Stewart, Elizabeth A.

Background Uterine leiomyomas, also known as fibroids (UF), are benign neoplasms of the uterus. UF consist of smooth muscle cells and fibroblasts, and display enriched extracellular matrix (ECM). Reports estimate a cumulative incidence of 70% in premenopausal women, with annual total health care costs attributed to UF exceeding 34 billion dollars in the United States alone. In spite of their prevalence, the underlying processes that drive UF development and growth are still poorly understood. Accumulating evidence implicates the canonical Hippo signaling pathway effectors Yes‐associated protein (YAP)/transcriptional coactivator with PDZ‐binding domain (TAZ) as key drivers of fibroblast biology. Thus, we seek to assess the differences in YAP/TAZ localization, ECM deposition, and gene expression between UF and normal myometrium (Myo). Methods Matched samples of UF and Myo were obtained from premenopausal women with IRB approval. Immunohistochemistry (N=5) was performed on OCT embedded tissue with YAP/TAZ antibody. For cell culture, UF and Myo were minced and digested with collagenase type 4. Cells (passages 1–4) were plated on plastic at confluence (N=4 for both). Immunostaining for YAP/TAZ, Fibronectin (FN), Collagen Type I (COL1), and Collagen Type III (COL3) were performed (N=4–5). Transcript levels were analyzed by treating cells with control or YAP/TAZ siRNA, and performing subsequent cDNA synthesis and qPCR reactions (N=4). Kruskall‐Wallis and Mann Whitney tests were used as appropriate. Results YAP/TAZ nuclear localization was upregulated in UF when compared to Myo tissue by immunofluorescence imaging. In freshly isolated cells cultured at confluence on tissue culture plastic, baseline YAP/TAZ nuclear localization was increased in UF when compared to Myo (p=0.03). UFs also exhibited a trend toward increased FN, COL1, and COL3 deposition when compared to Myo. UF displayed increased transcript levels for several matrix and pro‐fibrotic signaling genes (COL1A1, COL3A1, CTGF, EDN1, FN1) compared to the matched Myo. Following siRNA treatment for YAP/TAZ gene expression was reduced 40% and 50% for EDN1 (encoding endothelin‐1), as well as 70% and 65% for CTGF (encoding connective tissue growth factor), in UF and Myo respectively. Conclusions UF nuclear YAP/TAZ is increased at baseline in vivo as well as in vitro . UF also display upregulated deposition of the ECM proteins. Additionally, there are increased transcript levels of profibrotic genes in UF compared to Myo. Moreover, the observed decreases in gene expression following siRNA knockdown of YAP/TAZ suggests a role for the Hippo signaling pathway in regulation of certain profibrotic genes in UF. Together these results suggest that enhanced baseline YAP/TAZ localization in UF may drive upregulated expression of pro‐fibrotic genes promoting UF progression. Support or Funding Information Center for Biomedical Discovery‐Department of Obstetrics and Gynecology Team Science Pilot Award Program This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Women with uterine fibroids (UF), benign tumors of the myometrium, have a greater prevalence of hypertension than women without UF. The mechanism of this predisposition is unclear. Individuals with borderline hypertension have endothelial dysfunction in the form of decreased endothelium‐dependent vasodilation in response to intra‐arterial acetylcholine infusion. It is possible that women with UF also demonstrate impaired endothelium‐dependent vasodilation prior to overt clinical hypertension. We measured forearm blood flow (FBF) using venous occlusion plethysmography at baseline and during intra‐arterial acetylcholine (ACH; 1.0‐4.0 μg/100 ml tissue/min) and sodium nitroprusside (SNP; 0.25‐2.00 μg/100 ml tissue/min) infusions in 4 normotensive women with UF (41±3 y) and 4 age‐matched normotensive women without UF. These women did not differ in body mass index or mean arterial pressure (97±4 vs. 103±6 mmHg, Control vs. UF; p>0.05). Baseline FBF was not different between the two groups (1.6±0.4 vs. 3.0±1.3 ml/100 ml tissue/min; Control vs. UF; p>0.05). FBF increased significantly from baseline in both groups at the highest doses of ACH and SNP; however, the change in FBF was not significantly different between groups (ACH: Δ8.6±1.6 vs. 9.5±4.1 ml/100 ml tissue/min; SNP: Δ9.8±3.0 vs. 13.1±4.6 ml/100 ml tissue/min; Control vs. UF; p>0.05). Our preliminary results suggest that FBF responses to ACH and SNP do not differ between women with and without UF; thus, impairments in endothelium‐dependent and ‐independent vasodilation do not appear to contribute to hypertension risk in normotensive women with UF. Funded by AHA and NIH.

