The healthy urinary microbiome in asymptomatic participants in the MAPP Network Study: Relation to gender, age, and menopausal status.

INTRODUCTION: To understand the role of the urinary microbiome in disease states and interpret non-culture-based diagnostic urine testing of midstream urine specimens, we must have a better understanding of the urinary microbiome in asymptomatic, healthy individuals. We examined the impact of gender, age, and menopausal status on the healthy human urinary microbiome in asymptomatic control subjects enrolled in the multi-institution National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Multidisciplinary Approach to the Study of Chronic Pelvic Pain Network (MAPP) study. METHODS: Asymptomatic, healthy controls, recruited to be ageand sex-matched to patients in the Trans-MAPP Epidemiology and Phenotyping Study, provided midstream urine collection for polymerase chain reaction (PCR)-electrospray ionization mass spectrometry identification of urinary microbiota. The microbiomes of male and female participants were described and analyzed for differences in composition and diversity at the species and genus level by sex, age, and, in females, by menopausal status. RESULTS: Sixty-six total species were detected with a mean of 1.2 species (standard deviation [SD] 1.1) per male (n=97; mean age=43) and 2.3 (SD 1.3) per female (n=110, mean age=38) in asymptomatic, healthy controls. Species and genera diversity analyses showed significantly greater richness and diversity in females. With regard to species, Bifidobacterium subtile, Lactobacillus crispatus, and Lactobacillus johnsonii were more predominant in females. The genera Bifidobacterium, Staphylococcus, Lactobacillus, and Corynebacterium were more predominant in females, while for males the most prevalent organisms included Staphylococcus and Propionibacterium; only Propionibacterium approached a significant difference between genders. No significant difference in the presence and/or diversity of micro-organisms with menopausal status could be observed. Sex-specific age trends, particularly diversity, were larger for females than males. CONCLUSIONS: These results suggest the urinary microbiome of healthy, asymptomatic subjects differed between genders and age in females, but not menopausal status. Gender differences may be attributable to the detection of urethral/vaginal organisms in females and prostate organisms in males. These findings will better allow us to interpret the results of microbiome reports in the midstream urine specimens of patients with urinary symptoms.

Characterization of a method to study urodynamics and bladder nociception in male and female mice.

OBJECTIVES: Abdominal electromyogram or visceromotor response (VMR) elicited by bladder distension is a validated as a measure of bladder nociception in mice, however it is not without its limitations. The aim of this study is to address some of these limitations and validate voiding evoked VMR as a measure of bladder nociception mice. METHODS: Using both male and female C57BL/6 mice we assessed the VMR response to cytometry- induced voiding before and after instillation of 0.5% acetic acid into the bladder. We then delivered intravesical lidocaine to confirm the VMR response as nociceptive. VMR and correlative cystometric bladder pressures were analyzed. RESULTS: We found that the VMR can be evoked by continuous fluid infusion into the bladder of both male and female mice. This response is potentiated after bladder injury and can be attenuated by administration of a local anesthetic, providing strong evidence that this method can be used to evaluate bladder nociception. Further, evaluation of cystometric pressure traces obtained during VMR recording revealed that intercontraction intervals were not altered after bladder injury in either male or female mice. However, we did observe a decrease in peak threshold pressures after bladder injury in female mice, which could be rescued by lidocaine administration. CONCLUSIONS: In conclusion, this technique can measure the VMR and bladder nociception associated with voiding in both female and male mice. Although confounds still exist with the use of anesthesia, further exploration of non-anesthetized voiding-evoked VMR is warranted.

Are three-day voiding diaries feasible and reliable? Results from the Symptoms of Lower Urinary Tract Dysfunction Research Network (LURN) cohort.

AIMS: The aims of this study were to assess the completeness of voiding diaries in a research context and to correlate diary data with patient-reported questionnaires. METHODS: Men and women enrolled in the Symptoms of Lower Urinary Tract Dysfunction Research Network (LURN) were given a 3-day voiding and fluid-intake diary to fill-out. Diaries were assessed for completeness and intake-output imbalances. They were assigned to one of four categories based on a percentage of missing data and fluid imbalance: no diary submitted, unusable (>40% missing void or intake volumes, or unphysiological fluid imbalance), usable but not complete, and complete. RESULTS: A total of 1064 participants were enrolled and 85% (n = 902) returned the bladder diary. Of the diaries returned, 94% (n = 845) had data on three separate days, 87% (n = 786) had no missing intake volumes, 61% (n = 547) had no missing voided volumes, and 70% (n = 635) had a fluid imbalance within 3 L across the 3-day time period, resulting in 50% (n = 448) of participants with 100% complete diaries. Younger age was associated with a higher likelihood of not submitting a diary, or submitting an unusable diary. Women had a higher likelihood of submitting an unusable diary or a usable but incomplete diary. CONCLUSION: Overall, 50% of LURN participants returned voiding diaries with perfectly complete data. Incomplete data for voided volumes was the most common deficiency. There was only a moderate correlation between diary data and questionnaire responses, indicating that diaries are a source of unique information.

Symptom Duration in Patients With Urologic Chronic Pelvic Pain Syndrome is not Associated With Pain Severity, Nonurologic Syndromes and Mental Health Symptoms: A Multidisciplinary Approach to the Study of Chronic Pelvic Pain Network Study.

