Unaffected Myometrium Research Articles

Current possibilities of ultrasonography in diagnosing local spread of endometrial cancer

Background. The incidence of oncologic pathology in the female reproductive system is continually rising, highlighting the importance of finding new methods for early and precise diagnosis. This study aimed to determine the invasion depth of endometrial cancer through contrast-enhanced ultrasonography.
 Materials and methods. The study included 38 patients who were confirmed to have endometrial cancer. All patients underwent complex ultrasound examinations in real-time and grayscale mode. The qualitative assessment of vascularization in color and energy Doppler mapping modes was performed using contrast enhancement with the assessment of qualitative and quantitative parameters of contrast.
 Results. The results were compared with histological examination results from postoperative materials. The study identified contrasting qualitative parameters that are most indicative of endometrial cancer and quantitative parameters that are statistically significant and reliably distinguished the myometrium affected by the tumor from the unaffected myometrium (including peak intensity, time to peak intensity, and contrast half-life).
 Conclusions. Contrast enhancement increases the effectiveness of comprehensive ultrasonography in determining the invasion depth of endometrial cancer.

Sex steroid hormone receptors of telocytes - potential key role in leiomyoma development.

Uterine leiomyoma is the most widespread benign tumor affecting women of childbearing age. There are still gaps in the understanding of its pathogenesis. Telocytes are unique cells found in more than 50 different locations inside the human body. The functional relationship between cells could clarify the pathogenesis of leiomyomata. Examination of membrane receptors on telocytes could explain their role in fibrosis, oxidative stress, and myometrial contractility. This research was conducted to assess the density of telocytes in terms of their putative role in leiomyoma formation by focusing on their correlation with the expression of estrogen and progesterone receptors. For gross evaluation of uterine tissue samples from leiomyoma, routine histology of adjacent and unaffected myometrium was performed. Immunohistochemical analysis of c-kit, tryptase, CD34, PDGFRα (telocyte-specific), and ER and PRs (estrogen and progesterone receptors) was performed to examine uterine telocytes and the expression of sex steroid receptors. The decline in telocyte density in leiomyoma foci was correlated with high progesterone expression and low estrogen receptor expression. The unchanged myometrium showed the opposite correlation and balance between both steroid hormone receptors. The difference in sex steroid receptor expression is correlated with the density of uterine telocytes, which emphasizes their conductor function. A reduction in telocyte density and the changes in examined marker expression demonstrate the involvement of telocytes in local homeostasis. The expression of membrane receptors explicitly indicates their functional potential in the human myometrium, focusing attention on contractility and local homeostasis.

1739 Ultrasound Elastography for Gynecological Applications: Preliminary Reliability Analysis

Study Objective This preliminary investigation aimed to evaluate the feasibility of using ultrasound elastography (UE) to evaluate the myometrial and uterine leiomyoma stiffness. Our objectives were to (1) assess the reliability of UE stiffness measures of non-neoplastic myometrium, and (2) compare UE stiffness measures of non-neoplastic myometrium and leiomyomas. Design This preliminary investigation used a prospective, observational design. Setting Motor Function Measurement Laboratory at the University of Ottawa. Patients or Participants Nine healthy women with no gynecologic history (control) and one woman (case) with two asymptomatic uterine leiomyomas (FIGO 6) were recruited from the local community. Interventions Three trials of a 2D UE cineloops were acquired transvaginally (Aixplorer, SuperSonic Imagine) at the frontal midsagittal myometrium and in the sagittal plane of each leiomyoma. The procedure was repeated twice at the first visit (V1S1, V1S2) and again 2-3 days later (V2S1). Participants completed the menstrual bleeding questionnaire (MBQ) at V1S1. Measurements and Main Results Descriptive statistics were used to evaluate normative peak myometrial stiffness, and the reliability of peak tissue stiffness (kPa), averaged across three trials (10 frames/trial), was evaluated using intraclass correlation coefficients (ICCs). The control group (age=30.2±4.4 years, MBQ=11±9) had median parity of 0 (range: 0-1), while the case participant (age=31.4 years, MBQ=13) had parity of one. Peak myometrial tissue stiffness (V1S1=35.9±9.6 kPa, V1S2=34.1±8.8kPa, V2S1=32.7±11.9kPa) exhibited excellent within-session reliability (ICCV1S1=0.89, ICCV1S2=0.92, ICCV2S1=0.96), and good between-session (ICC=0.71) and between-day (ICC=0.76) reliability. The 95% prediction interval for normative myometrial tissue, computed using values recorded from the control group was 18.5-50.0kPa. The peak stiffness of both leiomyomas (Fibroid A = 111.4kPa; Fibroid B = 84.0kPa) well exceeded the 95% prediction interval for unaffected myometrium. Conclusion UE stiffness exhibited good to excellent reliability in non-neoplastic myometrial tissue, and much higher stiffness was observed in the two asymptomatic fibroids, suggesting that UE may have clinical value in this gynecologic population.

