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Have you read? Best of the best: basic research

Female sexual dysfunction

Author: Nuno Louro
Urology Department of Centro Hospitalar e Universitário do Porto (CHUP),
University of Porto (ICBAS-UP)
Porto, Portugal

Alvisi S, Baldassarre M, Gava G, et al. Structure of Epithelial and Stromal Compartments of Vulvar and Vaginal Tissue From Women With Vulvo-Vaginal Atrophy Taking Ospemifene. J Sex Med 2018. 15: 1776-1784.
The morphological and physiological changes in vulvar and vaginal epithelium of menopausal women lead to a number of symptoms including sexual dysfunction and low quality of life. Selective estrogen receptor modulators (SERM) have proven effective to reduce vulvo-vaginal atrophy (VVA) symptoms. Ospemifene is the only SERM approved by the Food and Drug Administration and European Medicines Agency for the treatment of moderate to severe VVA in menopausal women and has been proved to restore the vaginal epithelium characteristics of post-menopausal women. In this study the authors wanted to extend current knowledge on the effects of ospemifene at vestibular level and to provide direct evidence of its impact on collagen content and composition at vaginal and vulvar epithelium levels. They included 20 women 50 to 80 years of age with the diagnosis of physiological menopause and without evidence of significant co-morbidities. Eleven women were taking ospemifene for at least one month, and 9, the control group, were not. Full-thickness tissue biopsies of the proximal and distal anterior vaginal wall and from the left and right vulvar vestibulum were taken for evaluation of histological features, glycogen content, total collagen and collagen type I/III ratio. They have shown that the effects of ospemifene extend to the vulvar vestibule, with growth and maturation of the epithelium. They have also shown an increase in the total collagen content in women taking ospemifene and a significantly increased collagen Type I/III ratio in their vaginal epithelium. These results should be confirmed in a larger sample of women under a prospective randomized design so that matched comparisons before and after therapy can be performed.

Caruso S, Cianci A, Cianci S, et al. Ultrastructural Study of Clitoral Cavernous Tissue and Clitoral Blood Flow From Type 1 Diabetic Premenopausal Women on Phosphodiesterase-5 Inhibitor. J Sex Med 2019. 16: 375-382.
Neurovascular factors, due to diabetes, can impair vulvar, clitoral and vaginal flow, contributing to sexual arousal disorder in diabetic women. Phosphodiesterase type 5 (PDE5) inhibitors are the first line of treatment in men with erectile dysfunction but are still struggling to find their place (if any) in the treatment of female sexual dysfunction. The current study aimed to evaluate the effects of PDE5 inhibitors on the in vivo histomorphologic structure of the clitoral cavernosal tissue of premenopausal type 1 diabetic women. They enrolled 38 women in a prospective randomized study, of which 28 completed the trial (13 taking tadalafil 5 mg daily and 15 in the control group). The women underwent microbiopsy of the clitoral body during surgical therapy of a benign gynecological pathology and the tissue was processed for electron microscopy. Translabial colour doppler ultrasound was used to measure the peak systolic velocity (PSV), the end diastolic velocity (EDV) and the pulsatility index (PI) of clitoral arteries. The investigation showed that diabetic women had ultrastructural abnormalities of smooth muscle cells (SMC) and that women on PDE5 inhibitors had a larger mean SMC thickness than women without drug intake. These changes correlated with colour-flow Doppler sonography – a better clitoral flow was recorded in women on PDE5 inhibitor. One of the important limitations of this study is the fact that sexual function of these women was not investigated, so it is impossible to correlate the findings with sexual behaviour. Nevertheless these data could suggest that a daily treatment (rather than on demand) could be a better approach for diabetic women with organic sexual dysfunction.

Erectile dysfunction

Yang BB, Hong ZW, Zhang Z, et al. Epalrestat, an Aldose Reductase Inhibitor, Restores Erectile Function in Streptozocin-induced Diabetic Rats. Int J Impot Res; 2019. 31: 97-104
Erectile dysfunction is very common in men with diabetes. Several pathologic processes can contribute to sexual dysfunction in this group but peripheral neuropathy is one of the most relevant. One of the proposed mechanisms of neural injury is the increasing of the polyol pathways with accumulation of sorbitol. The subsequent depletion of myoinositol reduces Na/KATPase activity, causing diabetic neuropathy. Epalrestat is an aldose reductase (AR) inhibitor, the rate-limiting enzyme of the polyol pathway. It has been approved to human use, improving the declined function of peripheral nerves and increasing the diameter of nerve fibres of diabetic patients. In this study the authors aimed to investigate if epalrestat could restore the erectile function of diabetic erectile dysfunction, using a rat model (diabetic streptozocin-induced). They gave intragastric epalrestat daily to a group of ten rats, and saline to other 10 (the control group). They assessed erectile function by measuring intracavernous pressure (ICP) and the ratio of the ICP/mean systemic arterial pressure (MAP). The levels of AR, nerve growth factor (NGF) neuronal nitric oxide synthase (nNOS), α-smooth muscle antigen (α-SMA) and vonWillebrand factor (vWF) were also measured. Epalrestat partly restored ICP and increased the ICP/MAP ratio. The content of α-SMA-positive smooth muscle cells and v-WF-positive endothelial cells in the corpora cavernous was decreased in diabetic rats and was partly restored after treatment with epalrestat. The treated rats also had a higher expression of NGF and n-NOS. It would be very interesting to see if these effects are also seen in the human corpus cavernosum, a dose-response validation and translation into a clinical trial.

