Adjacent Myometrium Research Articles

The Potential of Transforming Growth Factor-Beta1 (TGFβ1) in Fibrotic Tissue of Leiomyoma Specimens

Background: Fibrosis can define as the development of excessive fibrous tissue in an organ or tissue. In the situation of the uterus, leiomyoma or scar tissue may involve fibrosis state. The main feature of fibrosis in myoma is the existence of disordered smooth muscle cells and collagen that can be recognized via histological examinations in addition to extracellular component deposition. Leiomyoma is the most public uterine disorder that is mainly consists of smooth muscle cells, myo-fibroblast, and an obvious content of extracellular matrix. The myofibroblast in myoma lesion produced an elevated content of ECM. This paper displays the fibrotic circumstantial in leiomyoma and the mechanisms related to deposition of ECM components. Methods: this study involved (50) patients of 18-80 years old women diagnosed with benign uterine fibroid. Tumor samples were attained by consent from women undergoing hysterectomy or myomectomy at the Al Karama Teaching Hospitals in Kut, Wasit Province, Iraq between October 2022 to March 2023. samples from normal and fibroid were subjected to routein histological processing and subsequently stained with routein stains for general description, Masson Trichrome stain, Van Gieson's stain to visualize collagen content and histochemical stain (Alcian Blue-Periodic Acid Schiff) to evaluate the profile of mucins secreted via tumor cells.Normal myometrium and myoma tissue expression of TGF β1 growth factor was compared using immunohistochemical staining. Qupath and Image J programs were used to analysis the content of fibrous tissue within normal and myoma tissue. Results: Microscopical results revealed that myoma have a proliferation of overlap fusiform cells organized in bundles alienated by variable amounts of connective tissue fibers, enclosed peripherally by a pseudo capsule. Special stained sections analysis revealed deposition of extracellular martix components specially collagen fibers that vary within each nodular fibroid (intrafascicular, interfascicular) enclosed myocytes that reflect the state of fibrosis that appears as blue – green colors enclosed in Masson's trichrome stain, while in Van Gieson's stain appears as red color. Proliferative tumor cells were revealed via Alcian Blue-PAS stain in dark blue color. results showed distribution of perivascular fibers in myoma as compared with normal adjacent myometrium, which appears in red color in Van Gieson's stain. Immunostaining recorded the significantly higher intensity of TGF-β in the leiomyoma tissue than in the normal myometrium (P < 0.05). The effects of the TGF-β1 inhibitor significantly differed between normal myometrium and leiomyoma tissue, with a greater decrease in cell survival in the leiomyoma tissue (P < 0.05). Conclusion: highly fibrous tissue contents showed within myoma and perivascular than adjacent myometrium, these fibrous tissues were detected via special colours in both Masson Trichrome and Van Gieson's stains. The fibrous tissue within leiomyoma showed higher intensity to TGF than normal myometrium.

Laparoscopic cornual resection for interstitial pregnancy: Staying in the Marginal Zone.

Fortunately, interstitial pregnancies are a rare early pregnancy presentation, yet they can be challenging to managed and are associated with a high risk of intra-abdominal haemorrhage. Once detected, surgical laparoscopic resection can be the preferred management method for both patient safety and for definitive treatment. The video presents a new technique for laparoscopic resection of an interstitial pregnancy which enables the procedure to be effectively bloodless. We report on a new technique for laparoscopic cornual resection. As shown in the video, staying in the marginal zone may result in the enblock resection of the gestational sac. Using meticulous applications of bipolar energy and cutting with scissors in the marginal zone, the operation may be completed with almost no blood loss and minimal damage to the adjacent healthy myometrium. The operation lasted 30 min with almost no blood loss. The patient had an uneventful recovery and was discharged on the first postoperative day. Staying in the marginal zone during dissection permits even less experienced laparoscopists to complete laparoscopic cornual resection with minimal blood loss concomitantly with minimal trauma to the adjacent myometrium.

Endometrial stromal tumor presenting as multiple endometrial polyps with limited infiltration; a novel presentation

