Access to male fertility preservation information and referrals at National Cancer Institute Cancer Centers.

Cancer treatment can lead to infertility, which is a significant source of financial and emotional distress for cancer patients and survivors. Given that future fertility and sexual function are critical quality of life issues, we hypothesise that access to subspecialist care is not uniformly distributed. Therefore, we sought to identify access gaps in male sexual health and infertility care at NCI cancer centres across US Census Regions. All 64 clinical NCI cancer centre websites were examined for language related to male sexual health and fertility. A phone-based survey was used to establish cancer centre referral patterns to andrologists and sperm banks. We utilised the Society for the Study of Male Reproduction member directory to determine geographic locations for andrologists relative to each centre. We found that the presence of information regarding male sexual health information was not associated with region. The presence of andrologists within 5-miles of a CC was significantly higher in the Northeast compared to all other census regions. Our work describes the access gap in fertility services at NCI cancer centres and how this differs by region of the country. These data can inform patients, and encourage centres to provide improved access to oncofertility care.

Advanced sperm testing.

PURPOSE OF REVIEW: The sensitivity of semen analysis for detection of infertility remains low. Many factors not measured in traditional semen analysis may contribute to male factor infertility. DNA fragmentation, oxidative stress, and sperm aneuploidy are three factors that may contribute further information to the evaluation when semen analysis is inconclusive. RECENT FINDINGS: DNA fragmentation measures the destruction of and failure to repair damage to DNA. Increased DNA fragmentation has been used as a marker for oxidative stress as well as toxic exposure. The oxidative stress adduct measures DNA aberrations, which sperm cannot repair and has been used to support use of antioxidants. Lastly, the aneuploid sperm frequency is a quantitative measure of deviation from the normal chromosomal complement. Although elevated sperm aneuploid frequency has been associated with recurrent pregnancy loss and implantation failures, barriers remain to its routine use. SUMMARY: We identified these three adjunctive tests, which have the potential to alter either management or counseling of patients with regards to male factor infertility. Elevated DNA fragmentation or significant sperm aneuploidy may suggest the need for further investigation or further preimplantation genetic testing prior to IVF. The oxidative stress adduct may lend further explanation and improved counseling of the infertile patient.

Impact of Abnormal Sperm Morphology on Live Birth Rates Following Intrauterine Insemination.

PURPOSE: We evaluated the live birth rate and the prevalence of congenital anomalies in couples undergoing intrauterine insemination with abnormal sperm morphology (less than 4% normal forms). MATERIALS AND METHODS: We retrospectively reviewed intrauterine insemination outcomes from January 2012 to March 2015. Patients who were found to have an ultrasound confirmed clinical pregnancy were contacted to determine the live birth rate and the prevalence of congenital abnormalities. We used chi-square analysis to assess categorical variables and the Student t-test to assess continuous variables. Logistic regression was done to assess the odds of achieving pregnancy and the risk of spontaneous abortion while assessing female age, the total motile count and sperm morphology. RESULTS: In 984 intrauterine insemination procedures performed in a total of 501 couples we found no difference in the ultrasound clinical pregnancy rate in couples with sperm morphology less than 4% vs 4% or greater (12.3% vs 13.6%, p=0.59). We collected live birth and birth abnormality data on 95 of the 130 couples with ultrasound confirmed clinical pregnancy for a 73% response rate. We found no difference in the live birth rate or the spontaneous abortion rate after an ultrasound confirmed clinical pregnancy in couples with abnormal sperm morphology (less than 4% normal forms). There was also no increased risk of birth abnormalities for patients with abnormal sperm morphology. CONCLUSIONS: Abnormal sperm morphology impacted neither the pregnancy rate nor the live birth rate in couples undergoing intrauterine insemination. These results can be used to reassure couples who undergo intrauterine insemination that there is a minimal impact of abnormal sperm morphology on the live birth rate and the prevalence of birth abnormalities.

Infection Prevention Considerations for Complex Penile Prosthesis Recipients.

