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.

Sperm proteins and cancer-testis antigens are released by the seminiferous tubules in mice and men.

Sperm develop from puberty in the seminiferous tubules, inside the blood-testis barrier to prevent their recognition as "non-self" by the immune system, and it is widely assumed that human sperm-specific proteins cannot access the circulatory or immune systems. Sperm-specific proteins aberrantly expressed in cancer, known as cancer-testis antigens (CTAs), are often pursued as cancer biomarkers and therapeutic targets based on the assumption they are neoantigens absent from the circulation in healthy men. Here, we identify a wide range of germ cell-derived and sperm-specific proteins, including multiple CTAs, that are selectively deposited by the Sertoli cells of the adult mouse and human seminiferous tubules into testicular interstitial fluid (TIF) that is "outside" the blood-testis barrier. From TIF, the proteins can access the circulatory- and immune systems. Disruption of spermatogenesis decreases the abundance of these proteins in mouse TIF, and a sperm-specific CTA is significantly decreased in TIF from infertile men, suggesting that exposure of certain CTAs to the immune system could depend on fertility status. The results provide a rationale for the development of blood-based tests useful in the management of male infertility and indicate CTA candidates for cancer immunotherapy and biomarker development that could show sex-specific and male-fertility-related responses.

Muscarinic receptors 2 and 5 regulate bitter response of urethral brush cells via negative feedback.

We have recently identified a cholinergic chemosensory cell in the urethral epithelium, urethral brush cell (UBC), that, upon stimulation with bitter or bacterial substances, initiates a reflex detrusor activation. Here, we elucidated cholinergic mechanisms that modulate UBC responsiveness. We analyzed muscarinic acetylcholine receptor (M1-5 mAChR) expression by using RT-PCR in UBCs, recorded [Ca2+]i responses to a bitter stimulus in isolated UBCs of wild-type and mAChR-deficient mice, and performed cystometry in all involved strains. The bitter response of UBCs was enhanced by global cholinergic and selective M2 inhibition, diminished by positive allosteric modulation of M5, and unaffected by M1, M3, and M4 mAChR inhibitors. This effect was not observed in M2 and M5 mAChR-deficient mice. In cystometry, M5 mAChR-deficient mice demonstrated signs of detrusor overactivity. In conclusion, M2 and M5 mAChRs attenuate the bitter response of UBC via a cholinergic negative autocrine feedback mechanism. Cystometry suggests that dysfunction, particularly of the M5 receptor, may lead to such symptoms as bladder overactivity.-Deckmann, K., Rafiq, A., Erdmann, C., Illig, C., Durschnabel, M., Wess, J., Weidner, W., Bschleipfer, T., Kummer, W. Muscarinic receptors 2 and 5 regulate bitter response of urethral brush cells via negative feedback.

High prevalence of urogenital infection/inflammation in patients with azoospermia does not impede surgical sperm retrieval.

Considering infection/inflammation to be an important risk factor in male infertility, the aim of this study was to make a comprehensive evaluation of the prevalence of urogenital tract infection/inflammation and its potential impact on sperm retrieval in azoospermic patients. In this prospective study, 71 patients with azoospermia were subjected to an extensive andrological workup including comprehensive microbiological diagnostics (2-glass test, semen, testicular swab and testicular tissue analysis) and testicular biopsy/testicular sperm extraction (TESE). Medical history suggested urogenital tract infection/inflammation in 7% of patients, 11% harboured STIs, 14% showed significant bacteriospermia, 15% had seminal inflammation, 17% fulfilled the MAGI definition, and 27% had relevant pathogens. At the testicular level, 1 patient had a swab positive for bacteria, no viruses were detected, tissue specimens never indicated pathogens, whereas histopathology revealed focal immune cell infiltrates in 23% of samples. Testicular sperm retrieval rate was 100% in obstructive and 46% in nonobstructive azoospermia. None of the infection/inflammation-related variables was associated with the success of sperm retrieval or inflammatory lesions in the testis. The high prevalence of urogenital infection/inflammation among azoospermic men underpins their role as significant aetiologic factors in male infertility. However, this observation does not refer to the chances of sperm retrieval at the time of surgery/TESE.

Prospective evaluation of scrotal ultrasound and intratesticular perfusion by color-coded duplex sonography (CCDS) in TESE patients with azoospermia.