Abstract Introduction/Objective The 2020 WHO classifies endometrial stromal tumors into endometrial stromal nodule (ESN), low-grade endometrial stromal sarcoma (LGESS), high-grade endometrial stromal sarcoma, and undifferentiated sarcoma. Minimal irregularity of the interface is allowed in ESN (up to 3 foci of <3 mm). In a small number of cases, the irregular interface exceeds the acceptable criteria for ESN but relatively limited compared to most LGESS. These cases are classified as endometrial stromal tumor with limited infiltration. These tumors may recur, and it is best managed as LGESS. Methods/Case Report We report a case of endometrial stromal tumor in a 42-year-old female who presented with abnormal vaginal bleeding. Imaging showed multiple fibroids. She underwent total abdominal hysterectomy and bilateral salpingectomy. On gross examination three endometrial polyps with sharp demarcation from the adjacent myometrium were identified (largest 4 cm). Histology revealed proliferation of uniform small cells with scant cytoplasm and round to oval nuclei with rare mitotic figures in a delicate network of arterioles. Immunostains showed positive staining for CD10 and estrogen receptor while negative for Caldesmon. These polyps were mainly confined to the endometrium with only small foci of irregular border with the myometrium. No vascular invasion, high grade cytologic atypia or necrosis were seen. The tumor showed JAZF1-SUZ12 gene fusion. This case was diagnosed as an endometrial stromal tumor with limited infiltration. Results (if a Case Study enter NA) NA Conclusion In conclusion, endometrial stromal tumors are commonly submucosal or intramural, we report a rare case that presented as multiple endometrial polyps, with very limited myometrium infiltration. To our knowledge, no cases have been reported so far. It is essential to be cognizant of this entity when dealing with polypoid lesions of the endometrium, they may recur and should be managed as LGESS.

New approaches to the correction of immune disorders in adenomyosis

ABSTRACT. A large number of studies have been devoted to the study of the problem of genital endometriosis, however, the etiology and mechanisms of the disease remain unresolved. Adenomyosis affects about 10% of women of reproductive age, occurs in 30% of women with pelvic pain syndrome and up to 50% of patients with female infertility. Numerous theories of the origin of endometriosis cannot explain the key points in the development of the disease, namely the penetration of the basal layer of the endometrium into the adjacent myometrium. This makes it difficult to choose an adequate treatment strategy for patients with adenomyosis. Treatment of adenomyosis is one of the most difficult issues in modern gynecology, one of the promising approaches in increasing the effectiveness of treatment of patients with adenomyosis is immunocorrective therapy. The aim of the work was to increase the effectiveness of treatment of adenomyosis patients by improving the diagnostic and prognostic tactics of patient management based on the study of pathogenetic aspects. Materials and methods. 115 patients of reproductive age were included in the study. The effectiveness of complex compared to traditional treatment was evaluated. Clinical and laboratory, instrumental and immunological indicators were evaluated in dynamics. Research results and their discussion. Taking into account the peculiarities of pathogenetic mechanisms, which indicate a violation of the cellular and macrophage links of immunity, cytokine imbalance, which contribute to the development of the disease and its persistence, we have developed a comprehensive approach to increasing the effectiveness of the treatment of patients with adenomyosis, which aims to reduce the pro-inflammatory immunological potential. Against the background of the comprehensive treatment, the activity of autoimmune reactions is reduced, the indicators of phagocytic and oxygen activity of neutrophils and monocytes are normalized, cytokine imbalance is reduced - the level of production of pro-inflammatory cytokines and VEGF is normalized. Conclusions. Based on the results of the work, the approaches to the treatment of adenomyosis patients were optimized, the feasibility of using corrective drugs in complex therapy was substantiated.

SAT632 The Functional Role And Regulatory Mechanism Of FTO M6A RNA Demethylase In Human Uterine Leiomyosarcoma

Abstract Disclosure: Q. Yang: None. A. Al-Hendy: None. Introduction: Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis, high rates of recurrence, and metastasis. However, the origin and molecular mechanism underlying driving its clinical and biological behavior remain to be determined. Recently, we and others have revealed the role of microRNAs, DNA methylation, and histone modifications contributing to the pathogenesis of uLMS. However, the connectivity between reversible RNA m6A methylation and uLMS remains unclear. In this study, we assessed the role and mechanism of the m6A demethylase fat mass- and obesity-associated protein (FTO) in the pathogenesis of uLMS. Materials and Methods: We performed immunohistochemistry (IHC) and immunoblot analysis to measure the m6A erasers FTO and ALKBH5 protein levels in LMS and adjacent myometrium tissues (n=8) and cell lines. We performed LMS cell proliferation and cell cycle phase distribution analysis. RNA-seq was performed to determine the molecular mechanism underlying m6A eraser inhibition in LMS cells (n=4 for each group). The R package limma+voom was used for this analysis. Hallmark pathway analysis was used to identify enriched pathways in response to FTO inhibition. Real-time PCR was performed to validate the differentially expressed genes. Ingenuity pathway analysis (IPA) was performed to determine the upstream regulators targeting cell cycle-related genes. We used a cutoff −1.5 > fold-change > 1.5 and an adjusted p-value of 0.05. In addition, data were analyzed by a two-tailed unpaired Student’s t-test between any two groups. Results: IHC analysis revealed that m6A demethylases FTO and ALKBH5 are aberrantly upregulated in uLMS tissues compared to adjacent myometrium (p<0.01, H-score). Furthermore, inhibition of FTO with its small, potent inhibitor (Dac51) decreased the uLMS proliferation dose-dependently via cell cycle arrest at G1. Notably, RNA-seq analysis revealed that inhibition of FTO with Dac51 exhibited a significant decrease in the expression of multiple CDK members and a significant increase in the expression of CDKN1A. Furthermore, IPA analysis revealed that transcription factors, including SP1 and E2F members are the upstream regulators targeting CDK members in response to Dac51 treatment. Moreover, Dac51 treatment altered the expression of several epigenetic regulators, including Sirt1 and 2, HDAC10 and SUV39H1, and rewiring of several critical pathways, including inflammation response, G2M checkpoint, and C-Myc signaling, among others, leading to suppression of uLMS phenotype. Conclusion: Our findings uncover the anti-uLMS function of a potent FTO inhibitor via reprogramming the oncogenic epitranscriptome. Therefore, targeted inhibition of FTO may provide an additional regulatory layer for a promising and novel strategy for treating patients with this aggressive uterine cancer. Presentation: Saturday, June 17, 2023

MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas.

Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.

Endometrium procurement and transplantation restores fertility in rats

Research questionCan rat endometrium be successfully procured and transplanted, and can a similar method be used to procure human endometrium? DesignRat endometrium was procured using an endometrium stripping method and transplanted into female Sprague–Dawley rats. Macroscopic and histological changes, endometrial receptivity-related protein concentrations and fertility were assessed. Additionally, a preliminary experiment was conducted to procure human endometrium using a similar method. ResultsEndometrium was successfully procured from both rats and humans, which contained intact endometrium and parts of the adjacent inner annulus myometrium. Endometrium auto-transplantation was conducted in rats and the procedure lasted a total of 41.3 ± 5.7 min with a mean blood loss of 0.09 ± 0.04 g. The transplanted endometrium survived well, but a fibrotic zone formed between the transplant and recipient tissue. Compared with sham rats, those with endometrium transplantation had similar endometrial thickness and endometrial gland numbers but reduced vascular density at 8 weeks after surgery. Endometrium transplantation also retained expression of the endometrial receptivity-related proteins leukaemia inhibitory factor and vascular endothelial growth factor. In contrast to non-pregnancy in the stripped horn, a mean of 5.0 ± 2.7 fetuses developed in the transplanted horn, and full-term live fetuses were conceived in the horns with transplanted endometrium. ConclusionsEndometrium procurement by stripping method can obtain an intact and functional endometrium, and endometrium transplantation can reconstruct the uterine cavity and restore fertility in rats.

The Functional Role and Regulatory Mechanism of FTO m6A RNA Demethylase in Human Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis and high rates of recurrence and metastasis. The origin and molecular mechanism underlying and driving its clinical and biological behavior remain largely unknown. Recently, we and others have revealed the role of microRNAs, DNA methylation, and histone modifications in contributing to the pathogenesis of uLMS. However, the connection between reversible m6A RNA methylation and uLMS pathogenesis remains unclear. In this study, we assessed the role and mechanism of FTO m6A RNA demethylase in the pathogenesis of uLMS. Immunohistochemistry analysis revealed that the levels of RNA demethylases FTO and ALKBH5 were aberrantly upregulated in uLMS tissues compared to adjacent myometrium with a significant change by histochemical scoring assessment (p < 0.01). Furthermore, the inhibition of FTO demethylase with its small, potent inhibitor (Dac51) significantly decreased the uLMS proliferation dose-dependently via cell cycle arrest. Notably, RNA-seq analysis revealed that the inhibition of FTO with Dac51 exhibited a significant decrease in cell-cycle-related genes, including several CDK members, and a significant increase in the expression of CDKN1A, which correlated with a Dac51-exerted inhibitory effect on cell proliferation. Moreover, Dac51 treatment allowed the rewiring of several critical pathways, including TNFα signaling, KRAS signaling, inflammation response, G2M checkpoint, and C-Myc signaling, among others, leading to the suppression of the uLMS phenotype. Moreover, transcription factor (TF) analyses suggested that epitranscriptional alterations by Dac51 may alter the cell cycle-related gene expression via TF-driven pathways and epigenetic networks in uLMS cells. This intersection of RNA methylation and other epigenetic controls and pathways provides a framework to better understand uterine diseases, particularly uLMS pathogenesis with a dysregulation of RNA methylation machinery. Therefore, targeting the vulnerable epitranscriptome may provide an additional regulatory layer for a promising and novel strategy for treating patients with this aggressive uterine cancer.