PURPOSE OF REVIEW: The purpose of this review is to critically analyze and summarize recent studies in the area of penile prosthesis surgery outcomes with a focus on infection prevention in high-risk patients. RECENT FINDINGS: Reduction of surgical time in complex prosthesis surgery may reduce infection risk. Concomitant implant surgery is not associated with increased infection risk. Certain immunocompromised patients may be more likely to have penile implant infections, but these may not include patients with well-controlled HIV, well-controlled diabetes, or transplant recipients. Substance abuse is correlated with increased risk of infection after penile implant surgery. Careful patient selection and preoperative optimization can reduce infection risk in spinal cord injury patients. In the last 5 years, there have been several important studies investigating the risk of penile prosthesis infection in complex patients, clarifying which patient categories are at increased risk and how that risk can be mitigated.

Advanced paternal age does not affect embryo aneuploidy following blastocyst biopsy in egg donor cycles.

PURPOSE: To study the impact of advanced paternal age on embryo aneuploidy. METHODS: This is a multicenter international retrospective case series of couples undergoing assisted reproduction via in vitro fertilization using donor eggs to control for maternal factors and preimplantation genetic testing for aneuploidy via next-generation sequencing at Igenomix reproductive testing centers. The main outcome measure was the prevalence of embryo aneuploidy in egg donor cycles. Semen analysis data was retrieved for a small subset of the male patients. RESULTS: Data from 1202 IVF/ICSI egg donor cycles using ejaculated sperm (total 6934 embryos) evaluated using PGT-A between January 2016 and April 2018 in a global population across all Igenomix centers were included. No significant association was identified between advancing paternal age and the prevalence of embryo aneuploidy overall and when analyzing for each chromosome. There was also no significant association between advancing paternal age and specific aneuploid conditions (monosomy, trisomy, partial deletion/duplication) for all chromosomes in the genome. CONCLUSIONS: This is the largest study of its kind in an international patient population to evaluate the impact of advancing paternal age on embryo aneuploidy. We conclude there is no specific effect of paternal age on the prevalence of embryo aneuploidy in the context of embryo biopsies from egg donor cycles.

Novel Therapy for Male Hypogonadism.

PURPOSE OF REVIEW: Hypogonadism and the treatment of low testosterone is increasingly a subject of medical inquiry and controversy. The few treatments that are FDA-approved such as testosterone topicals, injections, and pellets create significant demand from patients for treatments with more convenient means of administration, fewer adverse effects, and the ability to maintain male fertility. Off-label drugs are in abundant use for low testosterone, including selective estrogen receptor modulators, gonadotropins, and aromatase inhibitors. RECENT FINDINGS: In this review, we summarize the evidence in support of commonly used treatments for male hypogonadism, as well as discuss recent updates in the development of novel therapeutics. New therapies include nasal testosterone, new oral testosterone formulations, anabolic-androgenic steroids, and selective androgen receptor modulators.

Multicenter Investigation of the Micro-Organisms Involved in Penile Prosthesis Infection: An Analysis of the Efficacy of the AUA and EAU Guidelines for Penile Prosthesis Prophylaxis.

INTRODUCTION: Penile prosthesis infections remain challenging despite advancements in surgical technique, device improvements, and adoption of antibiotic prophylaxis guidelines. AIM: To investigate penile prosthesis infection microbiology to consider which changes in practice could decrease infection rates, to evaluate current antibiotic prophylaxis guidelines, and to develop a proposed algorithm for penile prosthesis infections. METHODS: This retrospective institutional review board-exempt multi-institutional study from 25 centers reviewed intraoperative cultures obtained at explantation or Mulcahy salvage of infected three-piece inflatable penile prostheses (IPPs). Antibiotic usage was recorded at implantation, admission for infection, and explantation or salvage surgery. Cultures were obtained from purulent material in the implant space and from the biofilm on the device. MAIN OUTCOME MEASURES: Intraoperative culture data from infected IPPs. RESULTS: Two hundred twenty-seven intraoperative cultures (2002-2016) were obtained at salvage or explantation. No culture growth occurred in 33% of cases and gram-positive and gram-negative organisms were found in 73% and 39% of positive cultures, respectively. Candida species (11.1%), anaerobes (10.5%) and methicillin-resistant Staphylococcus aureus (9.2%) constituted nearly one third of 153 positive cultures. Multi-organism infections occurred in 25% of positive cultures. Antibiotic regimens at initial implantation were generally consistent with American Urological Association (AUA) and European Association of Urology (EAU) guidelines. However, the micro-organisms identified in this study were covered by these guidelines in only 62% to 86% of cases. Antibiotic selection at admissions for infection and salvage or explantation varied widely compared with those at IPP implantation. CONCLUSION: This study documents a high incidence of anaerobic, Candida, and methicillin-resistant S aureus infections. In addition, approximately one third of infected penile prosthesis cases had negative cultures. Micro-organisms identified in this study were not covered by the AUA and EAU antibiotic guidelines in at least 14% to 38% of cases. These findings suggest broadening antibiotic prophylaxis guidelines and creating a management algorithm for IPP infections might lower infection rates and improve salvage success. Gross MS, Phillips EA, Carrasquillo RJ, et al. Multicenter Investigation of the Micro-Organisms Involved in Penile Prosthesis Infection: An Analysis of the Efficacy of the AUA and EAU Guidelines for Penile Prosthesis Prophylaxis. J Sex Med 2017;14:455-463.