OBJECTIVE: To evaluate if patients with urologic chronic pelvic pain syndromes (UCPPS) with longer duration of symptoms experience more severe pain and urologic symptoms, higher rates of chronic overlapping pain conditions (COPC) and psychosocial comorbidities than those with a more recent onset of the condition. We evaluated cross-sectional associations between UCPPS symptom duration and (1) symptom severity, (2) presence of COPC, and (3) mental health comorbidities. METHODS: We analyzed baseline data from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain. Symptom severity, COPC, and mental health comorbidities were compared between patients with symptom duration of < 2 vs ≥ 2 years. Symptom severity was assessed by the Genitourinary Pain Index, the Interstitial Cystitis Symptom and Problem Index, and Likert scales for pelvic pain, urgency, and frequency. Depression and anxiety were evaluated with the Hospital Anxiety and Depression Scale and stress with the Perceived Stress Scale. RESULTS: Males (but not females) with UCPPS symptom duration ≥2 years had more severe symptoms than those with <2 years. Participants with short (<2 years) and longer (≥2 years) symptom duration were as likely to experience COPC. CONCLUSION: Longer UCPPS symptom duration was associated with more severe symptoms only in limited patient subpopulations. Symptom duration was not associated with risk for COPC or mental health comorbidities. Females with longer UCPPS duration had decreased distress, but the association was largely attributable to age.

Validation of an Expanded Carrier Screen that Optimizes Sensitivity via Full-Exon Sequencing and Panel-wide Copy Number Variant Identification.

BACKGROUND: By identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess the risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the prevalence of the respective diseases, and the screen's analytical sensitivity for identifying disease-causing variants. Disease-level analytical sensitivity is often <100% in ECS tests because copy number variants (CNVs) are typically not interrogated because of their technical complexity. METHODS: We present an analytical validation and preliminary clinical characterization of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide CNV calling, and specialized assays for technically challenging genes. Next-generation sequencing, customized bioinformatics, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precluded novel variant identification or interpretation. RESULTS: Screening of 36859 patients' blood or saliva samples revealed the substantial impact on fetal disease-risk detection attributable to novel CNVs (9.19% of risk) and technically challenging conditions (20.2% of risk), such as congenital adrenal hyperplasia. Of the 7498 couples screened, 335 were identified as at risk for an affected pregnancy, underscoring the clinical importance of the test. Validation of our ECS demonstrated >99% analytical sensitivity and >99% analytical specificity. CONCLUSIONS: Validated high-fidelity identification of different variant types-especially for diseases with complicated molecular genetics-maximizes at-risk couple detection.

Development and validation of a 36-gene sequencing assay for hereditary cancer risk assessment.

The past two decades have brought many important advances in our understanding of the hereditary susceptibility to cancer. Numerous studies have provided convincing evidence that identification of germline mutations associated with hereditary cancer syndromes can lead to reductions in morbidity and mortality through targeted risk management options. Additionally, advances in gene sequencing technology now permit the development of multigene hereditary cancer testing panels. Here, we describe the 2016 revision of the Counsyl Inherited Cancer Screen for detecting single-nucleotide variants (SNVs), short insertions and deletions (indels), and copy number variants (CNVs) in 36 genes associated with an elevated risk for breast, ovarian, colorectal, gastric, endometrial, pancreatic, thyroid, prostate, melanoma, and neuroendocrine cancers. To determine test accuracy and reproducibility, we performed a rigorous analytical validation across 341 samples, including 118 cell lines and 223 patient samples. The screen achieved 100% test sensitivity across different mutation types, with high specificity and 100% concordance with conventional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). We also demonstrated the screen's high intra-run and inter-run reproducibility and robust performance on blood and saliva specimens. Furthermore, we showed that pathogenic Alu element insertions can be accurately detected by our test. Overall, the validation in our clinical laboratory demonstrated the analytical performance required for collecting and reporting genetic information related to risk of developing hereditary cancers.

Activation of spinal extracellular signal-regulated kinases (ERK) 1/2 is associated with the development of visceral hyperalgesia of the bladder.

Activation of extracellular signal-regulated kinases (ERK) 1/2 in dorsal horn neurons is important for the development of somatic hypersensitivity and spinal central sensitization after peripheral inflammation. However, data regarding the roles of spinal ERK1/2 in the development of visceral hyperalgesia are sparse. Here we studied the activation of ERK1/2 in the lumbosacral spinal cord after innocuous and noxious distention of the inflamed (cyclophosphamide-treated) and noninflamed urinary bladder in mice. We also correlated the spinal ERK1/2 activation to distention-evoked bladder nociception as quantified by the abdominal visceromotor response (VMR). Cyclophosphamide treatment (bladder inflammation) evoked increased bladder hyperalgesia and allodynia to bladder distention, as evident from an upward and leftward shift of the VMR stimulus-response curve compared with that of noninflamed mice. Development of bladder hyperalgesia was associated with robust enhancement of ERK1/2 activation in the dorsal horn and deeper laminae bilaterally in the L6-S1 spinal cord. Functional blockade of spinal ERK1/2 activity via intrathecal administration of the upstream MEK inhibitor U0126 attenuated distention-evoked bladder nociception and caused a significant downward shift of the VMR stimulus-response curve. In summary, we have provided functional and immunohistochemical evidence that activation of lumbosacral spinal ERK1/2 is associated with the development of primary visceral (bladder) hyperalgesia. Our results suggest that aberrant processing of visceral nociceptive information at the level of the lumbosacral spinal cord via activation of ERK1/2 signaling may contribute to chronic bladder pain in the context of inflammation.