Campbell JD, Alenezi H, DeYoung L, et al. Hydro-Jet Dissection of the Cavernous Nerves Preserves Erection Function in a Radical Prostatectomy Animal Model. J Sex Med. 2019; 7; 104-10
Many factors affect rerecovery of erectile function after radical prostatectomy (RP) for prostate cancer, including preoperative function, age, the degree of nerve-sparing (NS) during surgery and the need for adjuvant treatment. Several maneuvers used during NS-RP can have a negative impact on erectile function (electrothermal injury, excessive traction, transection or devascularization of the cavernous nerves (CN). Hydro-jet dissection (HJD) technology involves the application of a thin high-pressure fluid stream to establish and expand surgical planes. It has already been used in NS-RP with mixed results and the authors of this article state that before supporting HJD as an acceptable alternative to current NS approaches, preclinical studies must elucidate a mechanistic and functional benefit. With that objective they designed this study, using 32 male Sprague-Dawley rats that were randomized to 4 groups: sham surgery, bilateral HJD of CN, blunt CN injury or stretch CN injury. After 4 weeks, erectile function was assessed by measuring intracavernous pressure (ICP), and penile tissues were harvested for immunohistologic studies. The HJD group had a higher mean peak and more sustainable rise in ICP than the other dissection groups and erectile function returned to baseline function in the HJD group, suggesting that this technique has minimal impact on the CN. They also noticed preservation of both α-SMA and n-NOS after HJD, demonstrating the potential neuroprotective role of this technology. It has already been proved that this technique is safe in humans but there’s a need to prove its erectile function protective role in well designed human studies.

Nunes da Silva C, Pedrosa Nunes K, De Marco Almeida F, et al. PnPP-19 Peptide Restores Erectile Function in Hypertensive and Diabetic Animals Through Intravenous and Topical Administration. J Sex Med. 2019; 16: 365-74.
Peptides from animal venoms, such as some arthropods including spiders, can cause priapism. One example is the toxin PnTx2-6, renamed δ-CNTX-Pn2a from the spider Phoneutria nigriventer, which improves erection in normotensive, hypertensive, and diabetic animals, although it is extremely toxic and induces many side effects such as edema, vascular congestion, cellular necrosis, nuclear condensation, or cytoplasmic vacuolation in the heart, lungs, kidneys, liver, and brain. The modeled structure of δ-CNTX-Pn2a enabled the design of a 19-amino acid synthetic peptide that this group named Phoneutria nigriventer potentiator peptide (PnPP-19). PnPP-19 improves the erectile function, apparently without eliciting side effects, showing low immunogenicity and no signs of toxicity. The aim of this study was to evaluate the efficacy of PnPP-19 in hypertensive and diabetic mouse/rat models in restoring erectile function, after topical administration; verify the biodistribution of PnPP-19 administration (topical and intravenous), permeation, and cyclic guanosine monophosphate (cGMP)/nitric oxide via implication. Corpus cavernosum relaxation was evaluated using cavernous strips from male spontaneous hypertensive rats (SHR) and from streptozotocin (STZ)-diabetic mice contracted with phenylephrine and submitted to electrical field stimulation before and after incubation with PnPP-19 (108 mol/L, 10 minutes) or vehicle. This procedure was also used to determine cGMP/nitric oxide levels, at 8 Hz and to check the effect of PnPP-19 with sildenafil citrate. Biodistribution assays were performed using iodine 123-radiolabeled PnPP-19. In vivo erectile function was evaluated using intracavernosal pressure/main arterial pressure (ICP/MAP) ratio in STZ-diabetic rats after PnPP- 19 topical administration. Their results suggest that PnPP-19 improves the erectile function in hypertensive and diabetic animals, because it relaxed corpus cavernosum strips from hypertensive (SHR) rats and from STZ diabetic mice and rats, independent of PDE5 inhibition, besides increasing the ratio ICP/MAP in SD rats. The concomitant treatment with sildenafil suggests an additive beneficial effect. These results support the idea of this peptide as a possible new drug to treat ED and the announced clinical studies in humans are awaited.

