diagnostic workflow.

Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies.

BACKGROUND: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. METHODS: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines' representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. RESULTS: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. CONCLUSION: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression.

Linking human Dead end 1 (DND1) variants to male infertility employing zebrafish embryos.

STUDY QUESTION: Is the vertebrate protein Dead end (DND1) a causative factor for human infertility and can novel in vivo assays in zebrafish help in evaluating this? SUMMARY ANSWER: Combining patient genetic data with functional in vivo assays in zebrafish reveals a possible role for DND1 in human male fertility. WHAT IS KNOWN ALREADY: About 7% of the male population is affected by infertility but linking specific gene variants to the disease is challenging. The function of the DND1 protein was shown to be critical for germ cell development in several model organisms but a reliable and cost-effective method for evaluating the activity of the protein in the context of human male infertility is still missing. STUDY DESIGN, SIZE, DURATION: Exome data from 1305 men included in the Male Reproductive Genomics cohort were examined in this study. A total of 1114 of the patients showed severely impaired spermatogenesis but were otherwise healthy. Eighty-five men with intact spermatogenesis were included in the study as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: We screened the human exome data for rare, stop-gain, frameshift, splice site, as well as missense variants in DND1. The results were validated by Sanger sequencing. Immunohistochemical techniques and, when possible, segregation analyses were performed for patients with identified DND1 variants. The amino acid exchange in the human variant was mimicked at the corresponding site of the zebrafish protein. Using different aspects of germline development in live zebrafish embryos as biological assays, we examined the activity level of these DND1 protein variants. MAIN RESULTS AND THE ROLE OF CHANCE: In human exome sequencing data, we identified four heterozygous variants in DND1 (three missense and one frameshift variant) in five unrelated patients. The function of all of the variants was examined in the zebrafish and one of those was studied in more depth in this model. We demonstrate the use of zebrafish assays as a rapid and effective biological readout for evaluating the possible impact of multiple gene variants on male fertility. This in vivo approach allowed us to assess the direct impact of the variants on germ cell function in the context of the native germline. Focusing on the DND1 gene, we find that zebrafish germ cells, expressing orthologs of DND1 variants identified in infertile men, failed to arrive correctly at the position where the gonad develops and exhibited defects in cell fate maintenance. Importantly, our analysis facilitated the evaluation of single nucleotide variants, whose impact on protein function is difficult to predict, and allowed us to distinguish variants that do not affect the protein's activity from those that strongly reduce it and could thus potentially be the primary cause for the pathological condition. These aberrations in germline development resemble the testicular phenotype of azoospermic patients. LIMITATIONS, REASONS FOR CAUTION: The pipeline we present requires access to zebrafish embryos and to basic imaging equipment. The notion that the activity of the protein in the zebrafish-based assays is relevant for the human homolog is well supported by previous knowledge. Nevertheless, the human protein may differ in some respects from its homologue in zebrafish. Thus, the assay should be considered only one of the parameters used in defining DND1 variants as causative or non-causative for infertility. WIDER IMPLICATIONS OF THE FINDINGS: Using DND1 as an example, we have shown that the approach described in this study, relying on bridging between clinical findings and fundamental cell biology, can help to establish links between novel human disease candidate genes and fertility. In particular, the power of the approach we developed is manifested by the fact that it allows the identification of DND1 variants that arose de novo. The strategy presented here can be applied to different genes in other disease contexts. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the German Research Foundation, Clinical Research Unit, CRU326 'Male Germ Cells'. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Validation of the Spanish Acute Cystitis Symptoms Score (ACSS) in native Spanish-speaking women of Europe and Latin America.

INTRODUCTION AND OBJECTIVES: The Acute Cystitis Symptom Score (ACSS) is a patient self-reporting questionnaire for clinical diagnostics and patient-reported outcome (PRO), which may assess the symptoms and the effect on the quality of life in women with acute cystitis (AC). The current study aimed to create a validated Spanish version of the ACSS questionnaire. MATERIAL AND METHODS: The process of linguistic validation of the Spanish version of the ACSS consisted of the independent forward and backward translations, revision and reconciliation, and cognitive assessment. Clinical evaluation of the study version of the ACSS was carried out in clinics in Spain and Latin America. Statistical tests included the calculation of Cronbach's α, split-half reliability, specificity, sensitivity, diagnostic odds ratio, positive and negative likelihood ratio, and area under the receiver-operating characteristic curve (AUC). RESULTS: The study was performed on 132 patients [age (mean;SD) 45.0;17.8 years] with AC and 55 controls (44.5;12.2 years). Cronbach's α of the ACSS was 0.86, and the split-half reliability was 0.82. The summary scores of the ACSS domains were significantly higher in patients than in controls, 16.0 and 2.0 (p < 0.001), respectively. The predefined cut-off point of ≥6 for a summary score of the "Typical" domain resulted in a specificity of 83.6% and a sensitivity of 99.2% for the Spanish version of the ACSS. AUC was 0.91 [0.85; 0.97]. CONCLUSIONS: The validated Spanish ACSS questionnaire evaluates the symptoms and clinical outcomes of patients with AC. It can be used as a patient's self-diagnosis of AC, as a PRO measure tool, and help to rule out other pathologies in patients with voiding syndrome.