Prospective Evaluation of Sexual Dysfunction in Men With Chronic Pancreatitis: A Pilot Study.

OBJECTIVE: Our study aimed to determine the prevalence of sexual dysfunction (SD) and its association with quality of life (QOL) in men with chronic pancreatitis (CP). MATERIALS AND METHODS: Male patients with CP were prospectively enrolled in our pancreas center and completed the following 4 validated questionnaires: International Index of Erectile Function 5, Erectile Hardness Score, Pancreatitis Quality of Life Instrument, and Short Form Survey. Patients were classified as having mild, moderate, or severe SD based on review of questionnaires. RESULTS: Thirty patients were enrolled in the study, of which 18 patients had SD (mild in 9, moderate in 1, and severe in 8 patients). No significant differences were seen demographic or clinical characteristics in patients with and without SD. Patients with SD had more abdominal pain compared with those without SD (94.4% vs 83.3%, P = 0.001). No significant differences were noted in QOL metrics between the 2 groups. CONCLUSIONS: This pilot study shows that SD is present in 60% males with CP. No difference was noted in the QOL of patients with and without SD, albeit limited by our small sample size. Physicians caring for CP patients should routinely inquire for symptoms of SD and offer a urology referral if indicated.

SNF8, a member of the ESCRT-II complex, interacts with TRPC6 and enhances its channel activity.

BACKGROUND: Transient receptor potential canonical (TRPC) channels are non-selective cation channels involved in receptor-mediated calcium signaling in diverse cells and tissues. The canonical transient receptor potential 6 (TRPC6) has been implicated in several pathological processes, including focal segmental glomerulosclerosis (FSGS), cardiac hypertrophy, and pulmonary hypertension. The two large cytoplasmic segments of the cation channel play a critical role in the proper regulation of channel activity, and are involved in several protein-protein interactions. RESULTS: Here we report that SNF8, a component of the endosomal sorting complex for transport-II (ESCRT-II) complex, interacts with TRPC6. The interaction was initially observed in a yeast two-hybrid screen using the amino-terminal cytoplasmic domain of TRPC6 as bait, and confirmed by co-immunoprecipitation from eukaryotic cell extracts. The amino-terminal 107 amino acids are necessary and sufficient for the interaction. Overexpression of SNF8 enhances both wild-type and gain-of-function mutant TRPC6-mediated whole-cell currents in HEK293T cells. Furthermore, activation of NFAT-mediated transcription by gain-of-function mutants is enhanced by overexpression of SNF8, and partially inhibited by RNAi mediated knockdown of SNF8. Although the ESCRT-II complex functions in the endocytosis and lysosomal degradation of transmembrane proteins, SNF8 overexpression does not alter the amount of TRPC6 present on the cell surface. CONCLUSION: SNF8 is novel binding partner of TRPC6, binding to the amino-terminal cytoplasmic domain of the channel. Modulating SNF8 expression levels alters the TRPC6 channel current and can modulate activation of NFAT-mediated transcription downstream of gain-of-function mutant TRPC6. Taken together, these results identify SNF8 as a novel regulator of TRPC6.

Epigenetic natural variation in Arabidopsis thaliana.

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F(2) families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.

