Human prostate organoid generation and the identification of prostate development drivers using inductive rodent tissues.

The reactivation of developmental genes and pathways during adulthood may contribute to pathogenesis of diseases such as prostate cancer. Analysis of the mechanistic links between development and disease could be exploited to identify signalling pathways leading to disease in the prostate. However, the mechanisms underpinning prostate development require further characterisation to interrogate fully the link between development and disease. Previously, our group developed methods to produce prostate organoids using induced pluripotent stem cells (iPSCs). Here, we show that human iPSCs can be differentiated into prostate organoids using neonatal rat seminal vesicle mesenchyme in vitro. The organoids can be used to study prostate development or modified to study prostate cancer. We also elucidated molecular drivers of prostate induction through RNA-sequencing analyses of the rat urogenital sinus and neonatal seminal vesicles. We identified candidate drivers of prostate development evident in the inductive mesenchyme and epithelium involved with prostate specification. Our top candidates included Spx, Trib3, Snai1, Snai2, Nrg2 and Lrp4. This work lays the foundations for further interrogation of the reactivation of developmental genes in adulthood, leading to prostate disease.

In sickness and in health: The functional role of extracellular vesicles in physiology and pathology in vivo: Part I: Health and Normal Physiology: Part I: Health and Normal Physiology.

Previously thought to be nothing more than cellular debris, extracellular vesicles (EVs) are now known to mediate physiological and pathological functions throughout the body. We now understand more about their capacity to transfer nucleic acids and proteins between distant organs, the interaction of their surface proteins with target cells, and the role of vesicle-bound lipids in health and disease. To date, most observations have been made in reductionist cell culture systems, or as snapshots from patient cohorts. The heterogenous population of vesicles produced in vivo likely act in concert to mediate both beneficial and detrimental effects. EVs play crucial roles in both the pathogenesis of diseases, from cancer to neurodegenerative disease, as well as in the maintenance of system and organ homeostasis. This two-part review draws on the expertise of researchers working in the field of EV biology and aims to cover the functional role of EVs in physiology and pathology. Part I will outline the role of EVs in normal physiology.

Telocytes of the male urogenital system: Interrelationships, possible functions, and pathological implications.

The male urogenital system is composed of the reproductive system and the urinary tract; they have an interconnected embryonic development and share one of their anatomical components, the urethra. This system has a highly complex physiology deeply interconnected with the circulatory and nervous systems, as well as being capable of adapting to environmental variations; it also undergoes changes with aging and, in the case of the reproductive system, with seasonality. The stroma is an essential component in this physiological plasticity and its complexity has increased with the description in the last decade of a new cell type, the telocyte. Several studies have demonstrated the presence of telocytes in the organs of the male urogenital system and other systems; however, their exact function is not yet known. The present review addresses current knowledge about telocytes in the urogenital system in terms of their locations, interrelationships, possible functions and pathological implications. It has been found that telocytes in the urogenital system possibly have a leading role in stromal tissue organization/maintenance, in addition to participation in stem cell niches and an association with the immune system, as well as specific functions in the urogenital system, lipid synthesis in the testes, erythropoiesis in the kidneys and the micturition reflex in the bladder. There is also evidence that telocytes are involved in pathologies in the kidneys, urethra, bladder, prostate, and testes.

Urogenital Abnormalities in Adenosine Deaminase Deficiency.

BACKGROUND: Improved survival in ADA-SCID patients is revealing new aspects of the systemic disorder. Although increasing numbers of reports describe the systemic manifestations of adenosine deaminase deficiency, currently there are no studies in the literature evaluating genital development and pubertal progress in these patients. METHODS: We collected retrospective data on urogenital system and pubertal development of 86 ADA-SCID patients followed in the period 2000-2017 at the Great Ormond Street Hospital (UK) and 5 centers in Italy. In particular, we recorded clinical history and visits, and routine blood tests and ultrasound scans were performed as part of patients' follow-up. RESULTS AND DISCUSSION: We found a higher frequency of congenital and acquired undescended testes compared with healthy children (congenital, 22% in our sample, 0.5-4% described in healthy children; acquired, 16% in our sample, 1-3% in healthy children), mostly requiring orchidopexy. No urogenital abnormalities were noted in females. Spontaneous pubertal development occurred in the majority of female and male patients with a few cases of precocious or delayed puberty; no patient presented high FSH values. Neither ADA-SCID nor treatment performed (PEG-ADA, BMT, or GT) affected pubertal development or gonadic function. CONCLUSION: In summary, this report describes a high prevalence of cryptorchidism in a cohort of male ADA-SCID patients which could represent an additional systemic manifestation of ADA-SCID. Considering the impact urogenital and pubertal abnormalities can have on patients' quality of life, we feel it is essential to include urogenital evaluation in ADA-SCID patients to detect any abnormalities, initiate early treatment, and prevent long-term complications.