Prevalence and clinical significance of co-existing mutations in MED12 and FH in uterine fibroids of Australian women.

Uterine fibroids are exceedingly common benign tumours of the female reproductive system and cause severe symptoms, including acute pain, bleeding, and infertility. Fibroids are frequently associated with genetic alterations affecting mediator complex subunit 12 (MED12), fumarate hydratase (FH), high mobility group AT-hook 2 (HMGA2) and collagen, type IV alpha 5 and alpha 6 (COL4A5-COL4A6). Recently, we reported MED12 exon 2 mutations in 39 out of 65 uterine fibroids (60%) from 14 Australian patients. The aim of this study was to evaluate the status of FH mutations in MED12 mutation-positive and mutation-negative uterine fibroids. FH mutation screening of altogether 65 uterine fibroids and corresponding adjacent normal myometrium (n = 14) was carried out by Sanger sequencing. Three out of 14 patients displayed somatic mutations in FH exon 1 in addition to harbouring MED12 mutation in uterine fibroids. This study is the first to report that the mutations in MED12 and FH co-exist in uterine fibroids of Australian women.

H3K4me3 mediates uterine leiomyoma pathogenesis via neuronal processes, synapsis components, proliferation, and Wnt/β-catenin and TGF-β pathways

BackgroundUterine leiomyomas (UL) are the most common benign tumor in women of reproductive age. Their pathology remains unclear, which hampers the development of safe and effective treatments. Raising evidence suggests epigenetics as a main mechanism involved in tumor development. Histone modification is a key component in the epigenetic regulation of gene expression. Specifically, the histone mark H3K4me3, which promotes gene expression, is altered in many tumors. In this study, we aimed to identify if the histone modification H3K4me3 regulates the expression of genes involved in uterine leiomyoma pathogenesis.MethodsProspective study integrating RNA-seq (n = 48) and H3K4me3 CHIP-seq (n = 19) data of uterine leiomyomas versus their adjacent myometrium. Differentially expressed genes (FDR < 0.01, log2FC > 1 or < − 1) were selected following DESeq2, edgeR, and limma analysis. Their differential methylation and functional enrichment (FDR < 0.05) were respectively analyzed with limma and ShinyGO.ResultsCHIP-seq data showed a global suppression of H3K4me3 in uterine leiomyomas versus their adjacent myometrial tissue (p-value< 2.2e-16). Integrating CHIP-seq and RNA-seq data highlighted that transcription of 696/922 uterine leiomyoma-related differentially expressed genes (DEG) (FDR < 0.01, log2FC > 1 or < − 1) was epigenetically mediated by H3K4me3. Further, 50 genes were differentially trimethylated (FDR < 0.05), including 33 hypertrimethylated/upregulated, and 17 hypotrimethylated/downregulated genes. Functional enrichment analysis of the latter showed dysregulation of neuron-related processes and synapsis-related cellular components in uterine leiomyomas, and a literature review study of these DEG found additional implications with tumorigenesis (i.e. aberrant proliferation, invasion, and dysregulation of Wnt/β-catenin, and TGF-β pathways). Finally, SATB2, DCX, SHOX2, ST8SIA2, CAPN6, and NPTX2 proto-oncogenes were identified among the hypertrimethylated/upregulated DEG, while KRT19, ABCA8, and HOXB4 tumor suppressor genes were identified among hypotrimethylated/downregulated DEG.ConclusionsH3K4me3 instabilities alter the expression of oncogenes and tumor suppressor genes, inducing aberrant proliferation, and dysregulated Wnt/β-catenin, and TGF-β pathways, that ultimately promote uterine leiomyoma progression. The reversal of these histone modifications may be a promising new therapeutic alternative for uterine leiomyoma patients.

A Cautionary Tale of the Tubal Stump

A 22 year-old G2A1 female, with a history of prior right salpingectomy for a ruptured ectopic pregnancy, presented to hospital with diffuse abdominal pain and vaginal spotting. Her b-HCG was over 16 000 mIU/mL. A pelvic ultrasound demonstrated a gestational sac with a fetal pole at the right utero-tubal junction, outside of the endometrial cavity (Figure 1), suggesting a pregnancy contained in the interstitium of the tubal stump remnant. Laparoscopy confirmed imaging findings (Figure 2). Dilute vasopressin was injected into the adjacent myometrium. A transverse incision was made along the most dependent area of the interstitial pregnancy, the products of conception were expressed from the uterine cornua, tubal stump removed, and the defect repaired (Figure 3). Figure 2Laparoscopy confirms an interstitial ectopic pregnancy within the tubal stump from prior salpingectomy. View Large Image Figure Viewer Download Hi-res image Figure 3Image of the pelvis after removal of ectopic pregnancy and repair View Large Image Figure Viewer Download Hi-res image

