Dynamic Changes in Uterine NK Cell Subset Frequency and Function Over the Menstrual Cycle and Pregnancy.

Uterine natural killer cells (uNK) play an important role in promoting successful pregnancy by regulating trophoblast invasion and spiral artery remodelling in the first trimester. Recently, single-cell RNA sequencing (scRNAseq) on first-trimester decidua showed that uNK can be divided into three subsets, which may have different roles in pregnancy. Here we present an integration of previously published scRNAseq datasets, together with novel flow cytometry data to interrogate the frequency, phenotype, and function of uNK1-3 in seven stages of the reproductive cycle (menstrual, proliferative, secretory phases of the menstrual cycle; first, second, and third trimester; and postpartum). We found that uNK1 and uNK2 peak in the first trimester, but by the third trimester, the majority of uNK are uNK3. All three subsets are most able to degranulate and produce cytokines during the secretory phase of the menstrual cycle and express KIR2D molecules, which allow them to interact with HLA-C expressed by placental extravillous trophoblast cells, at the highest frequency during the first trimester. Taken together, our findings suggest that uNK are particularly active and able to interact with placental cells at the time of implantation and that uNK1 and uNK2 may be particularly involved in these processes. Our findings are the first to establish how uNK frequency and function change dynamically across the healthy reproductive cycle. This serves as a platform from which the relationship between uNK function and impaired implantation and placentation can be investigated. This will have important implications for the study of subfertility, recurrent miscarriage, pre-eclampsia, and pre-term labour.

Isolation of single cells from human uterus in the third trimester of pregnancy: myometrium, decidua, amnion and chorion.

During pregnancy, interactions between uterine immune cells and cells of the surrounding reproductive tissues are thought to be vital for regulating labour. The mechanism that specifically initiates spontaneous labour has not been determined, but distinct changes in uterine immune cell populations and their activation status have been observed during labour at term gestation. To understand the regulation of human labour by the immune system, the ability to isolate both immune cells and non-immune cells from the uterus is required. Here, we describe protocols developed in our laboratory to isolate single cells from uterine tissues, which preserve both immune and non-immune cell populations for further analysis. We provide detailed methods for isolating immune and non-immune cells from human myometrium, chorion, amnion and decidua, together with representative flow cytometry analysis of isolated cell populations present. The protocols can be completed in tandem and take approximately 4-5 h, resulting in single-cell suspensions that contain viable leucocytes, and non-immune cells in sufficient numbers for single-cell analysis approaches such as flow cytometry and single cell RNA sequencing (scRNAseq).

Left renal vein nutcracker syndrome and concurrent IVC duplication: Surgical treatment.

Multiple vascular anomalies may be encountered in patients with nutcracker syndrome; further compounding the surgical complexity in managing this condition. A 28-year-old male presented with persistent flank pain and hematuria. Imaging revealed narrowing of the left renal vein at the aortomesenteric junction, and a dilated vein consistent with the left gonadal vein. On surgical exploration, a duplicated IVC was found. The patient underwent a right caval-to-left caval bypass using a cryopreserved femoral vein homograft. The surgery was well tolerated and completely resolved the patient's symptoms.

Post-vasectomy semen analysis compliance with use of a home-based test.

INTRODUCTION Although the importance of post-vasectomy semen analysis (PVSA) is well known, compliance with this test has historically been low. We sought to compare compliance with PVSA when using a home-based testing kit with traditional office based microscopy, and to estimate the impact of compliance differences on the risk of undetected vasectomy failure. MATERIALS AND METHODS: A retrospective review of vasectomies performed by three providers was performed. Patients were prescribed either traditional office-based PVSA testing (Group 1) or home-based PVSA testing (Group 2). Compliance with PVSA testing was defined as completion of at least one PVSA test. Decision analysis methodology was applied to estimate the risk of undetected vasectomy failure in each group. RESULTS: A total of 226 vasectomies were reviewed, 141 in Group 1 and 85 in Group 2. The compliance rate was 65.96% in Group 1 compared to 76.47% in Group 2 (p = .095). When utilizing a single home-based test, the estimated risk of undetected vasectomy failure was 3.65% in Group 1 compared to 4.09% in Group 2. When utilizing two serial home-based tests, the estimated risk in Group 2 decreased to 2.87%. CONCLUSION: As home-based PVSA tests become more widely available, it is important to understand their impact. The availability of such tests may lead to improved compliance with PVSA testing. In turn, increased compliance may offer increased detection of vasectomy failure. Further study is needed with regard to the impact of home-based tests.