Deep learning enables automatic quantitative assessment of puborectalis muscle and urogenital hiatus in plane of minimal hiatal dimensions.

OBJECTIVES: To measure the length, width and area of the urogenital hiatus (UH), and the length and mean echogenicity (MEP) of the puborectalis muscle (PRM), automatically and observer-independently, in the plane of minimal hiatal dimensions on transperineal ultrasound (TPUS) images, by automatic segmentation of the UH and the PRM using deep learning. METHODS: In 1318 three- and four-dimensional (3D/4D) TPUS volume datasets from 253 nulliparae at 12 and 36 weeks' gestation, two-dimensional (2D) images in the plane of minimal hiatal dimensions with the PRM at rest, on maximum contraction and on maximum Valsalva maneuver, were obtained manually and the UH and PRM were segmented manually. In total, 713 of the images were used to train a convolutional neural network (CNN) to segment automatically the UH and PRM in the plane of minimal hiatal dimensions. In the remainder of the dataset (test set 1 (TS1); 601 images, four having been excluded), the performance of the CNN was evaluated by comparing automatic and manual segmentations. The performance of the CNN was also tested on 117 images from an independent dataset (test set 2 (TS2); two images having been excluded) from 40 nulliparae at 12 weeks' gestation, which were acquired and segmented manually by a different observer. The success of automatic segmentation was assessed visually. Based on the CNN segmentations, the following clinically relevant parameters were measured: the length, width and area of the UH, the length of the PRM and MEP. The overlap (Dice similarity index (DSI)) and surface distance (mean absolute distance (MAD) and Hausdorff distance (HDD)) between manual and CNN segmentations were measured to investigate their similarity. For the measured clinically relevant parameters, the intraclass correlation coefficients (ICCs) between manual and CNN results were determined. RESULTS: Fully automatic CNN segmentation was successful in 99.0% and 93.2% of images in TS1 and TS2, respectively. DSI, MAD and HDD showed good overlap and distance between manual and CNN segmentations in both test sets. This was reflected in the respective ICC values in TS1 and TS2 for the length (0.96 and 0.95), width (0.77 and 0.87) and area (0.96 and 0.91) of the UH, the length of the PRM (0.87 and 0.73) and MEP (0.95 and 0.97), which showed good to very good agreement. CONCLUSION: Deep learning can be used to segment automatically and reliably the PRM and UH on 2D ultrasound images of the nulliparous pelvic floor in the plane of minimal hiatal dimensions. These segmentations can be used to measure reliably UH dimensions as well as PRM length and MEP. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Envejecimiento del sistema urogenital y disfunción sexual femenina / Aging of the urogenital system and female sexual dysfunction

Resumen El sistema genitourinario presenta una serie de cambios micro y macroanatómicos desde el nacimiento, pasando por la pubertad, período reproductivo y por último con la menopausia, en la cual, se desarrolla un conjunto de síntomas sistémicos que incluyen los vasomotores, del sueño, cognitivos, del estado de ánimo y cambios sexuales, asociados a la presencia de disminución de la lubricación, estrechamiento y distensibilidad vaginal, atrofia vaginal, entre otros, que llevan a presentar una clínica de dispareunia, prurito, resequedad, además de síntomas urinarios. Su examinación conlleva la realización de una historia clínica, examen físico y ginecológico. Para confirmar los cambios se puede hacer una medición de pH y una citología para determinar un índice de maduración vaginal. Cuando estos síntomas se asocian a angustia y molestia se puede estar ante un caso de disfunción sexual, del cual se conocen factores de riesgo para su presencia como la edad como tal, problemas de pareja, estado de salud, autoestima, entre otros. Es por esto que a razón del tratamiento es importante hacer un abordaje interdisciplinario.

Abstract The genitourinary system presents a series of micro and macroanatomical changes from birth, through puberty, reproductive period and finally with menopause, in which a set of systemic symptoms are developed, including vasomotor, sleep, cognitive, mood and sexual changes, associated with the presence of decreased lubrication, narrowing and vaginal distensibility, vaginal atrophy, among others, leading to clinical symptoms of dyspareunia, pruritus, dryness, and urinary symptoms. Its examination involves the realization of a clinical history, physical and gynecological examination. To confirm the changes, a pH measurement can be made and a cytology to determine a vaginal maturation index. When these symptoms are associated with anguish and discomfort, there may be a case of sexual dysfunction, of which there are known risk factors for their presence such as age as such, couple problems, health status, self-esteem, among others. That is why, as to treatment, it is important to make an interdisciplinary approach.