Distribution of Mast Cells in Uterine Leiomyoma and Adjacent Myometrium: A Cross-sectional Study

Introduction: Mast cells are involved in various physiological and pathological processes. Their role in neoplasm angiogenesis remains an enigma. Mast cells have been observed in association with smooth muscle tumours of the uterus. The role of mast cells in leiomyomas is conflicting, with some studies favoring tumour development and others showing the opposite. Aim: To assess the distribution of mast cells in uterine leiomyomas and the adjacent myometrium. Materials and Methods: This cross-sectional study included 100 consecutive hysterectomy specimens from patients who underwent surgery for leiomyoma over a period of one year and five months at PES Institute of Medical Sciences and Research, Kuppam, Andhra Pradesh, India. Sections from the leiomyoma and adjacent myometrium were stained with Hematoxylin and Eosin (H&amp;E) and toluidine blue stains. Mast cell distribution was calculated. The data was collected and analysed using the Statistical Package for the Social Sciences (SPSS) (Version 23.0; SPSS Inc., Chicago, IL, USA). Mast cell counts were expressed as mean and standard deviation. The comparison between the means of different groups was performed using the “Independent sample t-test.” A p-value&lt;0.05 was considered statistically significant. Results: A total of 100 leiomyoma cases were included in the study. The mean age of the study participants who underwent hysterectomy was 46.3±6.73 years, and the majority (32%) of them were in the age group of 46-50 years. The average number of mast cells in the leiomyoma and adjacent myometrium was 11.65±15.813 and 37.16±23.008, respectively. A statistically significant difference was observed between leiomyoma and adjacent myometrium (p-value&lt;0.0001). Conclusion: A higher distribution of mast cells was observed in the myometrium adjacent to the leiomyoma. This finding may indicate that mast cells play a role in the growth of leiomyoma by providing appropriate growth factors.

Targeting Class I Histone Deacetylases in Human Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis, high rates of recurrence, and metastasis. Currently, the molecular mechanism of the origin and development of uLMS is unknown. Class I histone deacetylases (including HDAC1, 2, 3, and 8) are one of the major classes of the HDAC family and catalyze the removal of acetyl groups from lysine residues in histones and cellular proteins. Class I HDACs exhibit distinct cellular and subcellular expression patterns and are involved in many biological processes and diseases through diverse signaling pathways. However, the link between class I HDACs and uLMS is still being determined. In this study, we assessed the expression panel of Class I HDACs in uLMS and characterized the role and mechanism of class I HDACs in the pathogenesis of uLMS. Immunohistochemistry analysis revealed that HDAC1, 2, and 3 are aberrantly upregulated in uLMS tissues compared to adjacent myometrium. Immunoblot analysis demonstrated that the expression levels of HDAC 1, 2, and 3 exhibited a graded increase from normal and benign to malignant uterine tumor cells. Furthermore, inhibition of HDACs with Class I HDACs inhibitor (Tucidinostat) decreased the uLMS proliferation in a dose-dependent manner. Notably, gene set enrichment analysis of differentially expressed genes (DEGs) revealed that inhibition of HDACs with Tucidinostat altered several critical pathways. Moreover, multiple epigenetic analyses suggested that Tucidinostat may alter the transcriptome via reprogramming the oncogenic epigenome and inducing the changes in microRNA-target interaction in uLMS cells. In the parallel study, we also determined the effect of DL-sulforaphane on the uLMS. Our study demonstrated the relevance of class I HDACs proteins in the pathogenesis of malignant uLMS. Further understanding the role and mechanism of HDACs in uLMS may provide a promising and novel strategy for treating patients with this aggressive uterine cancer.

The Expression of the Immunoproteasome Subunit PSMB9 Is Related to Distinct Molecular Subtypes of Uterine Leiomyosarcoma.