PURPOSE: The objective of this study was to assess whether CCDS might improve the outcome of testicular sperm retrieval in patients with azoospermia. Furthermore, we evaluated potential sonographic alterations of the testis before and after trifocal and Micro-TESE. METHODS: 78 patients were enrolled prospectively: 24 with obstructive azoospermia (OA) and 54 with non-obstructive azoospermia (NOA). 31 of 54 patients in the NOA group had negative surgical sperm retrieval. Testicular volume, hormonal parameters and sonographical findings were compared before and after TESE. The spermatogenetic score was determined for all retrieval sites. CCDS was performed at the upper, middle and lower segment of the testis. Ultrasound parameters and peak systolic velocity (PSV) were measured pre- and post-operatively. RESULTS: Testicular volume and epididymal head size were significantly increased in OA patients compared to NOA patients. Ultrasound parameters were comparable between NOA patients with and without successful sperm retrieval. A higher intratesticular PSV was significantly correlated with a better spermatogenic score in the corresponding sonographic position. However, after adjustment for other clinical confounders, PSV does not show a significant influence on the spermatogenic score. Testicular volume decreased significantly in all patients post-operatively after 6 weeks (p < 0.001). Finally, the PSV significantly increased in all patients 24 h after surgery and nearly returned to baseline levels after 6 weeks (p < 0.001). CONCLUSIONS: A higher intratesticular PSV may be helpful as a pre-operative diagnostic parameter in mapping for better sperm retrieval, but CCDS does not help to predict successful testicular sperm retrieval after adjustment for other clinical confounders.

Chronic Prostatitis Affects Male Reproductive Health and Is Associated with Systemic and Local Epigenetic Inactivation of C-X-C Motif Chemokine 12 Receptor C-X-C Chemokine Receptor Type 4.

Background/Aims/Objectives: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has detrimental effects on the quality of life including the aspect of sexual dysfunction. The aim of the study was to identify if there was an adverse effect on the male genital compartment and if there are systemic or compartment-specific local signals for epigenetic dysregulation of inflammatory factors in CP/CPPS patients. METHODS: One hundred five NIH IIIb CP/CPPS patients and 41 healthy men were recruited and underwent investigations of urines, semen and blood. Promoter methylation and expression of the chemokine C-X-C motif chemokine 12 and its receptor C-X-C chemokine receptor type 4 (CXCR4) (involved in the recruitment of mast cells) were analyzed in prostate epithelial cell lines and in healthy volunteers' and patients' blood, ejaculate cell pellets, and separated ejaculate fractions (sperm and seminal somatic cells). RESULTS: Independently from age, CP/CPPS NIH IIIb was associated with significant impairment of sperm motility, morphology and semen pH (p < 0.001). Patients older than 33 years showed significantly increased seminal interleukin-8 and serum prostate specific antigen values. In patients, the CXCR4 mRNA-expression was significantly decreased in whole blood and ejaculate cell pellets due to promoter hypermethylation. Analyses on separated fractions of sperm and seminal somatic cells revealed that sperm DNA was unaffected, whereas somatic cell DNA was differentially methylated. CONCLUSIONS: NIH IIIb CP/CPPS has negative effects on surrogate parameters of male fertility and is associated significantly with systemic and local epigenetic inactivation of CXCR4.

Mapping the testicular interstitial fluid proteome from normal rats.

Communication between the testicular somatic (Sertoli, Leydig, peritubular myoid, macrophage) and germ cell types is essential for sperm production (spermatogenesis), but the communicating factors are poorly understood. We reasoned that identification of proteins in the testicular interstitial fluid (TIF) that bathes these cells could provide a new means to explore spermatogenic function. The aim of this study was to map the proteome of TIF from normal adult rats. Low-abundance proteins in TIF were enriched using ProteoMiner beads and identified by MALDI-MS/MS, recognizing 276 proteins. Comparison with proteomic and genomic databases showed these proteins originated from germ cells, somatic cells (Sertoli, peritubular myoid, Leydig), and blood plasma. In silico analysis revealed homologues of >80% TIF proteins in the human plasma proteome, suggesting ready exchange between these fluids. Only 36% of TIF proteins were common with seminiferous tubule fluid that transports mature spermatids to the epididymis, indicating these two fluids are quite different. This TIF proteome provides an important new resource for the study of intercellular communication in the testis.

Murine and Human Spermatids Are Characterized by Numerous, Newly Synthesized and Differentially Expressed Transcription Factors and Bromodomain-Containing Proteins.