Role of Androgens in Female Genitourinary Tissue Structure and Function: Implications in the Genitourinary Syndrome of Menopause.

INTRODUCTION: Genitourinary conditions in women increase in prevalence with age. Androgens are prerequisite hormones of estrogen biosynthesis, are produced in larger amounts than estrogens in women, and decrease throughout adulthood. However, research and treatment for genitourinary complaints have traditionally focused on estrogens to the exclusion of other potential hormonal influences. AIM: To summarize and evaluate the evidence that androgens are important for maintaining genitourinary health in women and that lack of androgenic activity can contribute to the development of symptoms of the genitourinary syndrome of menopause. METHODS: The role of androgens in the pathophysiology, diagnosis, and treatment of genitourinary syndrome of menopause was discussed by an international and multidisciplinary panel during a consensus conference organized by the International Society for the Study of Women's Sexual Health. A subgroup further examined publications from the PubMed database, giving preference to clinical studies or to basic science studies in human tissues. MAIN OUTCOME MEASURES: Expert opinion evaluating trophic and functional effects of androgens, their differences from estrogenic effects, and regulation of androgen and estrogen receptor expression in female genitourinary tissues. RESULTS: Androgen receptors have been detected throughout the genitourinary system using immunohistochemical, western blot, ligand binding, and gene expression analyses. Lower circulating testosterone and estradiol concentrations and various genitourinary conditions have been associated with differential expression of androgen and estrogen receptors. Supplementation of androgen and/or estrogen in postmenopausal women (local administration) or in ovariectomized animals (systemic administration) induces tissue-specific responses that include changes in androgen and estrogen receptor expression, cell growth, mucin production, collagen turnover, increased perfusion, and neurotransmitter synthesis. CONCLUSION: Androgens contribute to the maintenance of genitourinary tissue structure and function. The effects of androgens can be distinct from those of estrogens or can complement estrogenic action. Androgen-mediated processes might be involved in the full or partial resolution of genitourinary syndrome of menopause symptoms in women. Traish AM, Vignozzi L, Simon JA, et al. Role of Androgens in Female Genitourinary Tissue Structure and Function: Implications in the Genitourinary Syndrome of Menopause. Sex Med Rev 2018;6:558-571.

Regional morphology and mucus composition in the urogenital papilla skin of the blackbelly rosefish Helicolenus dactylopterus (Delaroche, 1809).

Blackbelly rosefish Helicolenus dactylopterus is a zygoparous fish whose males are equipped with the copulating organ named urogenital papilla (UP). This study deals with the morphology and the glycoconjugate pattern of the UP epidermis, which is the male tissue interacting with the female internal body during copulation. The carbohydrate content was studied by means of conventional and lectin histochemistry. The epidermis was shown to be a stratified cuboidal epithelium and to exhibit characteristic intraepithelial pits in the apical zone. The mucous cells are scattered in the epidermis. The epidermal cell layers and their thickness as well as the size of mucous cells varied along the UP. Conventional histochemistry showed that the mucous cells contained i) only neutral glycoproteins in the basal zone; ii) both neutral and acidic non-sulphated glycans as well as only acidic non-sulphated or sulphated glycoconjugates in the intermediate zone; iii) neutral and sulphated glycoconjugates in the apical zone. The mucous cells in the basal region expressed O-linked (mucin type) glycans terminating with αGalNAc, Galß1,3GalNAc which could be α2,3-linked to sialic acid, and high mannose type N-linked glycans terminating with fucose, lactosamine, and sialic α2,6-linked to galactose/N-acetylgalactosamine; terminal Gal and terminal/internal GlcNAc were also found. The mucous cells in the intermediate zone lacked Galß1,3GalNAc and showed less terminal α2,3-linked sialic acid, lactosamine, fucose, galactose, and internal N-acetylglucosamine residues. In the apical region, mucous cells only exhibited O-glycans terminating with GalNAc and N-acetylglucosamine. The demonstrated region-specific differences in the UP skin provide new insights into the reproductive biology of fishes with internal fertilization.

Female striated urogenital sphincter contraction measured by shear wave elastography during pelvic floor muscle activation: Proof of concept and validation.