Simple SummaryUterine leiomyosarcoma (uLMS) is a rare, aggressive, and highly heterogeneous tumor. Knockout female mice for the catalytic subunit of the immunoproteasome PSMB9 develops spontaneous uLMS. In this study, we used molecular data from 3 non-related uLMS cohorts that were integrated and analyzed by proteotranscriptomics. We observed overexpression of the immunoproteasome pathway in uLMS, and then further classified the samples as low or high PSMB9 gene expression levels and we provide evidence that; (i) in the group high there is an enrichment of pathways related to the immune system and in the group low, the ECM formation; (ii) samples with high CD8+/PSMB9 ratio shows better OS; and (iii) the main regulator in the high group is IFNγ and in the low, the proto-oncogene SRC. These findings contribute to the understanding of potential therapeutic or prognostic markers in uLMS.Background: Uterine leiomyosarcoma (uLMS) are rare and malignant tumors that arise in the myometrium cells and whose diagnosis is based on histopathological features. Identifying diagnostic biomarkers for uLMS is a challenge due to molecular heterogeneity and the scarcity of samples. In vivo and in vitro models for uLMS are urgently needed. Knockout female mice for the catalytic subunit of the immunoproteasome PSMB9 (MIM:177045) develop spontaneous uLMS. This study aimed to analyze the role of PSMB9 in uLMS tumorigenesis and patient outcome. Methods: Molecular data from 3 non-related uLMS cohorts were integrated and analyzed by proteotranscriptomic using gene expression and protein abundance levels in 68 normal adjacent myometrium (MM), 66 uterine leiomyoma (LM), and 67 uLMS. Results: the immunoproteasome pathway is upregulated and the gene PMSB9 shows heterogeneous expression values in uLMS. Quartile group analysis showed no significant difference between groups high and low PSMB9 expression groups at 3-years overall survival (OS). Using CYBERSORTx analysis we observed 9 out of 17 samples in the high group clustering together due to high M2 macrophages and CD4 memory resting, and high CD8+/PSMB9 ratio was associated with better OS. The main pathway regulated in the high group is IFNγ and in the low is the ECM pathway dependent on the proto-oncogene SRC. Conclusion: these findings suggest 2 subtypes of uLMS (immune-related and ECM-related) with different candidate mechanisms of malignancy.

Possible significance of degeneration and decreased expression of progesterone receptor in postmenopausal uterine leiomyoma

BackgroundThe growth of uterine leiomyomas is dependent on the levels of sex steroid hormones, and they usually shrink after menopause. However, there are cases in which leiomyomas continue to grow and/or surgery is required after menopause. In addition to estrogen, progesterone has recently been implicated in leiomyoma enlargement, but its relevance to postmenopausal leiomyoma remains unknown. Therefore, we investigated whether hormone receptor expression is associated with postmenopausal leiomyoma enlargement and characterized pathological findings of postmenopausal leiomyoma, which have not been clarified yet.MethodsNine cases that required total hysterectomy for leiomyomas after menopause were examined. Surgeries were conducted because of pelvic pressure, pelvic pain, suspected malignancy, or growing leiomyoma. Six cases of leiomyomas being incidentally found during total hysterectomy for postmenopausal uterine prolapse, and six patients who underwent hysterectomy for leiomyomas before menopause, were examined as controls. We evaluated the expression of estrogen receptor, progesterone receptor B, and progesterone receptor AB by immunohistochemical staining among the cases. We also analyzed the pathological findings of leiomyomas.ResultsIn postmenopausal leiomyomas, the expression of progesterone receptor was higher than that in the adjacent myometrium. Compared with premenopausal leiomyomas, the expression of progesterone receptor decreased. Postmenopausal leiomyomas that required surgery did not show elevated sex steroid hormone receptor expression, compared with the leiomyomas that did not require surgery.The degeneration frequency of leiomyomas was 92% in the group that underwent surgery for postmenopausal leiomyomas, 65% in the group that underwent surgery for reasons other than the presence of leiomyomas after menopause, and 47% in the group operated for leiomyomas before menopause.ConclusionsThese results suggest that sex steroid hormones are unlikely to be associated with the growth of leiomyomas after menopause. Since leiomyoma degeneration with increased extracellular matrix is likely to occur in postmenopausal women, the degeneration of leiomyomas may be the main mechanism for the growth of postmenopausal leiomyomas.

Uterine leiomyoma as useful model to unveil morphometric and macromolecular collagen state and impairment in fibrotic diseases: An ex-vivo human study

Collagen is one of the main components of the extracellular matrix (ECM), involved, among all, in the maintenance of the structural support of tissues. In fibrotic diseases, collagen is overexpressed, and its production determines the formation of a significantly stiffer ECM. The cross-linking of high-resolution analytical tools, able to investigate both the tridimensional organization and the secondary structure of collagen in fibrotic diseases, could be useful to identify defined markers correlating the status of this protein with specific pathological conditions. To this purpose, an innovative multidisciplinary approach based on Phase-Contrast MicroComputed Tomography, Transmission Electron Microscopy, and Fourier Transform Infrared Imaging Spectroscopy was exploited on leiomyoma samples and adjacent myometrium to characterize microstructural collagen features. Uterine leiomyoma is a common gynecological disorder affecting women in fertile age. It is characterized by a massive collagen production due to the repairing processes occurring at myometrium level, and, hence, it represents a valuable model to investigate collagen self-organization in a pathological condition. Moreover, to evaluate the sensitivity of this multidisciplinary approach, the effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) omega-3 fatty acids in collagen reduction were also investigated.