Background Uterine leiomyomas (fibroids; UF) are benign myometrial neoplasms that produce clinically‐relevant symptoms in >25% of all women including excessive menstrual bleeding and infertility, and represent the leading cause of hysterectomy (~600,000/year in the US). Despite their importance, the mechanisms that promote UF growth are still not well‐understood. UFs are comprised of smooth muscle cells and fibroblasts with substantial extracellular matrix (ECM). While cells within UFs produce ECM, the role of ECM per se in modulating cell properties towards overall tumor growth is not known. This study tests the hypothesis that UF cells are mechanically sensitive, and that the ECM provides a positive feedback mechanism which further promotes UF growth. Methods Cells were enzymatically isolated from UFs of patients undergoing myomectomy at Mayo Clinic Rochester (IRB approved). Fibroid cells were plated onto soft (0.2kPa) vs. stiff (64kPa) polydimethylsiloxane (PDMS) elastomer substrates or standard tissue culture plastic substrates (~100GPa) for 24 h in DMEM containing 10% FBS prior to RNA isolation and qPCR analysis. Transcript levels for known ECM and profibrotic genes were determined. Nuclear localization of YAP and TAZ was visualized using immunofluorescence and quantified using a Cytation5 imaging system. Separately, 2‐color fluorescence immunohistochemistry for YAP and TAZ was performed on formalin fixed; paraffin embedded normal myometrium and fibroid tissue sections. Transciption level and nuclear localization calculations were performed with Kruskall‐Wallis and Wilcoxon Sign‐Rank tests, respectively. Results Significant increases in transcripts for ECM gene Collagen 1 ( COL1A1 ) were noted for UF cells plated on stiff PDMS and standard plastic compared to soft PDMS. The pro‐fibrotic genes Connective Tissue Growth Factor ( CTGF ), alpha smooth muscle actin ( ACTA2 ), Endothelin 1 ( EDN1 ), and plasminogen activator inhibitor 1 ( SERPINE1 ) were progressively increased in expression going from soft PDMS substrate to plastic. The anti‐fibrotic gene, cyclooxygenase 2 ( PTGS2 ) was decreased on stiff PDMS and plastic compared to soft PDMS. In cells plated on plastic, there was a significant decrease in YAP/TAZ nuclear localization in cells treated with the Rho‐kinase inhibitor Y‐27632, consistent with a mechanobiologic feedback. Immunohistochemistry showed a two‐fold increase in YAP/TAZ nuclear localization for fibroid tissue compared to normal myometrium. Conclusions Based on the growing appreciation that ECM mechanical properties contribute to UF pathobiology and fibrotic matrix deposition, we show enhanced ECM and profibrotic gene expression by fibroid cells as a function of substrate stiffness. These results demonstrate a feedback mechanism by which UFs progress through matrix stiffness‐mediated ECM deposition. Here, the mechanosensitive pathways of YAP and TAZ may be involved in transducing ECM signals to UF growth. Support or Funding Information None This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background Uterine leiomyomas (fibroids) are benign smooth muscle cell tumors of the uterus that affect a large percentage of reproductive aged women, contribute to infertility, excessive menstrual bleeding, and represent the leading cause of hysterectomy (~70%). Despite their importance, the mechanisms that promote fibroid growth are still not well‐understood. Fibroids are comprised of smooth muscle cells (SMCs) and fibroblasts and their growth is modulated by hormones including estrogen and progesterone. Intracellular calcium [Ca 2+ ] i signaling is a key aspect of SMC physiology in promoting contractility, cell proliferation, and fibrosis: aspects relevant to fibroid biology. While [Ca 2+ ] i regulation in normal myometrium is well characterized, this has not