AIMS: Investigation of the function of the striated urogenital sphincter (SUS) is challenging because it is difficult to access and requires invasive measures. Ultrasound shear wave elastography (SWE) is a non-invasive real-time technique used to estimate tissue stiffness. As muscle stiffness can be used as an estimate of muscle force, SWE provides an opportunity to study contraction of the peri-urethral musculature. Validation of SWE to study SUS during functional tasks, such as pelvic floor muscle contractions, is required prior to application in clinical populations. METHODS: Ten healthy females (34[5] years) participated. Stiffness in a region expected to contain the SUS was quantified using SWE at rest and during a pelvic floor muscle contractions performed at 10%, 25%, and 50% of maximal voluntary contraction (MVC). Two repetitions were performed for 10 s. RESULTS: During contraction, stiffness increased in the region of the SUS in all participants and at all contraction intensities. Multiple regions of increased stiffness were detected, with 95.8% of regions situated ventral to the mid-urethra within the anatomical area of the SUS. The increase in stiffness was greater for 50% MVC than both 10% and 25% MVC contraction intensities (P < 0.01). CONCLUSIONS: Stiffness increased within the anatomical region of the SUS during voluntary pelvic floor muscle contractions with predictable response to changes in contraction intensity. These observations support the potential for ultrasound SWE to study SUS function non-invasively.

The temporal relationship between activity of the pelvic floor muscles and motion of selected urogenital landmarks in healthy nulliparous women.

AIMS: The purpose of this study was to investigate the relationship between pelvic floor muscle (PFM) electromyographic (EMG) activation and urogenital landmark motion measured using 2D transperineal ultrasound (US) imaging. METHODS: Eight healthy, nulliparous women performed maximum voluntary PFM contractions while EMG and transperineal US images were acquired simultaneously. Changes in the levator plate length (LPL), bladder neck (BN) position and urethral position were determined by visual inspection. The relative timing of EMG onset and the onset of landmark motion, and the correlation coefficients between EMG activation and landmark motion were computed. Comparisons between the correlation coefficients and onset latencies of each landmark motion were made using one-way analysis of variance models. RESULTS: Despite generally good reliability metrics for the onset of EMG and the onset of landmark motion, the relative timing of EMG onset and the onset of landmark motion demonstrated no systematic patterns of activation onset. That said, the overall motion of the different urogenital landmarks throughout the MVC task was highly correlated with the PFM EMG data; correlation coefficients were generally good (>0.90) and highly significant (p < .001). The correlation between PFM EMG and LPL motion was significantly better than the correlation between PFM EMG and motion of the other landmarks. The relevance of one outlier is discussed. CONCLUSIONS: The motion of all urogenital landmarks seen on US imaging is highly correlated with PFM EMG activation during maximum voluntary PFM contractions and may therefore be of value in the context of biofeedback training to infer that PFM activation has occurred. The motion of the BN, however, may require more cautious interpretation than the reduction of LPL. The timing of the onset of urogenital landmark motion on transperineal US imaging does not precede nor follow the timing of PFM activation, while the high variance of relative onset timing makes the onset of landmark motion of questionable value when training or evaluating co-ordination or motor control.

The menopausal woman: The need for an individualized plan of care.

With life expectancy increasing, women will spend one-third of their lives in and beyond menopause. A collaborative discussion with the clinician facilitates informed decision-making and should include evidence-based discussion of physiologic changes, assessment of symptoms and treatment options, review of screening recommendations, and discussion of disease risk-reduction strategies and psychosocial issues.

Genetic Manipulation of the Avian Urogenital System Using In Ovo Electroporation.

One of the advantages of the avian embryo as an experimental model is its in ovo development and hence accessibility for genetic manipulation. Electroporation has been used extensively in the past to study gene function in chicken and quail embryos . Readily accessible tissues such as the neural tube, somites, and limb bud, in particular, have been targeted. However, more inaccessible tissues, such as the embryonic urogenital system , have proven more challenging to study. Here, we describe the use of in ovo electroporation of TOL2 vectors or RCASBP avian viral vectors for the rapid functional analysis of genes involved in avian sex determination and urogenital development . In the context of the developing urogenital system , these vectors have inherent advantages and disadvantages, which will be considered here. Either vector can both be used for mis-expressing a gene and for targeting endogenous gene knockdown via expression of short hairpin RNAs (shRNAs). Both of these vectors integrate into the genome and are hence spread throughout developing tissues. Going forward, electroporation could be combined with CRISPR/Cas9 technology for targeted genome editing in the avian urogenital system .