The Functional Role and Regulatory Mechanism of Bromodomain-Containing Protein 9 in Human Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is the most common type of uterine sarcoma associated with poor prognosis, high rates of recurrence, and metastasis. There is currently limited information about uLMS molecular mechanisms of origin and development. Bromodomain (BRD)-containing proteins are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD protein dysfunction has been linked to many diseases including tumorigenesis. However, the role of BRD proteins in the pathogenesis of uLMS is unknown. Here, we show for the first time that BRD9 is aberrantly overexpressed in uLMS tissues compared to adjacent myometrium. BRD9 expression is also upregulated in uLMS cell lines compared to benign uterine fibroid and myometrium cell lines. Inhibition of BRD9 using the specific inhibitor (TP-472) suppressed uLMS cell proliferation via inducing apoptosis and cell cycle arrest. To further characterize the mechanistic basis for TP-472 inhibition of uLMS cell growth, we performed a comparative RNA-seq analysis of vehicle-treated and TP-472-treated uLMS cells (n = 4 each). Bioinformatics analysis revealed that TP-472 treatment distinctly altered the uLMS cell transcriptome. Gene set enrichment analysis identified critical pathways altered by BRD9 inhibition, including interferon-alpha response, KRAS signaling, MYC targets, TNF-a signaling via NFkB, and MTORC1 signaling. Parsimonious gene correlation network analysis identified nine enriched modules, including cell cycle and apoptosis modules. Moreover, the ENCODE Histone Modifications gene set and TargetScan microRNA analysis in Enrichr suggested that TP-472-induced BRD9 inhibition may alter the uLMS cell transcriptome by reprograming the oncogenic epigenome and inducing miRNA-mediated gene regulation. Therefore, BRD9 constitutes a specific vulnerability in malignant uLMS, and targeting non-BET BRD proteins in uLMS may provide a promising and novel strategy for treating patients with this aggressive uterine cancer.

P-526 Targeting histone modifications: H3K27 acetylation regulates the expression of genes involved in key processes of uterine leiomyoma pathogenesis

Abstract Study question Does histone mark H3K27ac regulate the expression of genes involved in uterine leiomyoma (UL) pathogenesis, and can histone deacetylation inhibition be a new therapeutic approach? Summary answer H3K27ac regulates genes implicated in UL pathogenesis through angiogenesis and extracellular matrix (ECM), and histone deacetylation reversion could offer a therapeutic approach to treat UL. What is known already UL is a benign tumor arising from myometrium (MM). Women present symptoms such as pelvic pain and infertility. There is no fully effective treatment with minimal side effects. Available medical options focus on the relief of symptoms and not in mechanisms implicated in UL development. Histone modifications are altered in tumors, particularly via histone acetylation which is correlated with gene activation. Herein, we aimed to identify if the modification H3K27ac is involved in UL pathogenesis, determining the relationship between H3K27ac and gene expression in UL versus MM and if its reversion may be a therapeutic option to treat UL. Study design, size, duration Prospective study comparing transcriptome (GEO:GSE192354 and GSE142332) by RNA sequencing (RNA-seq) (n = 48) and H3K27ac profile (GEO:GSE142332) by Chromatin Immunoprecipitation Sequencing (CHIP-seq) (n = 19) in UL versus adjacent MM tissue. Human uterine leiomyoma primary (HULP) cells obtained from individual patients (n = 10) were treated with 0.01% DMSO (control) and 10 µM SAHA, a histone deacetylases inhibitor, for 48 hours. Participants/materials, setting, methods Samples were collected from 48 women aged 31-54 years. Bioinformatics analysis were performed within the R/Bioconductor (4.1.1.). Differential expressed genes (DEGs) were analysed using three methods: DESeq2, edgeR and limma. Common DEGs (FDR&amp;lt;0.01,log2FC&amp;gt;1 or &amp;lt;-1) were considered for further analysis. Differential H2K27ac peak enrichment analysis of selected genes was performed with limma and functional enrichment analysis (FDR&amp;lt;0.05) with Shiny Go (version 0.741). SAHA effect on hypoacetylated/downregulated genes was assessed in HULP cells by qRT-PCR. Main results and the role of chance CHIP-seq analysis showed a lower amount of global H3K27ac peak enrichment level in UL versus MM (p-value&amp;lt;2.2e-16). RNA-seq showed 922 common DEGs in UL versus MM, being 559 up-regulated and 363 down-regulated (FDR&amp;lt;0.01, log2FC&amp;gt;1 or &amp;lt;-1). Integration of CHIP-seq and RNA-seq data showed that among 922 selected genes, 482 also presented the histone mark H3K27ac. Differential peak enrichment analysis demonstrated that 82 of them presented differential acetylation (FDR&amp;lt;0.05) in UL versus MM, being 29 hyper-acetylated/up-regulated and 53 hypo-acetylated/down-regulated. Functional enrichment analysis of 82 DEGs regulated by H3K27ac showed biological processes deregulated in UL that were related to angiogenesis. Additionally, we found cellular components enriched in UL, which were related to an alteration of ECM, one of the key features of UL. We studied further these 82 genes controlled by H3K27ac and found hyperacetylation/upregulation of oncogenes (NDP, TFAP2C, HOXA13, COL24A1 and IGFL3) and hypoacetylation/downregulation of tumor suppressor genes (CD40, GIMAP8, IL15, GPX3 and DPT) in UL, which are related to immune system, angiogenesis, invasion, metabolism, ECM, TGFβ3 and Wnt/β-catenin pathway dysregulation. Finally, SAHA treatment in HULP cells significantly increased expression of the tumor suppressor genes that were hypoacetylated/downregulated in UL versus MM (CD40, GIMAP8, IL15, GPX3 and DPT) (p &amp;lt; 0.05). Limitations, reasons for caution This is a preliminary study including only 19 participants in ChIP-seq analysis, thereby we should be cautious extrapolating our results to the general population. Further studies are necessary to determine the effectiveness of histone deacetylases inhibition, SAHA dose and adverse effects on UL in vivo. Wider implications of the findings H3K27 acetylation regulates the expression of oncogenes and tumor suppressor genes involved in key processes of UL pathogenesis, such as angiogenesis and ECM formation. Histone deacetylation reversion by SAHA upregulated the expression of tumor suppressor genes in HULP cells, suggesting histone deacetylation as a potential therapeutic approach for UL patients. Trial registration number Not applicable