Hypogonadism

Lee DS, Choi JB, Sohn DW. Impact of Sleep Deprivation on the Hypothalamic-Pituitary-Gonadal Axis and Erectile Tissue. J Sex Med. 2019; 16: 5-16.
Sleep deprivation (SD) is a common problem in modern society and it has been implicated in several pathophysiological processes in the human organism. However, the effect of SD on sex-related hormones has not yet been well explained. SD has been shown to activate many stress-related pathways, including the hypothalamic- pituitary-adrenal axis. In addition, testosterone (T) contributes to the intracellular signalling pathway in erectile tissue, and low T levels can lead to penile tissue degeneration. Thus, if SD induces low T levels, these could exert a negative influence on the cavernosal tissue in terms of oxidative stress. The authors investigated the target in the HPG axis that is mainly affected by SD and whether the subsequent SD-induced reduction in T levels was responsible for triggering oxidative stress in the cavernosal tissue. They used 56 male Wistar rats for this experiment. First, 16 rats were subjected to 72 hours of SD, and the following were compared with 16 control rats: (i) levels of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and cortisol; (ii) the expression of the kisspeptin mRNA in the brain; and (iii) assessment of immunohistochemistry (IHC) of brain and testis. To further investigate whether testosterone reduction due to SD could affect erectile tissue, an additional 24 rats were divided into 3 groups (control, SD, and SD with T supplementation [SDT]) and compared: (i) T and cortisol levels were quantified, and (ii) endothelial nitric oxide synthase (eNOS)/ neuronal nitric oxide synthase (nNOS)/NOX-2 expression in cavernosal tissue was assessed by measuring mRNA levels and performing Western blotting and IHC. In cavernosal tissues, levels of the eNOS/nNOSmRNAs and proteins tended to be lower, and NOX-2 levels (mRNA and protein) tended to be higher in the SD group than those in the control group and SDT group. IHC for eNOS/nNOS revealed lower-intensity staining in the SD group than in the control and SDT groups, whereas the NOX- 2 intensity was higher in the SD group than in the other groups. A lower cortisol level was observed in the control group than in the SD and SDT groups, whereas the level was similar between the SD and SDT groups. The ICP/MAP values were also decreased in the SD group but not on testosterone injection. This study could imply that even short-term SD can produce secondary hypogonadism, impairing men’s health. Apart from its potential value in the knowledge of SD consequences to men, it also has a great value as a potential educational tool for a better sleep hygiene.

Premature ejaculation

Wayman C, Russell R, Tang K, et al. Cligosiban, a Novel Brain Penetrant Selective Oxytocin Receptor Antagonist, Inhibits Ejaculatory Physiology in Rodents. J Sex Med. 2018; 15: 1699-706.
Although in recent years the targeting of neurotransmitters and receptors has changed the treatment of ejaculation disorders, namely premature ejaculation, the ideal drug is far from available. Evidence suggests a major role for serotonin and dopamine in the control of ejaculation reflex, but also other neurotransmitters including oxytocin (OT) are likely to play a role. Epelsiban, an OT receptor antagonist was reported to inhibit ejaculation in rats on central or spinal dosing but showed only limited effects after systemic dosing, consistent with impaired brain penetration. It was studied in a clinical setting and it was reported that it had no clinically or statistically significant change in intra-vaginal ejaculatory latency time (IELT). The authors speculated that maybe a more centrally penetrant OT receptor antagonist might have different results. The aim of the current study was to evaluate if cligosiban, a new OT receptor antagonist selectively inhibits human OT receptors, penetrates central nervous system (CNS), shows pharmacology in the CNS, and effects ejaculatory physiology in pre-clinical systems. In vitro potency and selectivity of cligosiban was assessed using recombinant and native OT receptor systems including both neuronal and non-neuronal cell types. Selectivity was determined over neighbouring V1A, V1B and V2 vasopressin receptors. To determine an effect on central OT receptors and on ejaculation, the substance was evaluated in 2 anesthetized rat models – the electromyography model of ejaculatory physiology and a model of OT-mediated CNS neuronal firing. The CNS penetration of cligosiban was also determined by measuring cerebrospinal fluid and plasma drug concentration following intravenous infusion in rats. The in vitro and in vivo testing showed that cligosiban is a potent selective antagonist at the OT receptor, and it demonstrated CNS penetration and pharmacology by modulating an OT-mediated response in the NTS. It was also shown that cligosiban interrupts the expulsion phase of ejaculation by reducing the normal bulbospongiosum burst pattern and reducing the expulsions that accompany bursts. It seems that it is a promising compound to test the hypothesis that antagonism of central OT receptors might be of therapeutic benefit in the treatment of premature ejaculation. We just hope that it will not have the same disappointing performance in clinical trials as epelsiban.