Kisspeptin signalling in the physiology and pathophysiology of the urogenital system.

Kisspeptin is a peptide hormone, which signals via the G-protein-coupled kisspeptin receptor (KISS1R). Kisspeptin-KISS1R signalling has been implicated in various physiological and pathophysiological processes in the urogenital system, including critical roles in ovarian function as a key player in the regulation of oocyte development. Kisspeptin also has roles in several different functions of the male reproductive tract, such as spermatogenesis and sperm capacitation, and is also thought to be involved in kidney physiology - studies in preclinical animal models have reported that expression of kisspeptin and/or KISS1R is altered in chronically impaired kidneys. The wider importance of kisspeptin action in the urogenital tract has been highlighted by the finding that it suppresses metastasis of urogenital carcinomas; besides the possible therapeutic potential of this finding, tissue and tumour-stage-specific alterations in kisspeptin and KISS1/KISS1R expression could potentially be used as biomarkers for the diagnosis and prognosis of urogenital carcinomas.

Hydrogen Sulfide and Urogenital Tract.

In this chapter the role played by H2S in the physiopathology of urogenital tract revising animal and human data available in the current relevant literature is discussed. H2S pathway has been demonstrated to be involved in the mechanism underlying penile erection in human and experimental animal. Both cystathionine-ß synthase (CBS) and cystathionine-γ lyase (CSE) are expressed in the human corpus cavernosum and exogenous H2S relaxes isolated human corpus cavernosum strips in an endothelium-independent manner. Hydrogen sulfide pathway also accounts for the direct vasodilatory effect operated by testosterone on isolated vessels. Convincing evidence suggests that H2S can influence the cGMP pathway by inhibiting the phosphodiesterase 5 (PDE-5) activity. All these findings taken together suggest an important role for the H2S pathway in human corpus cavernosum homeostasis. However, H2S effect is not confined to human corpus cavernosum but also plays an important role in human bladder. Human bladder expresses mainly CBS and generates in vitro detectable amount of H2S. In addition the bladder relaxant effect of the PDE-5 inhibitor sildenafil involves H2S as mediator. In conclusion the H2S pathway is not only involved in penile erection but also plays a role in bladder homeostasis. In addition the finding that it involved in the mechanism of action of PDE-5 inhibitors strongly suggests that modulation of this pathway can represent a therapeutic target for the treatment of erectile dysfunction and bladder diseases.

Urine--a waste or the future of regenerative medicine?

In recent years, urine has emerged as a source of urine cells. Two different types of cells can be isolated from urine: urine derived stem cells (USCs) and renal tubular cells called urine cells (UCs). USCs have great differentiation properties and can be potentially used in genitourinary tract regeneration. Within this paper, we attempt to demonstrate that such as easily accessible source of cells, collected during completely non-invasive procedures, can be better utilized. Cells derived from urine can be isolated, stored, and used for the creation of urine stem cell banks. In the future, urine holds great potential to become a main source of cells for tissue engineering and regenerative medicine.

Physiological roles for mafr-1 in reproduction and lipid homeostasis.

Maf1 is a conserved repressor of RNA polymerase (Pol) III transcription; however, its physiological role in the context of a multicellular organism is not well understood. Here, we show that C. elegans MAFR-1 is functionally orthologous to human Maf1, represses the expression of both RNA Pol III and Pol II transcripts, and mediates organismal fecundity and lipid homeostasis. MAFR-1 impacts lipid transport by modulating intestinal expression of the vitellogenin family of proteins, resulting in cell-nonautonomous defects in the developing reproductive system. MAFR-1 levels inversely correlate with stored intestinal lipids, in part by influencing the expression of the lipogenesis enzymes fasn-1/FASN and pod-2/ACC1. Animals fed a high carbohydrate diet exhibit reduced mafr-1 expression and mutations in the insulin signaling pathway genes daf-18/PTEN and daf-16/FoxO abrogate the lipid storage defects associated with deregulated mafr-1 expression. Our results reveal physiological roles for mafr-1 in regulating organismal lipid homeostasis, which ensure reproductive success.

Zebrafish as a model to study live mucus physiology.

Dysfunctional mucus barriers can result in important pulmonary and gastrointestinal conditions, but model systems to study the underlying causes are largely missing. We identified and characterized five mucin homologues in zebrafish, and demonstrated a strategy for fluorescence labeling of one selected mucin. These tools can be used for in vivo experiments and in pharmacological and genetic screens to study the dynamics and mechanisms of mucosal physiology.