been explored in fibroids. The goal of this study was to explore [Ca 2+ ] i regulation in fibroid SMCs and the effect of sex steroids towards obtaining insights into tumor progression. Methods Cells were enzymatically isolated from normal myometrium and select fibroids of different sizes from patients undergoing myomectomy at Mayo Clinic, Rochester, MN (IRB approved). [Ca 2+ ] i was measured using fura‐2/AM and real‐time fluorescence microscopy. The contribution of Ca 2+ influx vs. intracellular pathways in the responses to 1uM oxytocin were assessed using pharmacological inhibitors/activators. Cells were pretreated with vehicle, 1nM estrogen, or 1nM progesterone to assess the effect of sex steroids. Results Fibroid‐derived cells showed a greater contribution of Ca 2+ influx pathways in their responses to oxytocin than normal myometrium. Here, the Ca 2+ ‐activated chloride channel TMEM16A (Ano1) was found to be contributory, with Western analysis showing higher levels of Ano1 in fibroids. Exposure to estrogen resulted in significantly higher [Ca 2+ ] i responses compared to cells treated with vehicle or progesterone, although progesterone did enhance [Ca 2+ ] i responses as well. Estrogen increased Ano1 expression. Cell proliferation was also increased in fibroid cells. Conclusions Exposure to estrogen and progesterone enhances oxytocin induced [Ca 2+ ] i responses of fibroid‐derived cells, potentially via increased TMEM16A (Ano1). Such increases may further contribute to the observed hormone‐induced enhancement of fibroid cell proliferation. Menstrual cycle fluctuations in reproductive aged women may thus contribute to the progression of fibroids. Greater understanding of calcium signaling pathways such as Ano1 in fibroid tissue may allow for novel therapeutic targets in uterine leiomyomas. Support or Funding Information Supported by NIH grants R25 GMO75148 (JLR), R01 HL088029 (YSP), and R01 HL56470 (YSP). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background: Quality of life (QOL) and psychological health has been reported to be decreased among women with gynecological conditions such as uterine fibroids (UFs). Materials and Methods: Women enrolled in the Comparing Options for Management: PAtient-centered REsults for Uterine Fibroids (COMPARE-UF) registry, receiving procedural therapy for symptomatic UFs, were eligible for this analysis if they completed a series of health-related QOL surveys administered at three time points (baseline, 6–12 weeks postprocedure, and 1 year postprocedure; n = 1486). Ethical approval for this study was obtained at each recruiting site and the coordinating center (NCT02260752, clinicaltrials.gov ). Results: More than 26% ( n = 393) of women reported moderate anxiety/depression on the baseline anxiety/depression domain of the Euro-QOL 5-dimension instrument. At both the 6–12 weeks and 1-year postprocedural follow-up, there was significant improvement in the UF QOL symptom severity score ( p < 0.001, p < 0.001), the total UF symptom QOL score ( p < 0.001, p < 0.001), and the Euro-QOL 5-dimension visual analog scale ( p < 0.001, p = 0.004) compared with the preprocedural baseline scores. The reporting of anxiety/depression decreased by 66.4% among women who were at baseline, whereas 5.6% of women previously reporting no anxiety/depression reported anxiety/depression at the 1-year follow-up. Conclusion: UF symptoms were more severe among women reporting anxiety/depression at baseline. At the 1-year follow-up, health-related QOL scores improved among all women and the prevalence of anxiety/depression decreased in most, but not all women, whereas severity of anxiety/depression worsened in a small percentage of women (5.6%). Overall, these results suggest that UF treatment improves symptoms of anxiety/depression associated with symptomatic UFs.