Much of spermatid differentiation takes place in the absence of active transcription, but in the early phase, large amounts of mRNA are synthesized, translationally repressed, and stored. Most nucleosomal histones are then degraded, and chromatin is repackaged by protamines. For both transcription and the histone-to-protamine transition in differentiating spermatids, chromatin must be opened. This raises the question of whether two different processes exist. It is conceivable that for initiation of the histone-to-protamine transition, the already accessible, actively transcribed chromatin regions are utilized or vice versa. We analyzed the enrichment of different canonical TATA-box-binding, protein-associated factors and their variants in murine spermatids, diverse bromodomain-containing proteins, and components of the Polycomb repressive complexes PRC1 and PRC2 using quantitative PCR. We compared the enrichment of corresponding proteins in human and murine spermatids and analyzed the time frame of postmeiotic transcription and expression of histones, transition proteins, and protamines in human and murine spermatids using immunohistology. We correlated the expression of different transcription factors and bromodomain-containing proteins and the pattern of acetylated histones to active transcription and to the histone-to-protamine transition in both human and murine spermatids. Our findings suggest that differentiating spermatids use both common and specific features to open chromatin first for transcription and subsequently for histone-to-protamine transition.

TET enzymes are successively expressed during human spermatogenesis and their expression level is pivotal for male fertility.

STUDY QUESTION: Are ten-eleven-translocation (TET) 1-3 family enzymes involved in human spermatogenesis and do they impact male fertility? SUMMARY ANSWER: TET1, TET2 and TET3 are successively expressed at different stages of human spermatogenesis, and their expression levels associate with male fertility. WHAT IS KNOWN ALREADY: Spermatogenesis is a complex cell differentiation process accompanied by a drastic epigenetic remodeling. TET1-3 dioxygenases are essential for active DNA demethylation in the paternal pronucleus and in embryonic stem cells. STUDY DESIGN, SIZE, DURATION: Expression of TET1-3 mRNAs and proteinss and 5-hydroxymethylcytosine (5-hmC) proteins were analyzed in human testis tissues from men with obstructive azoospermia and exhibiting histologically normal spermatogenesis. Ejaculated spermatozoa from normozoospermic healthy volunteers, the 'controls' (TET1: n = 58; TET2-3: n = 63), and subfertile men who participated with their female partners in an ICSI-program, the 'patients' (TET1: n = 66; TET2-3: n = 64), were analyzed concerning the stored TET1-3 mRNAs, and the values were correlated to semen parameters and ICSI-outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis sections were used for in situ hybridization (ISH) and immunohistochemical (IHC) studies to determine TET1-3 mRNA and protein expression, and for immunofluorescence (IF) detection of 5-hmC. Sperm samples from controls were analyzed by western blot, immunocytochemistry (ICC) and RT-PCR concerning the presence of non-degraded TET1-3 protein and mRNA. Sperm samples from controls and patients were used for quantitative TET1-3 mRNA analyses (reverse transcription-polymerase chain reaction) and for comparative statistical evaluations under consideration of semen parameters and ICSI-outcome (pregnancy). MAIN RESULTS AND THE ROLE OF CHANCE: During human spermatogenesis TET1-3 proteins are successively expressed: TET2 is expressed in the cytoplasm of late pachytene spermatocytes of Stage V, TET1 starts to be expressed in the nuclei of Step 1 round spermatids at Stage I, and TET3 starts to be expressed in the nuclei of Step 3 round spermatids at Stage III. Five-hmC appears only in Step 5 elongated spermatids. All three TETs are still detectable at the mRNA and protein level in sperm cells in considerable amounts. Control men generally exhibited higher levels of TET1-3 in sperm. TET1- and TET3-mRNA levels in sperm were significantly negatively correlated with age (P = 0.0025 and P = 0.0343) and positively correlated with progressive sperm motility (P = 0.0007 and P = 0.018). All TETs showed a significant association with sperm concentration (P < 0.03). Patients diagnosed with oligozoospermia and/or asthenozoospermia (TET1: n = 35; TET2-3: n = 32) showed significantly reduced TET1-3 in sperm in comparison to controls (P = 0.003, P = 0.041 and P = 0.028), but not compared with normozoospermic patients. Levels of TET3 in sperm was significantly associated with high-fertilization rates (P = 0.009). Concerning ICSI-outcome, the lowest levels of TET1-3 mRNAs in sperm were found in the non-pregnant group. Increased TET2 in sperm was significantly associated with pregnancy (P = 0.006). LIMITATIONS, REASONS FOR CAUTION: Our results concerning the association of the mRNA level of TETs in ejaculated sperm cells to different fertility parameters are descriptive. Further studies clarifying the reasons for decreased TET1-3 levels in subfertile men and their effect on their sperm methylome are essential. WIDER IMPLICATIONS OF THE FINDINGS: The study gives a substantial indication that in human spermiogenesis, an active DNA demethylation process occurs with an involvement of TET enzymes, and that the level of TET1-3 expression is pivotal for male fertility. STUDY FUNDING: Research grant from the German Research Foundation (DFG) to U.S. (SCHA1531/1-1 and SCHA1531/2-1). COMPETING INTERESTS: None.

