Search

The aim of this study was to assess the molecular effect of ulipristal acetate (UPA) on gene expression in myometrium and endometrium of patients with symptomatic fibroids. Tissues isolated from four women treated preoperatively with UPA (5 mg) were compared to those from untreated controls using NanoString platform to assess the expression of 75 candidate genes modulated by UPA and ovarian steroids. Deregulated genes were then validated by real-time PCR. In myometrium, UPA exerted an antagonistic effect similar to that observed in fibroids. In UPA-treated endometrium, six genes were identified as highly and significantly upregulated, including matricellular genes CCN1 (54-fold, P = 0.0018) and CCN2 (11-fold, P = 0.00044), Krüppel-like factor 4 (>3-fold, P = 0.0036), and mast cell markers including tryptases TPSAB1/TPSB2 (31-fold, P = 0.023) and carboxypeptidase A ( CPA3 , 17-fold, P = 0.05). In endometrium, UPA induced the expression of genes involved in fibrogenesis and mast cell function-some of them being widely involved in hepatic injury, which could explain the marked fibrosis and inflammatory cell infiltration observed in explanted livers from patients under UPA treatment.

Organotypic cultures of tissue slices have been successfully established in lung, prostate, colon, gastric and breast cancer among other malignancies, but until now an ex vivo model based on tissue slices has not been established for uterine leiomyoma. In the present study, we describe a method for culturing tumour slides onto an alginate scaffold. Morphological integrity of tissue slices was maintained for up to 7 days of culture, with cells expressing desmin, estrogen and progesterone receptors. Driver mutations were present in the ex vivo slices at all-time points analyzed. Cultivated tumour slices responded to ovarian hormones stimulation upregulating the expression of genes involved in leiomyoma pathogenesis. This tissue model preserves extracellular matrix, cellular diversity and genetic background simulating more in-vivo-like situations. As a novelty, this platform allows encapsulation of microspheres containing drugs that can be tested on the ex vivo tumour slices. After optimizing drug release rates, microspheres would then be directly tested in animal models through local injection.

Objective: To study the expression levels of tachykinins and tachykinin receptors in uterine leiomyomas and matched myometrium., Design: Laboratory study., Setting: University research laboratories and academic hospital., Patient(s): Women undergoing hysterectomy for symptomatic leiomyomas., Intervention(s): Quantitative polymerase chain reaction, immunohistochemistry and Western blot., Main Outcome Measure(s): Expression and tissue immunostaining of substance P, neurokinin A, hemokinin-1, neurokinin 1 receptor full-length (NK1R-Fl) and truncated (NK1R-Tr) isoforms, and neurokinin 2 receptor (NK2R) in paired samples of leiomyoma and adjacent normal myometrium., Result(s): TAC1 messenger RNA (mRNA) was significantly up-regulated in leiomyomas, whereas intense immunoreaction for the three peptides was particularly abundant in connective tissue cells. Differential regulation of TACR1 mRNA was observed, and at the protein level there was a significant increased expression of NK1R short isoform (NK1R-Tr). TACR2 mRNA was significantly up-regulated in leiomyomas, although levels of NK2R protein were similar in normal and tumor cells., Conclusion(s): These and our previous data demonstrate that the whole tachykinin system is differentially regulated in leiomyomas. The increased expression of NK1R-Tr might stimulate leiomyoma growth in a similar way to that observed in other steroid-dependent tumors., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Study Question: Are the vasoactive peptide neurokinin B (NKB) and its preferred NK3 receptor (NK3R) differentially expressed in leiomyomas compared with normal myometrium?, Summary Answer: In leiomyomas, NKB is up-regulated and delocalized, while its preferred NK3R is also differentially regulated., What Is Known Already: The expression of NKB/NK3R in the central nervous system is essential for proper function of the human reproductive axis. Additionally, this system is also widely expressed throughout the female genital tract. Leiomyomas impair fertility and are a major source of abnormal uterine bleeding. The aberrant synthesis of local factors can contribute to the pathological symptoms observed in women with leiomyomata. NKB could be one of these factors, since a vasoactive role of this peptide at a peripheral level has been observed in different systems and species, including humans. NK3R is strongly regulated by estrogens and its activation leads to nuclear translocation affecting chromatin structure and gene expression., Study Design, Size, Duration: Samples were obtained between 2006 and 2012 from 28 women of reproductive age at different stages of the menstrual cycle by hysterectomy. Leiomyomas and matched macroscopically normal myometrium from each woman were analysed in vitro., Participants/materials, Setting, Methods: RT-PCR, quantitative real time, immunohistochemistry and in situ hybridization were used to investigate the pattern of expression of NKB/NK3R in tissue samples., Main Results and the Role of Chance: Expression of the gene encoding NKB (TAC3) was up-regulated 20-fold in leiomyomas, compared with matched myometrium (P = 0.0008). In tumour tissue, not only connective cells, but also myometrial, endothelial and vascular smooth muscle cells express TAC3 mRNA. Immunoreactivity to NKB was preferentially located in the smooth muscle cell nuclei from normal myometrium in the secretory phase, unlike matched leiomyoma, which showed a predominant cytoplasmic expression pattern. In the normal myometrium, TACR3 mRNA showed variable expression throughout the menstrual phases, with samples showing strong, reduced or no amplification. In leiomyoma, TACR3 was significantly up-regulated compared with matched myometrium (P = 0.0349)., Limitations, Reasons for Caution: This study is descriptive and although we observed clear differential regulation of the NKB/NK3R system at mRNA and immunohistochemical staining levels in leiomyoma, future functional studies are needed to determine the precise role of NKB in the myometrium in normal and pathological conditions. In addition, further analysis (e.g. in cell culture models) will be required to determine the role of NKB in the nucleus of normal smooth muscle cells, whether nuclear translocation is mediated by NK3R and the consequences of the cytoplasmic expression of NKB in tumour cells., Wider Implications of the Findings: The NKB/NK3R system dysregulation observed in leiomyoma may contribute to the pathological symptoms observed in women with leiomyomata.