Cryptozoospermia Associated With Genital Tucking Behavior in a Transwoman.

Transwomen may elect to pursue fertility preservation prior beginning hormonal treatment or proceeding with gender-affirming surgery. To date, there has been little research specifically investigating factors influencing fertility and preservation thereof among transwomen. Here, we review the case of a transwoman who engaged in genital tucking behavior presenting with severe oligospermia, and we review the literature regarding transgender fertility preservation and the role of the heat stress hypothesis with regards to this common behavior.

Infection Prevention Strategies Prior to Penile Implant Surgery.

CONTEXT: Over the last 4 decades, penile implant surgery has advanced significantly. Penile implant infection prevention continues to be an active area of research due to the significant patient morbidity associated with infection. OBJECTIVE: To summarize current approaches and supporting data for infection prevention. EVIDENCE SYNTHESIS: Common approaches to infection prevention include careful patient selection and preoperative evaluation, broad-spectrum antimicrobial prophylaxis, and modifications to surgical technique that minimize skin contact and postoperative hematoma formation. CONCLUSION: Many of these measures have proven efficacious, but our knowledge regarding the pathophysiology of penile prosthesis infection continues to evolve and demands continued efforts towards infection prevention.

Transurethral ablation of a prostatic utricle cyst with the use of a holmium laser.

OBJECTIVE: To demonstrate a safe and effective approach to the treatment of obstructing midline prostate utricle cyst with the use of a holmium laser. DESIGN: Video presentation. SETTING: University hospital. PATIENT(S): A 33-year-old man presented with chronic pelvic pain, pain with ejaculation, and infertility. Semen analysis demonstrated oligoasthenospermia with poor viability and computerized tomographic scan identified the presence of a midline 2-3-cm prostatic cyst with dilated seminal vesicles bilaterally. Transrectal ultrasound in the office confirmed the diagnosis of midline obstructing prostatic utricle cyst and estimated the distance from the urethra. INTERVENTION(S): Transurethral ablation of a midline prostate utricle cyst with the use of a holmium laser. MAIN OUTCOME MEASURE(S): Intraoperative technique highlighting the main steps for a transurethral ablation of a midline prostate utricle cyst with the use of a holmium laser. RESULT(S): This video highlights the technique for transurethral ablation of a midline prostate utricle cyst with the use of a holmium laser to unroof the cyst. Retrograde vesiculography was performed to confirm patency of the ejaculatory ducts. Outpatient surgery was tolerated well and the patient was discharged. After surgery at 4 weeks, his symptoms had abated and semen analysis revealed normozoospermia. CONCLUSION(S): We demonstrate safe and effective transurethral ablation of a midline prostate utricle cyst with the use of a holmium laser. Preoperative transrectal ultrasound or cross-sectional imaging can be useful for operative planning. When the orifices of the ejaculatory ducts can be identified, vesiculography can be performed to confirm patency of the ducts and seminal vesicles after relief of the obstructing cyst.

Using microscope for onco-testicular sperm extraction for bilateral testis tumors.

OBJECTIVE: To demonstrate a step-by-step approach to the use of the operating microscope for onco-testicular sperm extraction. DESIGN: Video presentation. SETTING: University hospital. PATIENT(S): A 34-year-old man (status post right orchiectomy at another institution for pT3 pure seminoma with negative preoperative tumor markers) was referred for contralateral orchiectomy for multifocal left testis mass and fertility preservation. A postoperative semen analysis for attempted cryopreservation of ejaculated semen identified azoospermia. INTERVENTION(S): Left radical orchiectomy, left microsurgical onco-testicular sperm extraction (TESE). MAIN OUTCOME MEASURE(S): Intraoperative technique with commentary highlighting tips for successful fertility preservation via microsurgical onco-TESE. Discussion of alternatives. RESULT(S): This video provides a step-by-step guide to microsurgical onco-TESE coordinated with radical orchiectomy for testis cancer as a means of fertility preservation in an azoospermic patient. Preoperative imaging with scrotal ultrasound can serve as a useful guide for targeting microdissection to areas of normal testicular parenchyma for extraction of seminiferous tubules likely to host normal spermatogenesis. This patient had successful recovery and cryopreservation of abundant testicular sperm following targeted ex-vivo testicular microdissection. CONCLUSION(S): Microsurgical onco-TESE may be offered to azoospermic patients when undergoing orchiectomy for testis cancer. Use of preoperative imaging and the surgical microscope guide surgical dissection and optimize sperm recovery.