Specific immune cell and cytokine characteristics of human testicular germ cell neoplasia.

STUDY QUESTION: Which immune cells and cytokine profiles are characteristic for testicular germ cell neoplasia and what consequences does this have for the understanding of the related testicular immunopathology? SUMMARY ANSWER: The unique immune environment of testicular germ cell neoplasia comprises B cells and dendritic cells as well as high transcript levels of IL-6 and other B cell supporting or T helper cell type 1 (Th1)-driven cytokines and thus differs profoundly from normal testis or inflammatory lesions associated with hypospermatogenesis. WHAT IS KNOWN ALREADY: T cells are known to be the major component of inflammatory infiltrates associated with either hypospermatogenesis or testicular cancer. It has previously been reported that B cells are only involved within infiltrates of seminoma samples, but this has not been investigated further. STUDY DESIGN, SIZE, DURATION: Immunohistochemical characterisation (IHC) of infiltrating immune cells and RT-qPCR-based analysis of corresponding cytokine microenvironments was performed on different testicular pathologies. Testicular biopsies, obtained from men undergoing andrological work-up of infertility or taken during surgery for testicular cancer, were used in this study. Samples were grouped as follows: (i) normal spermatogenesis (n = 18), (ii) hypospermatogenesis associated with lymphocytic infiltrates (n = 10), (iii) samples showing neoplasia [germ cell neoplasia in situ (GCNIS, n = 26) and seminoma, n = 18]. PARTICIPANTS/MATERIALS, SETTING, METHODS: IHC was performed using antibodies against T cells (CD3+), B cells (CD20cy+), dendritic cells (CD11c+), macrophages (CD68+) and mast cells (mast cell tryptase+). Degree and compartmental localisation of immune cells throughout all groups analysed was evaluated semi-quantitatively. RT-qPCR on RNA extracted from cryo-preserved tissue samples was performed to analyse mRNA cytokine expression, specifically levels of IL-1ß, IL-6, IL-17a, tumour necrosis factor (TNF)-α (pro-inflammatory), IL-10, transforming growth factor (TGF)-ß1 (anti-inflammatory), IL-2, IL-12a, IL-12b, interferon (IFN)-γ (Th1-driven), IL-4, IL-5, IL-13, IL-23a (Th2-driven), CXCL-13, CXCL-10 and CCL-5 (chemokines). MAIN RESULTS AND THE ROLE OF CHANCE: This is the first study showing a direct linkage between the distribution pattern of immune cells in hypospermatogenesis versus testicular cancer and analysis of a wide range of 17 related cyto- and chemokines. A fundamental difference between testicular inflammation patterns associated with different testicular inflammatory conditions either containing or lacking neoplastic cells was demonstrated. In hypospermatogenesis, T cells were detected, whereas B cells and dendritic cells were almost absent. Within GCNIS and seminoma, in addition to T cells, high numbers of dendritic cells and B cells were found, the latter additionally organised in cell clusters, whereas mast cells were absent. Transcripts encoding pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α), anti-inflammatory cytokines (TGF-ß1), Th1-driven cytokines (IL-2 and IFN-γ) as well as chemokines (CXCL-13, CXCL-10 and CCL-5) were all significantly increased in testicular germ cell neoplasia (P ≤ 0.01), suggesting the presence of a pro-tumorigenic environment. In contrast, Th2-related cytokines (IL-5, IL-13 and IL-23a) characterised the environment within samples showing normal spermatogenesis as well as hypospermatogenesis. One of the most important outcomes is the pivotal role of IL-6 in testicular cancer that opens potential novel diagnostic and/or immune-therapeutic perspective for testis cancer. LIMITATIONS, REASONS FOR CAUTION: Testicular tissue composed of immune cells as well as other somatic cells and germ cells does not allow identification of specific cytokine sources or single cell types, being responsible for establishing the overall cytokine environment. In this study, laser-assisted microdissection did not reach the required efficiency for RT-qPCR analyses. Therefore, in vitro models would be suggested for addressing the above-mentioned issue. Conclusions about cytokine levels in testes with GCNIS are based on a small number of samples. WIDER IMPLICATIONS OF THE FINDINGS: The unique B cell presence and the significantly increased expression level of IL-6 in testicular germ cell neoplasia (P < 0.001) strengthen its special role in this disease. In line with current knowledge on other types of cancer, these results underline the relevance of further investigating the potential of IL-6 as early biomarker and target for therapeutic intervention in testicular germ cell neoplasia. STUDY FUNDING/COMPETING INTERESTS: This study (and B.K. in person) was supported by the Deutsche Forschungsgemeinschaft (DFG) as part of the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) on 'Molecular pathogenesis on male reproductive disorders'. T.H., H.-C.S. and M.B. were supported by the LOEWE focus group 'MIBIE' (male infertility during infection & inflammation)-an excellence initiative of the German state government of Hessen. From the Australian side, K.L. was supported by NHMRC grants (Fellowship, ID1079646 and Project, ID1081987); K.L., S.I. and M.H. received scholarship (S.I.) and research funding (K.L., M.H.) from Monash University. The project also drew support from the Victorian Government's Operational Infrastructure Support Program. The authors have no competing interests to declare.

