Clay Mechlin

Gli2 Expression and Human Bladder Transitional Carcinoma Cell Invasiveness

PURPOSE Hedgehog signaling regulates Gli transcription factors. Aberrant hedgehog signaling can be oncogenic and drugs that block hedgehog are being tested as anticancer agents. We considered whether hedgehog/Gli signaling may be involved in human bladder transitional cell carcinoma proliferative or invasive behavior. MATERIALS AND METHODS We stratified the human bladder transitional cell carcinoma lines RT4 (ATCC), 253JP, 253BV, UMUC6 and UMUC3 for relative growth rate by cell counting and for in vitro invasiveness by Matrigel invasion assay. Cells were tested for growth inhibition by the hedgehog blocking drug cyclopamine or the inactive mimic tomatidine. Cell RNA was characterized for hedgehog signaling component expression, including ligands, receptors and signaling mediators, by quantitative reverse transcriptase-polymerase chain reaction. Gli2 expression or activity was modified by Gli2 expression lentiviruses or the Gli inhibitor GANT61. We measured effects on proliferation and invasiveness. RESULTS Cell growth rates and invasiveness were stratified into an equivalent order (RT4 <243JP <253BV <UMUC6 <UMUC3). All cells were weakly growth inhibited by tomatidine and cyclopamine. Gli2 was the only hedgehog signaling molecule of which expression correlated with stratification. Manipulation of Gli2 expression or activity significantly affected cell invasiveness. CONCLUSIONS Weak growth suppression by cyclopamine suggests that hedgehog signaling is not involved in bladder cancer cell proliferation but Gli2 expression strongly correlated with invasive behavior. Increased Gli2 expression increased low Gli2 cell invasiveness while Gli inhibition by GANT61 decreased high Gli2 cell invasiveness. Results suggest that Gli2 expression by noncanonical signaling contributes to bladder cancer cell invasiveness.

A Pilot Study to Determine Penile Oxygen Saturation Before and After Vacuum Therapy in Patients with Erectile Dysfunction After Radical Prostatectomy

INTRODUCTION Provoked and spontaneous nocturnal erections are thought to play a role in maintenance of male sexual health through oxygenation of the corpus cavernosa. Conversely, hypoxia is thought to be an etiological factor in the pathogenesis of cavernosal fibrosis and long-term erectile dysfunction. It has been hypothesized that the early penile hypoxia after radical prostatectomy (RP) may lead to fibrosis and consequently a decrease in stretched penile length and long-term erectile dysfunction. AIM The aim of this study was to assess the changes in penile tissue oxygenation with vacuum erection device (VED) use. METHODS Twenty men between 2 and 24 months following RP were enrolled prospectively. Each man cycled a VED to achieve full erection 10 consecutive times over a period of approximately 2 minutes without constriction ring. MAIN OUTCOME MEASURES Tissue oximetry was measured at baseline and immediately after VED using a tissue oximeter at five sites: right thigh, right corpora, glans, left corpora, and left thigh. Additional measurements were captured over the course of an hour. RESULTS Mean age and time from surgery was 58.2 years and 12.6 months, respectively, and the average Sexual Health Inventory for Men score was 7. Use of the VED significantly increased both glanular and corporal oximetry relative to the baseline values for the entire 60 minutes. An initial increase of 55% was seen in corporal oxygenation with VED use. CONCLUSIONS This is the first study demonstrating that a single, brief application of the VED without a constriction ring results in significant improvement in penile oxygen saturation. The use of a VED has significant benefits for patients both with regard to cost and invasiveness when compared with other penile rehabilitation protocols.

A Randomized Prospective Double-Blind Comparison Trial of Clomiphene Citrate and Anastrozole in Raising Testosterone in Hypogonadal Infertile Men