Protein Expression Profiles among Lichen Sclerosus Urethral Strictures-Can Urethroplasty Success be Predicted?

PURPOSE: Urethroplasty of lichen sclerosus strictures has a significantly higher failure rate than strictures due to other causes. We sought to determine predictors of urethroplasty failure in men with lichen sclerosus urethral stricture disease by evaluating protein expression profiles. MATERIALS AND METHODS: Urethral tissue was excised from patients with lichen sclerosus who were undergoing urethroplasty of urethral stricture disease at a single institution. A tissue microarray was created with cores from each sample. Immunohistochemistry was performed to compare protein expression related to inflammation, cell cycle disruption, oxidative stress, hormone receptor status and infection. Stricture recurrence was defined by the need for a subsequent unanticipated procedure for urethral stricture disease. RESULTS: We evaluated 50 men with lichen sclerosus urethral stricture disease, including 31 with successful reconstruction and 19 with recurrent stricture. Recurrent strictures expressed lower levels of several inflammatory markers and had a lower Ki-67 mitotic index and significantly higher vascular endothelial growth factor levels than nonrecurrent strictures. CONCLUSIONS: To our knowledge this is the first study to use tissue protein expression to identify risk factors for urethroplasty failure among men with lichen sclerosus urethral stricture disease. Our findings suggest that recurrent lichen sclerosus strictures demonstrate a suppressed inflammatory response, a decreased cell turnover rate, and poor oxygenation and nutrient delivery. Prospective studies are needed to clarify the role of these pathways in the pathophysiology of lichen sclerosus urethral stricture disease, determine whether preoperative biopsy can predict urethroplasty success, help counsel patients and develop future treatments.

Insights into the Pathophysiology of Urethral Stricture Disease due to Lichen Sclerosus: Comparison of Pathological Markers in Lichen Sclerosus Induced Strictures vs Nonlichen Sclerosus Induced Strictures.

PURPOSE: We evaluated the pathophysiology of lichen sclerosus and nonlichen sclerosus urethral stricture disease by comparing protein expression related to inflammation, cell cycle disruption, oxidative stress, hormone receptor status and infection. MATERIALS AND METHODS: Tissue samples were collected from the urethral strictures of 81 patients undergoing urethroplasty. Clinical and demographic data were obtained by chart review. After identifying areas pathognomonic for lichen sclerosus a tissue microarray was created with cores from each sample and immunohistochemistry was performed. RESULTS: Patients had similar baseline demographics and comorbidities. Of the 81 strictures 58 were and 23 were not due to lichen sclerosus. Lichen sclerosus strictures were significantly longer and showed higher levels of inflammation. The proportion of T cells which stained positive for CD8 was significantly higher in strictures due to lichen sclerosus (50% vs 13%, p = 0.004). CCL-4 was expressed significantly more in strictures due to lichen sclerosus (76% vs 42%, p = 0.01). Several other inflammatory markers were only found in strictures due to lichen sclerosus. Block-like p16, a surrogate for high risk human papillomavirus infection, and varicella zoster virus were found only in lichen sclerosus urethral stricture disease samples, although both were rare. Epstein-Barr virus RNA was found in significantly more lichen sclerosus samples (37% vs 10%, p = 0.024). CONCLUSIONS: To our knowledge this is the first study to evaluate protein expression in lichen sclerosus urethral stricture disease. These strictures demonstrate increased inflammation compared to nonlichen sclerosus urethral strictures. Markers of oxidative stress, cell cycle dysregulation and the androgen receptor do not appear to be uniquely associated with lichen sclerosus urethral stricture disease. Positive staining for several viruses in samples of lichen sclerosus urethral stricture disease suggests a possible infectious etiology.

MicroRNA Expression Profiles in Upper Tract Urothelial Carcinoma Differentiate Tumor Grade, Stage, and Survival: Implications for Clinical Decision-Making.

OBJECTIVE: To evaluate microRNA (miRNA) biomarkers for upper tract urothelial carcinoma (UTUC) to improve risk stratification. METHODS: miRNA was isolated from 157 radical nephroureterectomy specimens from 2 institutions. The relative expression of miRNA was examined for high grade vs low grade tumors as well as muscle invasive vs nonmuscle invasive tumors. Recurrence free survival (RFS) and overall survival (OS) were also stratified using relative expression of specific miRNA. RESULTS: The optimized model to identify high grade UTUC included miR-29b-2-5p, miR-18a-5p, miR-223-3p, and miR-199a-5p, generating a sensitivity of 83%, specificity of 85%, and generated a receiver operating characteristic (ROC) curve with area-under-the-curve of 0.86. Similarly, the model classifier for predicting ≥pT2 disease incorporated miR-10b-5p, miR-26a-5p-5p, miR-31-5p, and miR-146b-5p, producing a sensitivity of 64%, specificity of 96%, and area-under-the-curve of 0.90. RFS was best reflected by a combination of miR-10a-5p, miR-30c-5p, and miR-10b-5p, while OS was best predicted by miR-10a-5p, miR-199a-5p, miR-30c-5p, and miR-10b-5p. CONCLUSION: High-grade vs low-grade as well as muscle invasive vs nonmuscle invasive UTUC can be reliable distinguished with unique miRNA signatures. Furthermore, differential expression of UTUC miRNA produces robust classifiers for predicting RFS and OS that may help identify patients who would most benefit from adjuvant therapies.

