Saneal Rajanahally

Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15

Oocyte-derived growth factors are critically involved in multiple ovarian processes via paracrine actions. Although recombinant proteins have been applied to dissect the physiological functions of these factors, variation of activities among different protein preparations remains an issue. To further elucidate the roles of one of these growth factors, bone morphogenetic protein 15 (BMP15), in mediating oocyte-regulated molecular and cellular events and to explore its potential clinical application, we engineered the human BMP15 sequence to efficiently produce bioactive recombinant human BMP15 (rhBMP15). The proteolytic cleavage site of the hBMP15 precursor was optimized to facilitate the production of the mature protein, and a FLAG-tag was placed at the N-terminus of the mature region to ease purification and avoid potential interference of the tag with the cystine knot structure. The rhBMP15 protein was purified using anti-FLAG M2 affinity gel. Our results demonstrated that the N-terminal tagged rhBMP15 was efficiently processed in HEK-293 cells. Furthermore, the purified rhBMP15 could activate SMAD1/5/8 and induce the transcription of genes encoding cumulus expansion-related transcripts (Ptx3, Has2, Tnfaip6 and Ptgs2), inhibitory SMADs (Smad6 and Smad7), BMP antagonists (Grem1 and Fst), activin/inhibin betaA (Inhba) and betaB (Inhbb) subunits, etc. Thus, our rhBMP15 containing a genetically modified cleavage sequence and an N-terminal FLAG-tag can be efficiently produced, processed and secreted in a mammalian expression system. The purified rhBMP15 is also biologically active and very stable, and can induce the expression of a variety of mouse granulosa cell genes.

Anabolic steroid induced hypogonadism in young men.

PURPOSEnThe use of anabolic androgenic steroids has not been traditionally discussed in mainstream medicine. With the increased diagnosis of hypogonadism a heterogeneous population of men is now being evaluated. In this larger patient population the existence of anabolic steroid induced hypogonadism, whether transient or permanent, should now be considered.nnnMATERIALS AND METHODSnWe performed an initial retrospective database analysis of all 6,033 patients who sought treatment for hypogonadism from 2005 to 2010. An anonymous survey was subsequently distributed in 2012 to established patients undergoing testosterone replacement therapy.nnnRESULTSnProfound hypogonadism, defined as testosterone 50 ng/dl or less, was identified in 97 men (1.6%) in the large retrospective cohort initially reviewed. The most common etiology was prior anabolic androgenic steroid exposure, which was identified in 42 men (43%). Because of this surprising data, we performed an anonymous followup survey of our current hypogonadal population of 382 men with a mean±SD age of 49.2±13.0 years. This identified 80 patients (20.9%) with a mean age of 40.4±8.4 years who had prior anabolic androgenic steroid exposure. Hypogonadal men younger than 50 years were greater than 10 times more likely to have prior anabolic androgenic steroid exposure than men older than 50 years (OR 10.16, 95% CI 4.90-21.08). Prior anabolic androgenic steroid use significantly correlated negatively with education level (ρ=-0.160, p=0.002) and number of children (ρ=-0.281, p<0.0001).nnnCONCLUSIONSnPrior anabolic androgenic steroid use is common in young men who seek treatment for symptomatic hypogonadism and anabolic steroid induced hypogonadism is the most common etiology of profound hypogonadism. These findings suggest that it is necessary to refocus the approach to evaluation and treatment paradigms in young hypogonadal men.

Patient Satisfaction with Testosterone Replacement Therapies: The Reasons Behind the Choices

INTRODUCTIONnTestosterone replacement therapy (TRT) for male hypogonadism is rapidly gaining popularity and acceptance. Options include gels, injections, and implantable subcutaneous pellets.nnnAIMSnThe aim of this study was to determine rates of patient satisfaction and reasons for patient preferences in hypogonadal men on TRT.nnnMETHODSnAn anonymous, prospective survey was distributed to men presenting for TRT at an academic urology clinic. The survey was organized into multiple domains including patient satisfaction and treatment motivation.nnnMAIN OUTCOME MEASURESnPatient satisfaction responses obtained via anonymous survey.nnnRESULTSnAverage patient age was 49 ± 0.7 years (n = 382). Injectable testosterone was chosen by 53%, gel-based regimens by 31%, and pellets by 17%. Overall, 70% of patients were satisfied with their TRT and 14% reported dissatisfaction. Satisfaction rates were similar between gels (68%), injections (73%), and implantable pellets (70%). Doctor recommendation was the sole significant reason for patients preferring gel-based TRT (66% vs. 37% injection users vs. 31% pellet users). Injectable TRT was favored because of lower cost (35% vs. 21% gel users vs. 19% pellet users). Pellets were favored for ease of use (64% vs. 44% injection users vs. 43% gel users) and convenience (58% vs. 26% injection users vs. 19% gel users). Pellets had increased rates of satisfaction within the first 12 months. Improvements in concentration and mood occurred at higher percentages in satisfied patients.nnnCONCLUSIONSnPatients are satisfied with TRT. Lower costs are important to patients on injections. Convenience and ease of use are central in choosing pellet therapy. Men on TRT should be questioned about mood and concentration because these factors exhibited the greatest improvements in satisfied patients.