AIM Clomiphene citrate (CC) and anastrozole (AZ) have been used off label to increase testosterone (T) in hypogonadal infertile men (HIM). Both medications have been shown to increase T with different effects on estradiol (E2) and T-to-E2 ratios. There are no reported randomized trials comparing CC and AZ to improve T levels in HIM. We aimed to establish equivalence of CC vs. AZ with respect to improvement in T levels in HIM. METHODS We randomized 26 HIM (T less than 350 ng/dL and normal luteinizing hormone [LH]). Patients were randomized to CC (25 mg/day) or AZ (1 mg/day) for 12 weeks. Hormones assayed were total T, free T, E2, LH, follicle stimulating hormone (FSH), and sex hormone binding globulin (SHBG). Patient-reported outcomes were the International Index of Erectile Function, Erection Hardness Scale, and the Androgen Deficiency in the Aging Male questionnaires. Blood tests and questionnaires were recorded at baseline, 6 and 12 weeks. Semen analyses were performed at baseline and 12 weeks. RESULTS T increased significantly from baseline in both groups at 6 and 12 weeks. There was a significantly larger increase in T and mean increase from baseline in CC vs. AZ (571 vs. 408 ng/dL, respectively). Whereas E-2 levels increased in the CC group, they decreased in the AZ group. Though both groups demonstrated an increase in T-to-E-2 ratio from baseline, statistic significance at 6 and 12 weeks was only achieved with AZ. Neither group demonstrated significant changes in seminal parameters or patient-reported outcomes. CONCLUSIONS We failed to demonstrate equivalence of CC vs. AZ. CC resulted in significantly higher T levels than AZ. AZ resulted in a significantly larger increase in T/E-2 ratio than CC. No significant differences between CC and AZ on seminal parameters or patient-reported outcomes were demonstrated.

Coadministration of Anastrozole Sustains Therapeutic Testosterone Levels in Hypogonadal Men Undergoing Testosterone Pellet Insertion

INTRODUCTION Current U.S. Food and Drug Administration-approved therapies for hypogonadism involve testosterone (T) replacement. Testosterone pellets (TP) require a minor office procedure every 3 to 4 months. The need for repeated insertions increases the likelihood of a complication. Anastrozole (AZ) is an aromatase inhibitor that has been used off-label for the treatment of male hypogonadism. AZ increases T levels by lowering serum estradiol (E2) levels and increasing gonadotropin (GTP) levels. AIM We hypothesized that the concomitant use of AZ with TP insertions would sustain therapeutic T levels and increase the interval between TP insertions. METHODS Men treated with TP for hypogonadism at an academic center were offered AZ (1 mg/day) at the time of TP reinsertion as a way of potentially decreasing the frequency of TP insertions. Total T (TT), free T (FT), sex hormone binding globulin, E2, luteinizing hormone (LH), and follicle-stimulating hormone FSH levels were obtained prior to T replacement and at 6 and 15 weeks from TP insertion. Men were re-implanted at 16 weeks if their TT levels were less than 350 ng/dL and their symptoms recurred. We retrospectively reviewed our records of men who underwent TP, TP, and AZ from 2011 to 2012. Demographics, TT, FT, LH, FSH, and E2 levels were recorded. Data were analyzed with anova and a Tukeys test. MAIN OUTCOME MEASURE TT level at 6, 15, or >15 weeks from TP insertion. RESULTS Thirty-eight men with 65 insertions were analyzed. The TP AZ group had significantly higher TT and FT levels than the TP group at >120 days (P < 0.05). The TP group had significantly higher E2 levels at all time points (P < 0.01). GTP levels remained stable in the TP AZ group. Average time to reinsertion in TP AZ was 198 days vs. 128 days in the TP group. CONCLUSION Men on TP AZ maintain therapeutic T levels longer than men on TP alone and have significantly less GTP suppression.

Mast cell numbers negatively correlate with fibrosis in cryptorchid testes

PURPOSE Mast cells have been found to play a role in fibrotic processes in multiple organ systems and are increased in number in the testes of infertile men. We have reviewed the literature and to date have found no studies investigating the role of mast cells in fibrosis of undescended testis. We examined the expression of mast cells in human cryptorchid testes and compared mast cell expression with testicular fibrosis in these testes. METHODS Testicular biopsies from cryptorchid testis were collected over 2 years. Biopsies from 78 patients were retrospectively sectioned, stained, and reviewed for the amount of fibrosis (graded 0-3) as well as mast cell number (MCN). MCNs were quantified by tryptase staining, and the average MCN per high-powered field (HPF) was determined. Statistical analysis was performed using a one-way ANOVA with a Kruskal-Wallis test and post hoc analysis with the Dunn test when significant. RESULTS Larger MCNs were significantly associated with lower fibrotic indices at the time of orchidopexy. The average MCNs were 2.06, 0.86, 0.37, and 0.58 for fibrotic indices of 0, 1, 2, and 3, respectively. MCNs were significantly higher in biopsies with a fibrotic index of 0 than all other groups (p < 0.05). CONCLUSION Mast cell expression correlates inversely with testicular fibrosis in cryptorchid testes. Further studies correlating mast cell expression with testicular function in boys with cryptorchidism are warranted.