An Analysis of Staging and Treatment Trends for Upper Tract Urothelial Carcinoma in the National Cancer Database.

BACKGROUND: The purpose of this study was to analyze contemporary trends for diagnosis and treatment of upper tract urothelial carcinoma (UTUC). PATIENTS AND METHODS: We identified all cases of UTUC in the National Cancer Database (NCDB) between 2004 and 2013. Data comprising tumor, patient, and facility factors were extracted. Treatment data for surgery and chemotherapy were also collected. Comparisons used χ2 testing. RESULTS: Over this 10-year period, the sex and age distribution of UTUC was stable at 60% male and median age of 72 years. Most tumors were < cT2 at diagnosis, with an upward trend over 10 years (66% to 72%; P < .001). However, presentation with clinical metastatic disease also rose, from 4.6% to 8.9% (P < .001). Primary tumor biopsy increased from 37% to 50%. Overall rate of nephroureterectomy decreased from 59.6% to 56.7% whereas endoscopic ablation increased from 9.8% to 11.5%. Ablation was much more common in < cT2 tumors than ≥ cT2 (18.3% vs. 3.7%) and for low-grade tumors than high-grade (22.6% vs. 5.9%). Neoadjuvant chemotherapy was significantly more used, but still at a low rate. CONCLUSION: Treatment of UTUC appears to be shifting toward conservative surgical management with tumor ablation, and increasing neoadjuvant chemotherapy use. More primary tumor biopsies are being performed, likely reflecting improved ureteroscopic instruments and training. The NCDB also reports an increase in metastatic disease, which must be interpreted cautiously and might be artifactual.

Management of Urethral Stricture and Bladder Neck Contracture Following Primary and Salvage Treatment of Prostate Cancer.

PURPOSE OF REVIEW: This article discusses the incidence, evaluation, and treatment of bladder outlet obstruction from urethral stricture, vesicourethral anastomotic stricture, and bladder neck contracture following primary and salvage treatment of prostate cancer. RECENT FINDINGS: Rates of stenosis after prostate cancer treatment appear similar across all primary treatment modalities including radical prostatectomy, radiation therapy, cryoablation, and high-intensity focused ultrasound in contemporary series. Urethral dilation and urethrotomy continue to report moderate patency rates. Urethroplasty achieves high patency rates even for long strictures, but more extensive reconstruction increases the risk of postoperative urinary incontinence. Recent AUA guidelines on urethral strictures provide new recommendations for management of these patients. All treatment options for prostate cancer carry a risk for bladder outlet obstruction, and intervention is often necessary to relieve long-lasting morbidity. Careful preoperative evaluation should be completed to assess location and extent of the stricture in order to choose optimal therapy. Endoscopic treatments, open reconstruction, and urinary diversion all play a role in relief of stenosis depending on stricture length, location, characteristics, and patient comorbidities.

Use of Alternative Techniques and Grafts in Urethroplasty.

The current management for complex urethral strictures commonly uses open reconstruction with buccal mucosa urethroplasty. However, there are multiple situations whereby buccal mucosa is inadequate (eg, pan-urethral stricture or prior buccal harvest) or inappropriate for utilization (eg, heavy tobacco use or oral radiation). Multiple options exist for use as alternatives or adjuncts to buccal mucosa in complex urethral strictures. This article reviews the current state of alternate techniques for urethral stricture treatment besides buccal mucosa, including injectable antifibrotic agents, augmentation urethroplasty with skin flaps, lingual mucosa, colonic mucosa, and new developments in tissue engineering for urethral graft material.

Short- and Long-term Complications and Outcomes of Radiation and Surgically Induced Rectourethral Fistula Repair With Buccal Mucosa Graft and Muscle Interposition Flap.