Erectile dysfunction among male adult entertainers: a survey.

BACKGROUND: Erectile dysfunction (ED) has not been evaluated among male adult entertainers. Our goal was to evaluate the prevalence of ED and use of erectile aids among men working in the adult entertainment industry. METHODS: An online survey was distributed to male adult entertainers through collaboration with the Free Speech Coalition (FSC), the North American Trade Association of the Adult Industry. Surveys were sent via email to those who met the criteria of being non-transgender men with biological penises and have experience as adult entertainers. The survey acquired baseline characteristics, use and frequency of erectile aids, and assessed erectile function using the International Index of Erectile Function (IIEF) survey. Statistical analysis was performed in Microsoft Excel. RESULTS: Of the 98 respondents, 62 met inclusion criteria. The median age was 36 (IQR 28.25-45) years. Of those included, 69.4% (43/62) used erectile aids. In men who used erectile aids, 58.1% (25/43) used for both work and personal reasons. Erectile aids were used most among young men aged 20-29 (84.2%). Overall, 38.7% (24/62) of men had ED as defined by the IIEF survey. Of note, 26.3% (5/19) of men aged 20-29 had moderate to severe ED. CONCLUSIONS: This is the first study to evaluate ED among adult male entertainers. Male entertainers between the ages of 20-29 had both the highest prevalence of moderate to severe ED and the highest erectile aid usage among all male adult entertainers.

Varicoceles: prevalence and pathogenesis in adult men.

Varicocele, or dilation of the pampiniform venous plexus, affects up to 15% of men. However, few of these men encounter problems with fertility. This discrepancy between men with varicocele and the number of adversely affected men has led to abundant research to identify the mechanisms for formation of varicocele as well as the pathologic mechanisms by which varicoceles affect fertility potential. In this review, we discuss the prevalence of varicocele in adults, the anatomic features of varicocele, the leading theories as to how varicocele can negatively affect fertility potential, and finally, the current literature on the impact of varicocele on testosterone production.

Active surveillance for low-risk prostate cancer: Need for intervention and survival at 10 years.

INTRODUCTION: To describe the need for treatment and cancer-specific and overall survival in a contemporary active surveillance (AS) cohort. PATIENTS AND METHODS: Historical cohort study of men diagnosed with localized prostate cancer between 1997 and 2009 and managed with AS at a tertiary care center. Inclusion criteria were Gleason score ≤ 6 (Gleason score of 7 in select patients),≤ 3/12 cores positive, and prostate-specific antigen (PSA) level< 20 ng/ml. Survival analyses were conducted using the Kaplan-Meier method. RESULTS: A total of 469 men with median age at diagnosis of 68.1 years (interquartile range [IQR]: 62.5-73.4) were followed up for a median of 4.8 years (IQR: 3.4-7.3). Median PSA level at diagnosis was 5.1 ng/ml (IQR: 4.0-6.9), with 94% of them having PSA level<10 ng/ml. Overall, 98.3% (461/469) of patients had a Gleason score of 6 and 1.7% (8/469) had a Gleason score of 3+4 = 7, and 94.0% (441/469) had T1c stage disease. Freedom from treatment was 77% at 5 years and 62% at 10 years. A total of 116 (24.7%) patients received treatment during the course of surveillance. Reasons for treatment included 44.8% (52/116) for pathologic reclassification, 30.2% (35/116) for PSA progression, 12.1% (14/116) for patient preference, 5.2% (6/116) for digital rectal examination progression, and 4.3% (5/116) for metastatic disease. Of the patients treated, 59 (50.1%) received radiation, 26 (22.4%) underwent surgery, 17 (14.7%) received brachytherapy, and 14 (12.1%) received androgen-deprivation therapy. Cancer-specific survival was 100% at 5 and 10 years. Overall survival was 95% at 5 years and 88% at 10 years. CONCLUSION: In a contemporary cohort of men with low-risk prostate cancer, AS allowed avoidance of treatment most of them. Common reasons for change in management were Gleason upgrading and volume progression on prostate rebiopsy.