Highly heterogeneous genomic landscape of uterine leiomyomas by whole exome sequencing and genome-wide arrays

To determine the genomic signatures of human uterine leiomyomas and prevalence of MED12 mutations in human uterine leiomyosarcomas. Retrospective cohort study. Not applicable. This study included a set of 16 fresh frozen leiomyoma and corresponding unaffected myometrium specimens as well as 153 leiomyosarcomas collected from women diagnosed with uterine leiomyomas or leiomyosarcomas who underwent clinically indicated abdominal hysterectomy. None. Whole exome sequencing and high-resolution X-chromosome and whole genome single nucleotide polymorphism microarray analyses were performed on leiomyoma samples negative for the known MED12 mutations and compared with their corresponding myometrium. Leiomyosarcoma specimens were examined for exon 2 MED12 mutations to evaluate the frequency of MED12 mutated leiomyosarcomas. Our results indicate remarkable genomic heterogeneity of leiomyoma lesions. MED12-negative leiomyomas contain copy number alterations involving the Mediator complex subunits such as MED8, MED18, CDK8, and long intergenic nonprotein coding RNA340 (CASC15), which may affect the Mediator architecture and/or its transcriptional activity. We also identified mutations in a number of genes that were implicated in leiomyomagenesis such as COL4A6, DCN, and AHR, as well as novel genes: NRG1, ADAM18, HUWE1, FBXW4, FBXL13, and CAPRIN1. Mutations in genes implicated in cell-to-cell interactions and remodeling of the extracellular matrix and genomic aberrations involving genes coding for the Mediator complex subunits were identified in uterine leiomyomas. Additionally, we discovered that ∼4.6% of leiomyosarcomas harbored MED12 exon 2 mutations, but the relevance of this association with molecular pathogenesis of leiomyosarcoma remains unknown.

Possible roles of oxytocin receptor and vasopressin-1α receptor in the pathomechanism of dysperistalsis and dysmenorrhea in patients with adenomyosis uteri

To investigate the expression of oxytocin (OTR) and/or vasopressin (VP1αR) receptor in patients with and without adenomyosis uteri. Retrospective nonrandomized study. University hospital endometriosis research center. Forty patients with histologically proven adenomyosis and 40 patients without adenomyosis who had undergone hysterectomy for dysmenorrhea, bleeding disorders, and fibroids. Immunohistochemical examination of both OTR and VP1αR expression in endometrium, myometrium, and adenomyotic lesions, and identification of smooth muscle cells using antibodies against OTR, VP1αR, and smooth muscle actin (sm-actin). The immunoreactive score (IRS) was used for expression of OTR, VP1αR, and sm-actin. Expression of OTR in epithelial cells of adenomyotic lesions and surrounding myometrial cells was detectable. VP1αR was expressed only in myometrial cells and blood vessels. Using a specific anti-sm-actin antibody, another spindle cell population was characterized to represent smooth muscle cells which are in direct contact with the adenomyotic stroma. Compared with the unaffected myometrium, the surrounding adenomyosis-associated myometrium overexpressed OTR and showed changes in morphology. In the uteri of patients with adenomyosis, the junctional zone was often seen to be quite fissured. In addition to the specific expression of VP1αR, OTR expression and morphologic changes in the myometrial architecture of uteri having adenomyosis support the hypothesis that dysperistalsis plays an essential role in the development of endometriosis and dysmenorrhea. In the near future, specific inhibition of this receptor might yield a promising treatment for therapy.

Comparison of Human and Rat Uterine Leiomyomata: Identification of a Dysregulated Mammalian Target of Rapamycin Pathway