OBJECTIVE: To analyze and report 30-day, 90-day, and long-term complications and surgical outcomes over a 17-year period for anterior transperineal repair of rectourethral fistulas (RUFs) resulting from pelvic radiation and surgery. MATERIALS AND METHODS: We performed a retrospective review of patients undergoing RUF repair between January 1, 1998 and February 28, 2015, at a single institution. All RUF were repaired using an anterior transperineal approach with an interposition muscle flap and selective use of a buccal mucosa graft onlay. RESULTS: Ninety-eight patients underwent repair with an anterior transperineal approach and muscle interposition flap (49 non-radiation induced and 49 radiation or ablation induced). Thirty- and 90-day complication rates were 29% and 2%, respectively, for non-radiated RUF, and 29% and 24%, respectively, for radiated RUF. Urethral diverticula, urinary incontinence, urethral stricture, and bowel problems were delayed complications requiring surgery. At a median follow-up of 14.5 months (range 3-144), 98% (48 of 49) of non-radiated RUF were closed with 1 procedure, whereas 86% (42 of 49) of radiated RUF were closed with 1 procedure. Gastrointestinal tract continuity was restored in 94% (45 of 48) of non-radiated RUF and in 65% (30 of 46) of radiated RUF. CONCLUSION: Successful RUF closure is possible in 98% of non-radiated and in 86% of radiated or ablated patients with 1 procedure. Most radiation-induced RUF, regardless of size, can be successfully repaired with minimal short-term complications. Delayed complications may arise and require surgery, and thus continued surveillance is recommended.

Management of Radiation Anterior Prostato-symphyseal Fistulas With Interposition Rectus Abdominis Muscle Flap.

OBJECTIVE: To describe a novel, organ-sparing approach for reconstruction of radiation-induced anterior prostato-symphyseal fistulas (PSFs) at our institution over a consecutive 10-year period. MATERIALS AND METHODS: We performed a retrospective review of patients undergoing surgical reconstruction for anterior PSF between January 1, 2006 and October 31, 2015. Patient demographics as well as preoperative, operative, and postoperative data were reviewed, including etiology of fistula, surgical management, and outcomes. RESULTS: A total of 4 patients with anterior PSF underwent organ-sparing reconstruction. All fistulas were the result of previous pelvic radiation. All 4 patients presented with pubic osteomyelitis. Patients underwent pubic symphysis debridement, fistula closure, and placement of an interposition rectus abdominis muscle flap. At a median follow-up of 27 months, 100% of the patients undergoing repair with interposition rectus flap were closed with 1 procedure. CONCLUSION: Radiation-induced PSF can be successfully reconstructed with pubic symphysis debridement and fistula closure using an adjunct rectus abdominis interposition flap, avoiding prostatectomy and urinary diversion.

Rho-kinase inhibition attenuates calcium-induced contraction in ß-escin but not Triton X-100 permeabilized rabbit femoral artery.

K+-depolarization (KCl) of smooth muscle has long been known to cause Ca2+-dependent contraction, but only recently has this G protein-coupled receptor (GPCR)-independent stimulus been associated with rhoA kinase (ROCK)-dependent myosin light chain (MLC) phosphatase inhibition and Ca2+ sensitization. This study examined effects of ROCK inhibition on the concentration-response curves (CRCs) generated in femoral artery by incrementally adding increasing concentrations of KCl to intact tissues, and Ca2+ to tissues permeabilized with Triton X-100, ß-escin and α-toxin. For a comparison, tissue responses were assessed also in the presence of protein kinase C (PKC) and MLC kinase inhibition. The ROCK inhibitor H-1152 induced a strong concentration-dependent inhibition of a KCl CRC. A relatively low GF-109203X concentration (1 µM) sufficient to inhibit conventional PKC isotypes also inhibited the KCl CRC but did not affect the maximum tension. ROCK inhibitors had no effect on the Ca2+ CRC induced in Triton X-100 or α-toxin permeabilized tissues, but depressed the maximum contraction induced in ß-escin permeabilized tissue. GF-109203X at 1 µM depressed the maximum Ca2+-dependent contraction induced in α-toxin permeabilized tissue and had no effect on the Ca2+ CRC induced in Triton X-100 permeabilized tissue. The MLC kinase inhibitor wortmannin (1 µM) strongly depression the Ca2+ CRCs in tissues permeabilized with Triton X-100, α-toxin and ß-escin. H-1152 inhibited contractions induced by a single exposure to a submaximum [Ca2+] (pCa 6) in both rabbit and mouse femoral arteries. These data indicate that ß-escin permeabilized muscle preserves GPCR-independent, Ca2+- and ROCK-dependent, Ca2+ sensitization.