5-Alpha-reductase inhibitors in diseases of the prostate.

PURPOSE OF REVIEW: To summarize the history of the use of 5-alpha-reductase inhibitors in the treatment of urologic diseases and discuss the current practices and indications for therapy. RECENT FINDINGS: 5-Alpha-reductase inhibitors (5-ARIs) are indicated in the treatment of benign prostatic hyperplasia (BPH) with obstructive urinary symptoms to reduce symptoms, reduce the risk of acute urinary retention, and reduce the risk of needing surgical management for this condition. These drugs have also been employed in the treatment of recurrent and refractory hematuria of prostatic origin because of their effect on the androgen-mediated angiogenic pathways in the prostate. Data support the use of 5-ARIs for this indication, though this is off-label use. There is no role for the use of 5-ARIs in primary prostate cancer chemoprevention and they may increase the risk of developing high-grade prostate cancer. Current investigations are underway to determine the efficacy of 5-ARIs in the secondary treatment of localized prostate cancer as adjunctive therapy, which may decrease the likelihood of prostate cancer progression and increase the likelihood that patients will remain on active surveillance. SUMMARY: 5-ARIs have significantly impacted the medical treatment of urologic disease, in particular BPH, and prostatic hematuria. Their use in the secondary treatment of prostate cancer is currently under investigation.

Evolution of a distinct genomic domain in Drosophila: comparative analysis of the dot chromosome in Drosophila melanogaster and Drosophila virilis.

The distal arm of the fourth ("dot") chromosome of Drosophila melanogaster is unusual in that it exhibits an amalgamation of heterochromatic properties (e.g., dense packaging, late replication) and euchromatic properties (e.g., gene density similar to euchromatic domains, replication during polytenization). To examine the evolution of this unusual domain, we undertook a comparative study by generating high-quality sequence data and manually curating gene models for the dot chromosome of D. virilis (Tucson strain 15010-1051.88). Our analysis shows that the dot chromosomes of D. melanogaster and D. virilis have higher repeat density, larger gene size, lower codon bias, and a higher rate of gene rearrangement compared to a reference euchromatic domain. Analysis of eight "wanderer" genes (present in a euchromatic chromosome arm in one species and on the dot chromosome in the other) shows that their characteristics are similar to other genes in the same domain, which suggests that these characteristics are features of the domain and are not required for these genes to function. Comparison of this strain of D. virilis with the strain sequenced by the Drosophila 12 Genomes Consortium (Tucson strain 15010-1051.87) indicates that most genes on the dot are under weak purifying selection. Collectively, despite the heterochromatin-like properties of this domain, genes on the dot evolve to maintain function while being responsive to changes in their local environment.

TRPC6 mutations associated with focal segmental glomerulosclerosis cause constitutive activation of NFAT-dependent transcription.

Mutations in the canonical transient receptor potential channel TRPC6 lead to an autosomal dominant form of human kidney disease characterized histologically by focal and segmental glomerulosclerosis. Several of these mutations enhance the amplitude and duration of the channel current. However, the effect of these mutations on the downstream target of TRPC6, the nuclear factor of activated T cell (NFAT) transcription factors, has not been previously examined. Here we demonstrate that all three TRPC6 mutations previously shown to enhance channel activity lead to enhanced basal NFAT-mediated transcription in several cell lines, including cultured podocytes. These effects are dependent on channel activity and are dominant when mutants are coexpressed with wild-type TRPC6. While TRPC6 mutants do not demonstrate an increase in basal channel currents, a subset of cells expressing the R895C and E897K mutants have elevated basal calcium levels as measured by Fura-2 imaging. Activation of NFAT by TRPC6 mutants is blocked by inhibitors of calcineurin, calmodulin-dependent kinase II, and phosphatidylinositol 3-kinase. PP2 partially inhibits NFAT activation by mutant TRPC6 independently of Src, Yes, or Fyn. Differences in channel glycosylation and surface expression do not explain the ability of mutants to enhance NFAT activation. Taken together, these results identify the activation of the calcineurin-NFAT pathway as a potential mediator of focal segmental glomerulosclerosis.