On the relationship between tumor structure and complexity of the spatial distribution of cancer cell nuclei: a fractal geometrical model of prostate carcinoma.

BACKGROUND: A risk of the prostate cancer patient is defined by both the objective and subjective criteria, that is, PSA concentration, Gleason score, and pTNM-stage. The subjectivity of tumor grading influences the risk assessment owing to a large inter- and intra-observer variability. Pathologists propose a central prostate pathology review as a remedy for this problem; yet, the review cannot eliminate the subjectivity from the diagnostic algorithm. The spatial distribution of cancer cell nuclei changes during tumor progression. It implies changes in complexity measured by the capacity dimension D0, the information dimension D1, and the correlation dimension D2. METHODS: The cornerstone of the approach is a model of prostate carcinomas composed of the circular fractals CF(4), CF(6 + 0), and CF(6 + 1). This model is both geometrical and analytical, that is, its structure is well-defined, the capacity fractal dimension D0 can be calculated for the infinite circular fractals, and the dimensions D0, D1, D2 can be computed for their finite counterparts representing distribution of cell nuclei. The model enabled both the calibration of the software and the validation of the measurements in 124 prostate carcinomas. The ROC analysis defined the cut-off D0 values for seven classes of complexity. RESULTS: The Gleason classification matched in part with the classification based on the D0 values. The mean ROC sensitivity was 81.3% and the mean ROC specificity 75.2%. Prostate carcinomas were re-stratified into seven classes of complexity according to their D0 values. This increased both the mean ROC sensitivity and the mean ROC specificity to 100%. All homogeneous Gleason patterns were subordinated to the class C1, C4, or C7. D0 = 1.5820 was the cut-off D0 value between the complexity class C2 and C3 representing low-risk cancers and intermediate-risk cancers, respectively. CONCLUSIONS: The global fractal dimensions eliminate the subjectivity in the diagnostic algorithm of prostate cancer. Those complexity measures enable the objective subordination of carcinomas to the well-defined complexity classes, and define subgroups of carcinomas with very low malignant potential (complexity class C1) or at a large risk of progression (complexity ass C7).

Expression of sperm-specific protamines impairs bacterial and eukaryotic cell proliferation.

Protamines are the predominant nuclear proteins in testicular spermatids and ejaculated spermatozoa. During spermiogenesis, protamine-DNA interaction induces a higher-order chromatin packaging which finally results in a complete transcriptional stop in elongating spermatids. Although numerous studies investigated the role of protamines in male fertility, to date, no study is available that investigates protamine function, particularly transcriptional silencing, in non-germ cells. Transcriptional stop due to the high binding affinity of arginine-rich protamines to the negatively charged DNA backbone, however, may be induced in somatic cells and may result in suppressing cell division in tumor cells. In the present study, we therefore analyzed whether a protamine-mediated chromatin condensation in somatic cancer cell lines can stop gene expression and arrest cancer cell proliferation. In contrast to terminally differentiated sperm, cancer cells represent immortalized cells that have modulated natural mechanisms for the regulation of apoptosis and cell proliferation. We expressed human protamines in two fast-growing cell systems, E. coli and HeLa cells. In both cases, protamine expression significantly attenuated cell proliferation when compared with control cells. To our knowledge, this is the first study that demonstrates a stop of cell proliferation in both E. coli and HeLa cells by protamine expression. Follow-up studies on the molecular effect of protamines on proliferative cells may, in the future, open new avenues to investigate effective and specific treatments of cancer cells.

Microsurgical Spermatic Cord Denervation as a Treatment for Chronic Scrotal Content Pain: A Multicenter Open Label Trial.