Uterine leiomyomata, or fibroids, are benign tumors of the uterine myometrium that significantly affect up to 30% of reproductive-age women. Despite being the primary cause of hysterectomy in the United States, accounting for up to 200,000 procedures annually, the etiology of leiomyoma remains largely unknown. As a basis for understanding leiomyoma pathogenesis and identifying targets for pharmacotherapy, we conducted transcriptional profiling of leiomyoma and unaffected myometrium from humans and Eker rats, the best characterized preclinical model of leiomyomata. A global comparison of mRNA from leiomyoma versus myometrium in human and rat identified a highly significant overlap of dysregulated gene expression in leiomyomata. An unbiased pathway analysis using a method of gene-set enrichment based on the sigPathway algorithm detected the mammalian target of rapamycin (mTOR) pathway as one of the most highly up-regulated pathways in both human and rat tumors. To validate this pathway as a therapeutic target for uterine leiomyomata, preclinical studies were conducted in Eker rats. These rats develop uterine leiomyomata as a consequence of loss of Tsc2 function and up-regulation of mTOR signaling. Inhibition of mTOR in female Eker rats with the rapamycin analogue WAY-129327 for 2 weeks decreased mTOR signaling and cell proliferation in tumors, and treatment for 4 months significantly decreased tumor incidence, multiplicity, and size. These results identify dysregulated mTOR signaling as a component of leiomyoma etiology across species and directly show the dependence of uterine leiomyomata with activated mTOR on this signaling pathway for growth.

Expression profile of several genes in human myometrium and uterine leiomyoma

To screen several genes that are differentially expressed in uterine leiomyoma and matched unaffected myometrium by using microarray-based hybridization and real-time polymerase chain reaction analyses. Screen by arrays for < or =2,400 known genes in leiomyoma and control myometrium. University clinical research laboratory. Four patients with leiomyoma scheduled for surgery during the proliferative phase. Four paired samples of leiomyoma and adjacent myometrium were obtained from patients undergoing hysterectomy. Fold-changes in expression of leiomyoma and matched myometrium (L/M). A comparison of expression patterns revealed 73 genes significantly up- or down-regulated in each paired tissue sample, of which 30 genes showed increased expression (mean L/M of >2) and 43 showed decreased expression (mean L/M of <0.5) in leiomyoma compared with normal myometrium. When considering only growth factors, pleiotropin (PTN) was expressed 3.5-fold more in leiomyomas compared with in myometrium. No other growth factors were similarly affected. In addition, real-time polymerase chain reaction analysis correlated well with microarray data. Data obtained from the present study suggest that several genes are selectively overexpressed in leiomyomas compared with in myometrium. Increased expression of growth factor PTN may represent a promising target for therapy.

Gene expression profile of leiomyoma and myometrium and the effect of gonadotropin releasing hormone analogue therapy.

To determine the profile of differentially expressed genes in leiomyoma and matched unaffected myometrium and to identify the genes whose expression are altered after gonadotropin releasing hormone analogue (GnRHa) therapy. Using total RNA isolated from untreated and GnRHa-treated leiomyoma and myometrium subjected to Clontech Atlas 1.2 K cancer microarray containing 1176 known genes, we found transcripts for many cytokines, growth factors and their receptors, adhesion molecules, extracellular matrix, proteases, signaling intermediates, and transcription factors in both tissues. Based on the overall normalization and reproducibility of the results, 328 differentially expressed and regulated genes with at least threefold change in their expression in untreated and GnRHa-treated tissues were chosen for further analysis. The expression value of the 328 genes subjected to significance analysis of microarray showed a total of 100 genes with relative significant change in expression. Of these genes, the expression of 18 was up-regulated and 82 down-regulated in untreated leiomyoma compared with myometrium. In GnRHa-treated tissues, the expression of 34 genes showed a significant decrease in leiomyoma, whereas 27 genes increased and 15 decreased in myometrium compared with their respective untreated tissues. There was no difference in the expression profile of these genes when comparing leiomyoma and myometrium from the GnRHa-treated group. Hierarchical cluster analysis revealed that these differentially expressed or regulated genes, classified based on their biologic functions, are involved in regulation of cell growth/cycle, signal transduction, transcription factors, and cell and tissue structure. Microarray analysis allowed us to identify the profile expression of several specific genes in leiomyoma and myometrium whose expression appears to be differentially regulated after GnRHa therapy.

Gonadotropin releasing hormone analogue therapy alters signal transduction pathways involving mitogen-activated protein and focal adhesion kinases in leiomyoma

Because gonadotropin releasing hormone analogue (GnRHa) therapy often causes leiomyoma regression, in part through alteration of growth factor and receptor expression, we determined whether GnRHa therapy alters the expression of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK), which are linked to intracellular signaling pathways activated by ovarian steroids, growth factors, and adhesion molecules. Leiomyoma and matched unaffected myometrium were collected from women who received GnRHa therapy (n = 5) and untreated women (n = 10). We determined the expression of ERK1, ERK2, FAK, phosphorylated ERK (pERK1/2), and pFAK using Western blotting and immunohistochemical analysis. Leiomyoma and myometrium expressed ERK1 (44 kD), ERK2 (42 kD), and FAK (125 kD) at variable levels with increased ERK2, pERK, and FAK expression in leiomyoma. We found that GnRHa therapy resulted in a noticeable decrease in ERK2 and FAK, with significant reduction in pERK1/2 and low or undetectable levels of pFAK in both leiomyoma and myometrium compared with the untreated group (P <.05). Immunohistochemically ERK1, ERK2, FAK, pERK1/2, and pFAK were localized in smooth muscle cells and connective tissue fibroblasts in GnRHa-treated and untreated leiomyoma and myometrium, with considerable reduction in their intensity as indicated by HScore in GnRHa-treated tissues. The data provide further evidence that leiomyoma regression induced by GnRHa is mediated in part through a mechanism involving suppression of signal transduction pathways involving growth factors or ovarian steroid and adhesion molecules.