Abstract Objective: The incidence of carpal tunnel syndrome (CTS) is higher in women, and peaks around the age of menopause. Therefore, we investigated whether bilateral oophorectomy is associated with an increased risk of severe CTS. Methods: We included all of the 1,653 premenopausal women who underwent bilateral oophorectomy for a nonmalignant indication between 1988 and 2007, and a random sample of 1,653 age-matched referent women who did not undergo bilateral oophorectomy in Olmsted County, MN. Diagnoses of CTS assigned to women over their entire lifetime were identified in these two cohorts. The risk of de novo severe CTS after bilateral oophorectomy (or index date) was evaluated using Cox proportional hazards models adjusted for potential confounders. Results: Bilateral oophorectomy was associated with an increased risk of severe CTS (adjusted hazard ratio 1.65, 95% confidence interval 1.20-2.25). The risk was suggestively greater in women with lower body mass index, nulliparity, and with a benign ovarian indication for oophorectomy (nonsignificant interactions). We did not observe a protective effect of estrogen therapy after the oophorectomy. The findings were similar in secondary analyses considering the incidence of CTS of any severity or idiopathic CTS. Conclusions: The risk of severe CTS, common in perimenopausal women, is increased after bilateral oophorectomy. The association may be causal or due to confounding. Therefore, the precise biological mechanisms explaining the association and the absence of a mitigating effect of estrogen therapy should be further investigated.

Background: To evaluate differences in the proportion of uterine fibroid (UF) treatments that are uterine-sparing between Black women and White women and identify factors that could explain disparities. Methods: Women at age 18–54 years who were enrolled from 10 clinical sites in the United States into the Comparing Options for Management: Patient-Centered Results for UFs (COMPARE-UF) treatment registry completed questionnaires before their UF procedure. UF symptoms and quality of life were assessed by questionnaires. Details on UF imaging and treatment (hysterectomy, myomectomy, or uterine artery embolization [UAE]) were collected from each patient's medical record. Random-effects logistic regression was used to assess the association between race and the odds of having a uterine-sparing procedure versus hysterectomy. Subgroup analyses compared each uterine-sparing procedure with hysterectomy. Results: In this cohort of 1141 White women and 1196 Black women, Black women tended to be younger (median 41.0 vs. 42.0 years) and report worse symptoms, pain, and function on every scale compared with White women. Black women were more likely to have had a prior UF treatment compared with White women (22.8% vs. 14.6%). White women had more hysterectomies (43.6% vs. 32.2%) and myomectomies (50.9% vs. 50.2%) versus Black women. Black women had more UAEs (15.1% vs. 4.7%) than White women. After adjusting for clinical site and other variables, Black women had greater odds than White women of having a myomectomy (odds ratio [OR] = 2.41, 95% confidence interval [CI] = 1.63–3.56) or a UAE versus hysterectomy (OR = 4.24, 95% CI = 2.41–7.46). Conclusion: In these participants, Black women were more likely to schedule a uterine-sparing UF treatment and a nonsurgical UF treatment than their White counterparts; this may not be true for all women. Longer comparative effectiveness studies are needed to inform women about the durability of UF treatments. Greater understanding of factors influencing treatment selection is needed as are studies that include women without access to tertiary care centers. Clinical Trial Registration: Clinicaltrials.gov, NCT02260752 (enrollment start: November 2015).

AbstractAdenomyosis is a poorly understood and clinically underappreciated gynecologic disorder associated with substantial morbidity including dysmenorrhea, pelvic pain, heavy menstrual bleeding, infertility, and poor pregnancy outcomes. Substantial gaps persist in our understanding of essentially all aspects of this disorder – epidemiology, risk factors, pathogenesis, pathophysiology, diagnosis, and treatment. In this article, we summarize current thoughts on future directions in basic, translational, and clinical adenomyosis research.