PURPOSE: We prospectively evaluated the results of microsurgical spermatic cord denervation in a series of patients with chronic scrotal content pain in a multicenter study, including 1 center in Germany and 3 centers in Chile. MATERIALS AND METHODS: A total of 50 patients with chronic scrotal content pain more than 3 months in duration were prospectively selected for standardized operative microsurgical spermatic cord denervation as pain treatment. In all patients preoperative management included a positive response to a spermatic cord block test with local anesthesia. Pain severity was assessed using an analog visual pain scale (range 0 to 10) for 30 consecutive days. A total of 52 testicular units were operated on using a subinguinal approach. In all cases a surgical microscope was used to identify the arteria testicularis. RESULTS: No intraoperative complications were observed and no testicular units were lost. Two reoperations were performed, including 1 for hematocele and 1 for hydrocele. Six months after surgery 40 patients (80%) were completely pain-free. In 6 patients (12%) intermittent testicular discomfort persisted, which could be managed by acetaminophen on demand. Four patients (8%) had no change in pain severity after surgery. CONCLUSIONS: After proper selection of patients microsurgical spermatic cord denervation seems to be a safe and efficient procedure to treat chronic scrotal content pain. Considering the limitations of the study, a randomized, controlled trial with longer followup is highly warranted.

Systemic atherosclerosis causes detrusor overactivity: functional and morphological changes in hyperlipoproteinemic apoE-/-LDLR-/- mice.

PURPOSE: The prevalence of systemic atherosclerosis and overactive bladder/detrusor overactivity increases almost simultaneously with age but an association between these diseases has not yet been proved. We evaluated changes in bladder function and morphology, including vascularization, in apoE(-/-)LDLR(-/-) double knockout mice with systemic atherosclerosis but without central nervous system involvement. MATERIALS AND METHODS: Cystometry was performed in awake, freely moving 60-week-old apoE(-/-)LDLR(-/-) mice and C57BL/6N controls. The mice were sacrificed and perfused with Microfil® contrast medium. The bladder was excised, dissected and scanned by nano-computerized tomography, including 3-dimensional reconstruction. Samples then underwent histomorphological analysis. RESULTS: In apoE(-/-)LDLR(-/-) mice cystometry revealed a significant decrease in the peak-peak interval, micturition interval, functional bladder capacity and micturition volume. However, maximum bladder pressure increased. Nano-computerized tomography revealed a significant reduction in bladder wall thickness, segment volume, vascular volume and the vascular volume fraction. Histomorphologically bladder specimens showed a thickened media of intramural vessels, activated endothelial cells and intramural inflammatory cells. CONCLUSIONS: To our knowledge this study presents a new in vivo mouse model of nonneurogenic detrusor overactivity caused by systemic atherosclerosis. Decreased bladder wall vascularization seems to be a major factor for detrusor overactivity onset. Capillaries are rarified with reduced lumina due to thickened media. Activated endothelial cells and the infiltration of inflammatory cells in apoE(-/-)LDLR(-/-) mice underlines once more that atherosclerosis is an inflammatory process that may also be relevant to the onset of detrusor overactivity.

A Randomized, Double-Blind, Parallel-Group, Multicenter Clinical Study of Escherichia coli-Lyophilized Lysate for the Prophylaxis of Recurrent Uncomplicated Urinary Tract Infections.

BACKGROUND: One strategy for managing recurrent uncomplicated urinary tract infections (UTIs) is prevention. This study tested OM-89S, a lyophilized lysate of 18 Escherichia coli strains manufactured using a modified lytic process. METHODS: This was a randomized, double-blind trial in 451 female subjects with recurrent uncomplicated UTIs. Period 1 of the study tested 6 mg of OM-89S versus placebo (3 months), plus a 3-month observation. Period 2 of the study was a 3-month treatment period (each monthly cycle consisted of 6 mg of OM-89S daily for 10 days and placebo for 20 days, vs. 50 mg nitrofurantoin daily for 30 days), plus a 3-month observation. RESULTS: There was no difference in the mean rate of UTI episodes between the OM-89S (0.66 ± 0.93) and placebo groups (0.63 ± 0.86; p = 0.95) in period 1. Similar findings were obtained for period 2. OM-89S was well-tolerated. CONCLUSIONS: Our results did not demonstrate a preventive effect of OM-89S compared to placebo. This may be due to the low number of UTIs that occurred during the study, the high number of protocol violations, and/or the modified manufacturing process used for OM-89S.

Urinary concentrations and antibacterial activities of nitroxoline at 250 milligrams versus trimethoprim at 200 milligrams against uropathogens in healthy volunteers.