Factors Influencing Patient Decisions to Initiate and Discontinue Subcutaneous Testosterone Pellets (Testopel) for Treatment of Hypogonadism

INTRODUCTIONnA variety of modalities for testosterone replacement therapy (TRT) are available, including topical gels, injections, and Testopel subcutaneous testosterone pellets (STP). STP are becoming more commonly utilized in the United States; however, patient preferences, expectations, and usage patterns regarding this therapy remain poorly characterized.nnnAIMnTo identify factors influencing patients decisions to initiate or discontinue STP.nnnMETHODSnA total of 175 men from an academic urology clinic who were currently using or who had previously used STP for hypogonadism received a 32-item electronic survey.nnnMAIN OUTCOME MEASURESnAssessment of the impact of convenience, efficacy, side effects, cost, and symptom relief on initiation and discontinuation of STP.nnnRESULTSnOne hundred and thirteen men (64.6% response rate) of mean age 51.4 years who previously underwent a mean of 2.8 STP implant procedures completed the survey. Fifty-nine (52.2%) and 40 (35.4%) men had switched to STP from topical gel and injection therapy, respectively, whereas 14 (12.4%) men initially started TRT with STP. Convenience (68.8%) was the most important factor in patients decision to start STP, while cost of the previous form of TRT (14.7%) was least important. At the time of the survey, 32 men (28.3%) had discontinued STP therapy. Cost of therapy (50%) was the primary factor in discontinuing STP. There was no difference in serum testosterone levels between men who continued STP and those who discontinued therapy (642.8 vs. 629.0u2009ng/dL, Pu2009=u20090.83). Overall, 68.1% of patients continued STP therapy at the time of survey completion.nnnCONCLUSIONSnConvenience is the most important factor in a patients decision to initiate STP; however, physician recommendation also plays a substantial role. Cost was the primary reason for discontinuation. Upon survey completion, greater than two-thirds of respondents elected to continue STP therapy. STP are a viable treatment option for hypogonadal men seeking a convenient and efficacious alternative modality of TRT.

Defective Gonadotropin-Dependent Ovarian Folliculogenesis and Granulosa Cell Gene Expression in Inhibin-Deficient Mice

Inhibin-α knockout (Inha-/-) female mice develop sex cord-stromal ovarian cancer with complete penetrance and previous studies demonstrate that the pituitary gonadotropins (FSH and LH) are influential modifiers of granulosa cell tumor development and progression in inhibin-deficient females. Recent studies have demonstrated that Inha-/- ovarian follicles develop precociously to the early antral stage in prepubertal mice without any increase in serum FSH. These studies suggest that in the absence of inhibins, granulosa cells differentiate abnormally and thus at sexual maturity may undergo an abnormal response to gonadotropin signaling contributing to tumor development. To test this hypothesis, we stimulated immature wild-type and Inha-/- female mice with gonadotropin analogs prior to tumor formation and subsequently examined gonadotropin-induced ovarian follicle development as well as preovulatory and human chorionic gonadotropin-induced gene expression changes in granulosa cells. We find that at 3 wk of age, inhibin-deficient ovaries do not show further antral development or undergo cumulus expansion. In addition, there are widespread alterations in the transcriptome of gonadotropin-treated Inha-/- granulosa cells, with significant changes in genes involved in extracellular matrix and cell-cell communication. These data indicate the gonadotropins initiate an improper program of cell differentiation prior to tumor formation in the absence of inhibins.