Increased expression of stromelysin 3 mRNA in leiomyomas (uterine fibroids) compared with myometrium.

To determine the level of expression of selected matrix metalloproteinases in uterine leiomyoma compared with unaffected myometrium in an effort to explain the abnormal accumulation of extracellular matrix in the leiomyoma. The levels of matrix metalloproteinase (MMP) mRNA in leiomyoma and myometrium were measured in samples from 22 patients during either proliferative (n = 6) or secretory phases (n = 16) of the menstrual cycle. Relative amounts of collagenase (MMP-1) and stromelysin (MMP-3) mRNAs were measured by Northern blot analysis, and amounts of stromelysin 3 (MMP-11) and matrilysin (MMP-7) mRNA from each sample were determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to beta-actin mRNA. The levels of MMP-1 and MMP-3 mRNAs were similar in both leiomyoma and unaffected myometrium. The levels of MMP-11 mRNA were twofold greater in leiomyoma compared with myometrium throughout the menstrual cycle, and the differences in the levels of MMP-11 were significantly different during the secretory phase. The level of MMP-7 mRNA expression was similar in leiomyoma, myometrium, and endometrium. Among the metalloproteinases examined in this study, only the levels of MMP-11 mRNA were elevated in leiomyoma compared with myometrium. The increased expression of MMP-11 in uterine leiomyoma compared with myometrium is analagous to previously reported elevations of MMP-11 mRNA in dermatofibromas compared with unaffected skin. The increased expression of MMP-11 mRNA in fibroid tumors suggests that this MMP may be involved in the formation of a more fibrous extracellular matrix in leiomyoma relative to unaffected myometrium.

Differential expression of matrix metalloproteinases and their tissue inhibitors in leiomyomata: a mechanism for gonadotrophin releasing hormone agonist-induced tumour regression.

Tissue remodelling involving extracellular matrix (ECM) turnover plays a major role in leiomyoma growth and regression, regulated by the combined action of matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs). We postulated that leiomyomata express MMP and TIMP mRNA and protein, and their expression is inversely regulated during tumour growth and gonadotrophin releasing hormone agonist (GnRHa)-induced regression. We therefore examined the expression of mRNA and protein for MMPs (interstitial collagenase, MMP-1; gelatinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and TIMPs (TIMP-1 and TIMP-2) in leiomyoma and matched unaffected myometrium from GnRHa (lupron)-treated and untreated patients. Reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme analysis revealed that leiomyomata and myometrium expressed MMP-1, -2, -3 and -9, as well as TIMP-1 and -2 mRNA. Quantitative RT-PCR indicated that leiomyomata and myometrium during the secretory phase of the menstrual cycle expressed higher levels of MMP and TIMP mRNA compared to the proliferative phase (P < 0.05), with low to undetectable levels of MMP-1, -2 and -3 mRNA in the tumours. GnRHa therapy induced an overall reduction in MMP and TIMP mRNA expression in both leiomyomata and myometrium, but a significant decrease in TIMP-1, and an increase in MMP mRNA expression compared with untreated tumours (P < 0.05). Immunohistochemically, MMP-1, -2, -3 and -9 and TIMP-1 and -2 proteins were localized in leiomyomata and myometrial smooth muscle cells, arteriole wall and connective tissue fibroblasts, with an overall increase in MMP and a decrease in TIMP staining intensity in GnRHa-treated groups. The results suggest that MMP and TIMP expression in leiomyoma and myometrium are hormonally regulated, and that GnRHa-induced tumour regression is accompanied by an increase in MMP expression with a concomitant decrease in TIMP-1 expression, which may potentially provide an environment favouring ECM degradation.

Suppression of transforming growth factor-beta (TGF beta) and TGF beta receptor messenger ribonucleic acid and protein expression in leiomyomata in women receiving gonadotropin-releasing hormone agonist therapy.