Because of the increasing bacterial resistance of uropathogens against standard antibiotics, such as trimethoprim (TMP), older antimicrobial drugs, such as nitroxoline (NTX), should be reevaluated. This randomized crossover study investigated the urinary concentrations of parent drugs and their metabolites and their antibacterial activities (urinary inhibitory titers [UITs] and urinary bactericidal titers [UBTs]) against uropathogens at three different urinary pH values within 24 h in six healthy volunteers after a single oral dose of NTX at 250 mg versus TMP at 200 mg. In three additional volunteers, urinary bactericidal kinetics (UBK) were studied after oral administration of NTX at 250 mg three times a day. The mean urinary concentrations of NTX and NTX sulfate in 24 h were 0.012 to 0.507 mg/liter and 0.28 to 27.83 mg/liter, respectively. The mean urinary concentrations of TMP were 18.79 to 41.59 mg/liter. The antibacterial activity of NTX was higher in acidic urine than in alkaline urine, and that of TMP was higher in alkaline urine than in acidic urine. The UITs and UBTs of NTX were generally lower than those of TMP except for a TMP-resistant Escherichia coli strain, for which NTX showed higher UITs/UBTs than did TMP. UBK showed mainly bacteriostatic activity of NTX in urine. NTX exhibits mainly bacteriostatic activity and TMP also shows bactericidal activity in urine against susceptible strains. NTX is a more active antibacterial in acidic urine, and TMP is more active in alkaline urine. The cumulative effects of multiple doses or inhibition of bacterial adherence could not be evaluated. (This study has been registered at EudraCT under registration no. 2009-015631-32.).

Urinary tract infections and bacterial prostatitis in men.

PURPOSE OF REVIEW: The purpose of this review is to highlight advances in research on urinary tract infections (UTIs) and bacterial prostatitis in men in the preceding year. RECENT FINDINGS: The antiseptic properties of the prostate secretions might be an important factor for prevention of recurrency. Risk factors for UTI in men include prostate enlargement and urological interventions, such as transrectal prostate biopsy. Preventive treatment of prostate enlargement has been demonstrated to reduce frequency of UTI. Ensuing infections after prostate biopsy, such as UTI and bacterial prostatitis, are increasing due to increasing rates of fluoroquinolone resistance. The increasing global antibiotic resistance also significantly affects management of UTI in men, and therefore calls for alternative strategies.Apart from prevention of complicating factors leading to UTI, a more thorough understanding of the pathophysiology may play a more important role in the future, to define new targets for treatment. Interesting results that might interfere with the intracellular mucosal bacterial load in the bladder wall have been found in the last years. SUMMARY: UTI in men and bacterial prostatitis are currently underrepresented in the medical literature. Increasing antibacterial resistance calls for novel strategies in the prevention and management of UTI and bacterial prostatitis in men.

Expression pattern of estrogen receptors α and ß and G-protein-coupled estrogen receptor 1 in the human testis.

Estrogen signaling is considered to play an important role in spermatogenesis, spermiogenesis and male fertility. Estrogens can act via the two nuclear estrogen receptors ESR1 (ERα) and ESR2 (ERß) or via the intracellular G-protein-coupled estrogen receptor 1 (GPER, formerly GPR30). Several reports on the localization and expression of all three receptors in the human testis have been published but are controversial particularly in case of ERα. Contrary to previous studies, we decided therefore to evaluate expression of all three receptors in the testis by a number of different methods and in comparison with MCF-7 cells. Using qPCR, we could show that mRNA expression of ERα is considerably lower and expression of ERß and GPER much higher in the testis than in MCF-7 cells. RT-PCR after laser-assisted microdissection of tubular and interstitial compartments from normal and Sertoli cell only syndrome testes plus in situ hybridization and immunohistochemical analyses of the same samples demonstrated that there is very low expression of ERα in germ cells and in single interstitial cells, very high expression of ERß in germ cells and Sertoli cells and high expression of GPER in interstitial cells and less in Sertoli cells.

Autoantibodies against protein disulfide isomerase ER-60 are a diagnostic marker for low-grade testicular inflammation.