Genetic evidence that SMAD2 is not required for gonadal tumor development in inhibin-deficient mice

BackgroundInhibin is a tumor-suppressor and activin antagonist. Inhibin-deficient mice develop gonadal tumors and a cachexia wasting syndrome due to enhanced activin signaling. Because activins signal through SMAD2 and SMAD3 in vitro and loss of SMAD3 attenuates ovarian tumor development in inhibin-deficient females, we sought to determine the role of SMAD2 in the development of ovarian tumors originating from the granulosa cell lineage.MethodsUsing an inhibin α null mouse model and a conditional knockout strategy, double conditional knockout mice of Smad2 and inhibin alpha were generated in the current study. The survival rate and development of gonadal tumors and the accompanying cachexia wasting syndrome were monitored.ResultsNearly identical to the controls, the Smad2 and inhibin alpha double knockout mice succumbed to weight loss, aggressive tumor progression, and death. Furthermore, elevated activin levels and activin-induced pathologies in the liver and stomach characteristic of inhibin deficiency were also observed in these mice. Our results indicate that SMAD2 ablation does not protect inhibin-deficient females from the development of ovarian tumors or the cachexia wasting syndrome.ConclusionsSMAD2 is not required for mediating tumorigenic signals of activin in ovarian tumor development caused by loss of inhibin.

The Fate of Transitional Urology Patients Referred to a Tertiary Transitional Care Center

OBJECTIVEnTo determine the changes in management of children with neurogenic bladder (NGB) or genitourinary congenital anomalies as they moved to our transitional care clinic at the Center for Restorative Pelvic Medicine, a multidisciplinary center led by an adult urologic team dedicated to the long-term care of these patients.nnnMATERIALS AND METHODSnWe retrospectively reviewed charts of patients with NGB or genitourinary congenital abnormalities referred between 2010 and 2013. Analysis included patient characteristics, causes of NGB, bladder management, recurrent urinary tract infection, stones, renal function, upper tract studies, video urodynamics, and change in management.nnnRESULTSnTwenty-four patients with an average age of 22.0 ± 2.7 years were included in analysis. Management was altered in 70.8% of patients (n = 17). Surgical management was instituted in 58.3% (n = 14 of 24) of patients and included bladder augmentation or urinary diversion (n = 7), intravesical botulinum toxin A injections (n = 5), cystolitholapaxy, or cystolithotomy (n = 2). Conservative management was changed in 12.5% (n = 3) of patients and included initiating anticholinergic medication (n = 2) or self-catheterization (n = 1). Follow-up was 8.9 ± 12.1 months.nnnCONCLUSIONnThere is an immense need for transitional care of patients with NGB or genitourinary congenital abnormalities as they grow into adulthood. Nearly 71% of our patients had a change in their bladder management with 38% undergoing a major surgery. This study emphasizes the necessity for a dedicated adult urologic team in conjunction with a comprehensive team to care for these complex patients because their urologic care and needs may vary significantly from their childhood.

Men regret anabolic steroid use due to a lack of comprehension regarding the consequences on future fertility

We examined whether men with anabolic‐steroid‐induced hypogonadism (ASIH) seeking testosterone supplementation therapy (TST) regretted their decision to use anabolic‐androgenic steroids (AAS) and what their reasons were for this regret. An anonymous, prospective survey was distributed to 382 men seeking follow‐up treatment for hypogonadism. Prior AAS use was confirmed by self‐report, and men were categorised based upon whether they regretted (R) or did not regret (NR) their use of AAS. The average patient age was 40 ± 0.9 years (n = 79) and 15.2% expressed regret over AAS use. No demographic differences were identified between those who regretted AAS use (n = 12) and those who did not (n = 67). Regret was not related to ASIH diagnosis or to AAS‐related side effects like increased aggression, mood disorders, erectile dysfunction, acne, fluid retention or dyslipidemia. Those who regretted AAS use were significantly more likely to have not comprehended the negative impact on future fertility (P < 0.030). Actual fertility issues were comparable in men who regretted AAS use (16.7%) and those who did not (13%). A total of 15.2% of men regretted using AAS. A lack of awareness regarding the negative long‐term effects on fertility was the primary factor related to regret of AAS use in men with ASIH.

Urethral diverticulum after midurethral sling erosion, excision, and subsequent management

Background Urethral diverticulum formation after midurethral sling placement is a very rare complication. Only 3 previous cases have been reported in the English literature. This is the first reported case of urethral diverticulum formation after midurethral sling excision for urethral erosion. Case A 43-year-old female patient presented with recurrent stress incontinence after midurethral tension-free vaginal tape sling removal for urethral erosion. Retrograde urethrogram demonstrated a urethral diverticulum. Video urodynamics showed a Valsalva leak point pressure of 50 cm H2O at 250 mL. She subsequently underwent urethral diverticulectomy with pubovaginal sling placement. Conclusions We present the first reported case of urethral diverticulum formation after midurethral sling excision for urethral erosion. Future consideration must be given to full excision of the sling tract during the sling removal operation in cases of urethral erosion to prevent this rare complication.