Deregulated steroids are involved in different hormone-dependent tumors, including benign and malignant uterine neoplasms. Leiomyomas (LM) are estrogen and progesterone-dependent benign tumors, whereas "bizarre or atypical LMs" (AL) are considered a subgroup of LM and clinically benign, although their malignant potential is suspect. Uterine leiomyosarcomas (LMS) are malignant smooth muscle tumors, and ovarian steroids may control their growth. Estrogen effects are mediated by 2 receptors, estrogen receptors (ER) α and β, and the ratio of both receptors seems to be a critical parameter in the estrogen-mediated carcinogenic process. Estradiol induces the expression of neurotensin (NTS), and the coupling of this peptide with its high-affinity receptor, NTS1, has been involved in the regulation of tumoral cell growth. Given the importance of these markers in tumor development, we aim to determine the status of ERα and ERβ in the myometrium and LM, AL, and LMS, concomitantly with the expression of NTS/NTS receptor 1 in these tumors. For that purpose, we use immunohistochemistry for all markers analyzed and in-situ hybridization to detect NTS mRNA. These data suggest that LMS are estrogen-dependent tumors, which may use NTS as an autocrine growth factor. In addition, the phenotype of AL with regard to ERα and ERβ status and NTS expression is closer to LMS than LM; thus, a potential malignization of this tumor is feasible.

Leiomyomas or fibroids are the most frequently diagnosed tumors of the female genital tract, and their growth seems to be steroid-hormone dependent by a yet undetermined cellular and molecular mechanism. Sexual hormones induce the secretion of growth factor peptides and the expression of their receptors, stimulating cell proliferation. One of these factors is neurotensin, and increasing evidence suggests that it can promote growth of different cancer cells. Since there are no data on neurotensin expression in normal and tumoral uterine tissue, we have analyzed the expression of NTS and NTSR1 receptor using immunohistochemistry for protein detection, in situ hybridization to detect cells expressing NTS mRNA, and RT-PCR to detect NTSR1 transcript as well as any of the alternative splice variants recently described for this receptor. We found that NTS and NTSR1 are expressed in connective cells of normal myometrium. In leiomyomas, immunoreactivity for NTS and NTSR1 receptor is colocalized in the smooth muscle cells that are also transcribing NTS. Women receiving high doses of steroids for in vitro fertilization showed tumor growth and increased immunoreactivity for neurotensin and NTSR1 receptor. Interestingly, alternative splice variants of NTSR1 receptor were detected only in tumoral tissue. These findings suggest a role of steroid hormones inducing neurotensin expression in leiomyoma smooth muscle cells. In these cells, NTS could act autocrinally through NTSR1 receptor, promoting their proliferation.

Objective: Cellular and subcellular localization of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in uterine leiomyomas., Design: Retrospective study., Setting: University of La Laguna (ULL) and Canary University Hospital (HUC)., Patient(s): Premenopausal and postmenopausal women with uterine leiomyomas., Intervention(s): Hysterectomy and myomectomy., Result(s): Estrogen receptor alpha was only present in smooth muscle cells with variation in the subcellular location in different leiomyomas. Estrogen receptor beta was widely distributed in smooth muscle, endothelial, and connective tissue cells with nuclear location in all cases studied; variations were only found in the muscle cells for this receptor., Conclusion(s): Estrogens operate in leiomyoma smooth muscle cells through different receptors, alpha and beta. However they only act through the ERbeta in endothelial and connective cells.