What lessons can be learned from testicular histology in undescended testes

Over the last 40-50 years studies involving thousands of testicular biopsies in boys with cryptorchidism have contributed to our knowledge of testicular histopathology and our understanding of the effects of cryptorchidism on the normal development of the germinal epithelium. Growth and maturation of germ cells and Leydig cells are crucial to allow boys to reach normal fertility potential. The following aberrations in testicular development are seen in cryptorchid testes: a decrease in total germ cell numbers, failure of fetal gonocytes (stem cells) to transform into adult dark (Ad) spermatogonia, failure for Ad spermatogonia to mature into primary spermatocytes, Leydig cell hypoplasia, and testicular fibrosis. All of these findings have been found to have a strong negative correlation with a boy’s age at the time of orchidopexy. Some of these findings have prognostic significance in regards to fertility potential especially when coupled with key clinical findings such as hormonal findings, age at orchidopexy, testicle size, laterality and location of cryptorchid testes. This review focuses on key lessons learned from testicular histology in cryptorchid testes.

Mast cells, estrogens, and cryptorchidism: A histological based review

If untreated, cryptorchidism leads to age dependent decreases in germ cell number (GCN) and testicular fibrosis. The pathophysiology of this process and its long-term effects on fertility are unclear. Mast cells are intricately involved in inflammation and fibrosis in a variety of organ systems. Their secretory products have mitogenic effects on fibroblasts and promote collagen deposition. Mast cell activation and migration are under the influence of estrogens and this interaction has been demonstrated in the testes in several animal models. Models of cryptorchidism have shown increased estrogen levels and expression of estrogen receptors in undescended testes compared to controls. Mast cell numbers have positively correlated with testicular fibrosis in human studies and decreased spermatogenesis as well. We found no human studies of mast cells in cryptorchid testes. However several animal models have investigated the effect of estrogens on mast cells and spermatogenesis in undescended testes. In this review we examine the possible links between estrogens, mast cells, and testicular fibrosis in cryptorchidism, focusing on histological studies.

A retrospective review of single-institution outcomes with robotic-assisted microsurgical varicocelectomy

We report the largest single-center experience with robotic-assisted microscopic varicocelectomy (RAMV) in male infertility. From August 2012 to February 2015, men with infertility of at least a year and varicoceles underwent RAMV by a single surgeon. Varicocele was diagnosed on physical examination and confirmed by ultrasound by a single ultrasonographer. Preoperative hormone panel, semen analyses, and testicular Doppler ultrasound were obtained from all men and repeated at 3 months. One hundred and forty consecutive men (258 varicocelectomies) were included. Mean age and duration of infertility was 36.4 and 2.8 years, respectively. Median total and free testosterone increased by 145 ng dl−1 and 4.3 pcg ml−1 (44.3%), respectively (P < 0.0001). Median sperm concentration increased by 37.3% (P < 0.03). Median sperm motility and morphology did not significantly change. Median left and right testicular volume increased by 22.3% (P < 0.0001) and 12.6% (P < 0.0006), respectively. Hydroceles occurred 0.8% of procedures. We had no testicular artery injuries. Persistence of varicocele by Doppler ultrasound was 9.6%. Only 37.3% of patients required pain medications postoperatively. We concluded that RAMV is a safe and effective alternative for varicocele repair with outcomes comparable to historical traditional microsurgical approach.

Preoperative PDE5i use is a prognostic metric for poor postoperative erectile function in men undergoing radical prostatectomy: An addition to patient counseling

Objective: In patients scheduled for radical prostatectomies (RP), preoperative (pre-op) erectile function (EF) characterization may be complicated by social and medical factors. We investigated pre-op use of phosphodiesterase type 5 inhibitor (PDE5i) as a simple metric for predicting long-term postoperative EF. Materials and methods: Electronic medical records (EMRs) for consecutive men who underwent RP between January 2004 and March 2009 at our institution were retrospectively reviewed. Data extracted included demographics, pre-op PDE5i use, cancer treatment details, post-op EF and ED treatment. Predictor variable data were categorical pre-op PDE5i use (pre-op PDE5i use vs. pre-op PDE5i naïve). ANOVA and Chi squared test were used. Results: A total of 250 individuals out of 436 charts met inclusion criteria. Mean follow-up length was 4.2 years (range 2–7). Thirty-seven men (15%) used PDE5i preoperatively. There were no differences in mean age at RP, type of nerve-sparing surgery (NSS), or medical comorbidities between groups. No men with pre-op PDE5i use regained unassisted EF but 37% regained PDE5i-assisted EF after bilateral nerve sparing (BNS). No men with pre-op PDE5i use regained unassisted or PDE5i-assisted EF after unilateral (UNS) or non-nerve-sparing surgery (NNS). Conclusions: Pre-op PDE5i use predicts poor long-term EF outcomes after RP and should be included in pre-op patient counseling.