The expression and cellular distribution of transforming growth factor-1 (TGF beta 1) through TGF beta 3 and TGF beta type I-III receptor messenger ribonucleic acid (mRNA) and protein were analyzed in leiomyomata from patients receiving GnRH agonist (GnRHa; leuprolide acetate) compared to those in untreated controls. Standard reverse transcription-PCR revealed that the unaffected myometrium and leiomyomata from leuprolide-treated and untreated patients express TGF beta 1-3 and TGF beta type I-III receptor mRNA. The myometrial and leiomyomata smooth muscle cells were the primary site of TGF beta 1-3 and TGF beta type I and II receptor mRNA and protein expression, as determined by in situ hybridization and immunohistochemical localization. These observations indicate that leiomyomata express a higher of level of TGF beta and TGF beta receptor mRNA and protein than unaffected myometrium during the secretory phase of the menstrual cycle, and women who received leuprolide acetate therapy had a substantially lower level of expression than untreated controls. Furthermore, competition-based quantitative reverse transcription-PCR using synthetic internal standards revealed that leiomyomata express a significantly higher number (copies per cell) of TGF beta type II receptor mRNA, followed by TGF beta 1, TGF beta type I receptor, TGF beta 2, and TGF beta 3 (P < 0.05). However, there was a significant decrease in the levels (copies per cell) of TGF beta 1, TGF beta 3, and TGF beta type I and type II receptor mRNA expression in leiomyomata from leuprolide-treated compared to untreated patients (P < 0.05). The data provide further evidence that leiomyomata express mRNA and protein for all components of the TGF beta system, and GnRHa therapy results in down-regulation of their expression. More specifically, these data suggest that TGF beta 1 and TGF beta 3 may play a more important role in leiomyomata growth than TGF beta 2, which leads us to propose that lowering TGF beta and receptor expression may have a direct effect on leiomyomata regression.

Gonadotropin-releasing hormone (GnRH) and GnRH receptor gene expression in human myometrium and leiomyomata and the direct action of GnRH analogs on myometrial smooth muscle cells and interaction with ovarian steroids in vitro.

The objective of the present study was to determine whether GnRH and GnRH receptor are expressed in myometrium and leiomyomata, and if GnRH analogs alone or in the presence of ovarian steroids can modulate the rate of DNA synthesis, proliferation, and transforming growth factor-beta 1 (TGF beta 1) production in myometrial smooth muscle cells in vitro. Reverse transcription-PCR revealed that leiomyomata, unaffected myometrium, and isolated myometrial smooth muscle cells express GnRH and GnRH receptor messenger ribonucleic acid. Furthermore, in a dose-dependent manner, GnRH agonist (leuprolide acetate) inhibited, but GnRH antagonist [D-pGlu1,D-Phe2,D-Trp3.6] (GnRH-Ant1) stimulated, the rate of [3H]thymidine incorporation into myometrial smooth muscle cells (P < 0.05), whereas GnRH-Ant2 (Ac-D-P-Cl-Phe1.2,D-Trp3,D-Arg6,D-Ala10) had no effect. 17 beta-Estradiol (E2) medroxyprogesterone acetate (MPA), and E2 plus MPA (1 micromol/L) stimulated the rate of DNA synthesis by smooth muscle cells (P < 0.05), which was inhibited by GnRH analogs used at 5 micromol/L (P < 0.05). GnRH analogs had no significant effect on myometrial smooth muscle cell proliferation, with the exception of GnRH-Ant1; however, they inhibited the stimulatory action of E2, MPA, and E2 plus MPA in a time-dependent manner (P < 0.05). These cells also synthesized and released approximately 1.32 +/- 0.02 ng/mL total (active plus latent) TGF beta 1, of which 0.73 +/- 0.02 ng/mL was in an active form. E2, MPA, E2 plus MPA, and GnRH analog treatments resulted in an increase in total TGF beta 1 production, whereas GnRH agonist and GnRH-Ant2, but not GnRH-An1, inhibited active TGF beta 1 (P < 0.05). GnRH analogs also inhibited the action of E2 plus MPA on total and active TGF beta 1 production, whereas GnRH-Ant1 further stimulated E2, MPA, or E2 plus MPA action on active TGF beta 1 production (P < 0.05). The data demonstrate for the first time that GnRH and GnRH receptor messenger ribonucleic acid are expressed in myometrium, leiomyomata, and myometrial smooth muscle cells. The local expression of GnRH and receptor along with the direct action of GnRH analogs on the smooth muscle cell DNA synthesis and TGF beta 1 production suggest an autocrine/paracrine role for GnRH in these tissues, a mechanism that may be involved in leiomyomata regression in women receiving GnRH agonist therapy.