Peyronie’s disease

Ilg MM, Mateus M, Stebbeds WJ, et al. Antifibrotic Synergy between Phosphodiesterase type 5 Inhibitors and Selective Oestrogen Receptor Modulators in Peyronie’s Disease Models. Eur Urol. 2019. 75: 329-40
Peyronie’s disease (PD) is a fibrotic disorder of the penile tunica albuginea (TA) characterized by the formation of plaques that can lead to deformity and erectile dysfunction. Myofibroblasts seem to be key cells in the pathogenesis of PD and inhibition of myofibroblastic transformation has been suggested as a therapeutic option. The authors developed a phenotyping screening assay capable of reproducibly measure transformation of human primary TA-derived fibroblasts to myofibroblasts in a high-throughput format and then tested 21 compounds/drugs that have been suggested to be efficacious in in vitro, in vivo animal and human studies of PD. They identified 2 classes of drugs that were able to inhibit myofibroblast transformation: phosphodiesterase type 5 (PDE5) inhibitors and selective oestrogen receptor modulators (SERM). When applied together these 2 classes showed synergistic activity both in vitro and in vivo. These results suggest that this combination might be beneficial in the acute phase of the disease and should be tested in a prospective randomized clinical trial.

Milenkovic U, Ilg MM, Zuccato C, et al. Simvastatin and the Rho-kinase Inhibitor Y-27632 Prevent Myofibroblast Transformation in Peyronie´s Disease-Derived Fibroblasts via Inhibition of YA/TAZ Nuclear Translocation. BJU Int. 2019; 123: 703-15
Currently available treatment options for Peyronie´s disease (PD) are merely symptomatic and do not target the underlying pathophysiological processes leading to plaque formation. The search for the molecular mechanisms that lead to the development of PD and ways to interfere with them are the goals of many researchers in this field. The objectives of the current study were to uncover the anti-myofibroblast (MFB) properties of Rho-kinase inhibitor Y-27632 and simvastatin in an in vitro and an in vivo model of PD. Tissue samples from tunica albuginea (TA) were collected prospectively from 5 patients receiving surgery for PD. Human fibroblasts were isolated from these samples. To induce MFB status, cells were stimulated with TGF-β1. Increasing doses of Y-27632 and simvastatin were added. RT-qPCR was used to assess mRNA expression of alpha-smooth muscle actin (α-SMA), collagen III, elastin and CTGF after 72 h. WB was used to quantify α-SMA protein contents and IF visualized MFB differentiation by staining for α-SMA after 72 h. Resazurin-based assay was performed to assess cell viability to ensure anti-MFB effect of the drugs. A mechanistic study was performed using IF staining for YAP/TAZ nuclear translocation. Unstimulated, harvested cells stained positive for vimentin and negative for α-SMA, while desmin mRNA levels were nearly undetectable. Stimulation of the cells with TGF-β1 caused a significant increase of α-SMA expression on mRNA and protein level, suggesting a myofibroblast phenotype. They also observed an increased level of extracellular matrix (ECM) (collagen I, III and elastin) and CTGF mRNA production, suggesting an upregulation of CTGF in TGF-β1 stimulated PD-derived fibroblasts. They also found that Y-27632 could significantly impede TGF-β1 induced myofibroblast transformation, as evidenced on an mRNA and protein level, while there was also a significantly reduced expression of CTGF and collagen III. This data suggest that ROCK-inhibitors can inhibit the formation of myofibroblasts, and hence inhibit their secretory capabilities. The authors suggest that TGF-β1 signalling in TA-derived fibroblasts occurs through ROCK-mediated YAP/TAZ nuclear translocation and that this nuclear activity of YAP/TAZ leads to transcription of several pro-fibrotic genes, including collagens and CTGF. Their findings also suggest that active plasma levels of simvastatin could be beneficial for inhibition of fibroblast to myofibroblast transformation and thus impeded fibrogenesis in a clinical setting. They have shown that simvastatin is a very potent inhibitor of myofibroblast transformation, production of ECM (collagen I, III and elastin) and CTGF mRNA. Additionally, the proliferation of PD-derived FBs was attenuated compared to controls or TGF-β1 stimulated cells. Further investigation of this pathway may eventually lead to novel treatment targets.