STUDY QUESTION: Is there a non-invasive biomarker for the diagnosis of testicular inflammatory lesions? SUMMARY ANSWER: In sera from infertile azoospermic patients with histologically confirmed low-grade testicular inflammation, significantly elevated titers of autoantibodies against disulfide isomerase family A, member 3 (ER-60) were found. WHAT IS KNOWN ALREADY: Infection and inflammation of the genital tract are supposed to be responsible for up to 15% of cases among infertile males. However, specific seminal or serological markers are not available to assess subacute or chronic inflammatory conditions in the testis. STUDY DESIGN, SIZE, DURATION: This study consisted of the identification of autoantibodies for testicular antigens in sera of patients with low-grade testicular inflammation, validation of candidates, development of an ELISA for the most promising target antigen and measurement of autoantibodies titers in healthy normozoospermic men (n = 20); male blood donors (n = 14); men with impaired semen quality without (n = 14) or with (n = 26) symptoms of genital tract infection/inflammation; azoospermic men with histologically confirmed testicular inflammatory lesions (n = 16); men after pharmacotherapy of genital tract infection/inflammation (n = 15) and men with acute epididymo-orchitis (n = 30). PARTICIPANTS/MATERIALS, SETTING, METHODS: Proteins in lysates of normal testicular tissue were separated by high-resolution 2D gel electrophoresis and probed with sera of 13 patients with histologically confirmed chronic testicular inflammation. There were 14 proteins that immunoreacted with a majority of these sera and could be identified by mass spectrometry. Of these 14 proteins, disulfide isomerase family A, member 3 (ER-60), transferrin and chaperonin containing TCP1 complex, subunit 5 (epsilon) (CCT5) were considered as specific. Since ER-60 reacted with 92% of patient sera, an ER-60-autoantibody ELISA was developed. MAIN RESULTS AND THE ROLE OF CHANCE: The newly established ELISA detected significantly elevated titers of autoantibodies against ER-60 in the sera from infertile men with histologically confirmed chronic testicular inflammation (median 8.6; P < 0.01) compared with the control groups. Moreover, elevated levels of anti-ER-60 titers were detected in patients suffering from acute epididymo-orchitis (median 3.3; P < 0.05) as compared with healthy normozoospermic men (median 2.13; P < 0.001), male blood donors with unknown fertility status (median 2.72; P < 0.01), patients with impaired semen quality but no infection/inflammation (median 2.59; P < 0.001) and patients with symptoms of genital tract infections and/or inflammation (median 2.18; P < 0.001). Significantly lower levels of anti-ER-60 antibodies were measured in sera from patients after application of anti-inflammatory pharmacotherapy (median 1.9; P < 0.01) compared with those with histologically confirmed chronic testicular inflammation. The cut-off value of the assay was set to 6.6 U/ml based on a calculated sensitivity of 100% and a specificity of 81.2%. LIMITATIONS, REASONS FOR CAUTION: The results obtained in this study showed statistically significant elevated titers of ER-60 antibodies in sera from patients with histologically confirmed testicular inflammatory lesions and from a few patients with acute epididymo-orchitis. However, the number of serum samples tested was limited. Severe testicular damage seen in azoospermic patients could represent a bias towards ER-60 reactivity, while the assay does not allow for different etiologies of the lesions to be distinguished. Due to ethical reasons, the prevalence of testicular inflammatory lesions among controls and non-azoospermic men cannot be studied at the histological level. WIDER IMPLICATIONS OF THE FINDINGS: Measurement of ER-60 autoantibody titers in serum could be a novel non-invasive marker for the diagnosis of asymptomatic testicular inflammation causing male fertility disturbances. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a grant of the Deutsche Forschungsgemeinschaft (ME 1323/4-4) and the Translational Science Fund (Wirtschafts-und Strukturbank Hessen-WI Bank). M.F., A.P., W.W., H.-C.S. and A.M. are supported by the LOEWE focus group 'MIBIE' (Male infertility during infection and inflammation). The ER-60 ELISA is protected by a patent to the Justus-Liebig-University of Giessen with A.M. and M.F. as inventors (patent no. DE 10 2008 053 503). T.Z. as employee of the DRG Company was responsible for the ELISA development.

The Integral Theory System Questionnaire: an anatomically directed questionnaire to determine pelvic floor dysfunctions in women.

PURPOSE: Anatomical damage to pelvic floor structures may cause multiple symptoms. The Integral Theory System Questionnaire (ITSQ) is a holistic questionnaire that uses symptoms to help locate damage in specific connective tissue structures as a guide to reconstructive surgery. It is based on the integral theory, which states that pelvic floor symptoms and prolapse are both caused by lax suspensory ligaments. The aim of the present study was to psychometrically validate the ITSQ. MATERIALS AND METHODS: Established psychometric properties including validity, reliability, and responsiveness were considered for evaluation. Criterion validity was assessed in a cohort of 110 women with pelvic floor dysfunctions by analyzing the correlation of questionnaire responses with objective clinical data. Test-retest was performed with questionnaires from 47 patients. Cronbach's alpha and "split-half" reliability coefficients were calculated for inner consistency analysis. RESULTS: Psychometric properties of ITSQ were comparable to the ones of previously validated Pelvic Floor Questionnaires. Face validity and content validity were approved by an expert group of the International Collaboration of Pelvic Floor surgeons. Convergent validity assessed using Bayesian method was at least as accurate as the expert assessment of anatomical defects. Objective data measurement in patients demonstrated significant correlations with ITSQ domains fulfilling criterion validity. Internal consistency values ranked from 0.85 to 0.89 in different scenarios. CONCLUSIONS: The ITSQ proofed accurate and is able to serve as a holistic Pelvic Floor Questionnaire directing symptoms to site-specific pelvic floor reconstructive surgery.