Integrative analysis of the DNA methylome and transcriptome in uterine leiomyoma shows altered regulation of genes involved in metabolism, proliferation, extracellular matrix, and vesicles.

Uterine leiomyomas (ULs) are the most common benign tumors in women of reproductive age. Despite the high prevalence, tumor pathology remains unclear, which hampers the development of safe and effective treatments. Epigenetic mechanisms appear to be involved in UL development, particularly via DNA methylation that regulates gene expression. We aimed to determine the relationship between DNA methylation and gene expression in UL compared with adjacent myometrium (MM) to identify molecular mechanisms involved in UL formation that are under epigenetic control. Our results showed a different DNA methylation profile between UL and MM, leading to hypermethylation of UL, and a different global transcriptome profile. Integration of DNA methylation and whole-transcriptome RNA-sequencing data identified 93 genes regulated by methylation, with 22 hypomethylated/upregulated and 71 hypermethylated/downregulated. Functional enrichment analysis showed dysregulated biological processes and molecular functions involved in metabolism and cell physiology, response to extracellular signals, invasion, and proliferation, as well as pathways related to uterine biology and cancer. Cellular components such as cell membranes, vesicles, extracellular matrix, and cell junctions were dysregulated in UL. In addition, we found hypomethylation/upregulation of oncogenes (PRL, ATP8B4, CEMIP, ZPMS2-AS1, RIMS2, TFAP2C) and hypermethylation/downregulation of tumor suppressor genes (EFEMP1, FBLN2, ARHGAP10, HTATIP2), which are related to proliferation, invasion, altered metabolism, deposition of extracellular matrix, and Wnt/β-catenin pathway dysregulation. This confirms that key processes of UL development are under DNA methylation control. Finally, inhibition of DNA methyltransferases by 5-aza-2'-deoxycitidine increased the expression of hypermethylated/downregulated genes in UL cells in vitro. In conclusion, gene regulation by DNA methylation is implicated in UL pathogenesis, and reversion of this methylation could offer a therapeutic option for UL. © 2022 The Pathological Society of Great Britain and Ireland.

MicroRNAs as potential indicators of the development and progression of uterine leiomyoma.

Recent studies demonstrated a significant role of several microRNAs (miRs) in the development of leiomyoma. Here, we investigated miR expression profiles using microarray and found a significantly higher expression of miRs in leiomyoma than in adjacent myometrium. We also confirmed the upregulation of five selected miRs including miR-181a-5p, 127-3p, 28-3p, 30b-5p and let-7c-5p in cellular proliferation, extracellular matrix turnover, and angiogenesis by RT-qPCR. Interestingly, the miRs showed a higher expression in cases of large leiomyoma or in patients with a history of transfusion due to anemia. We then analyzed the expression of the miR target molecules including Transforming Growth Factor Beta Receptor 2 (TGFBR2) and Insulin-like Growth Factor 2 mRNA Binding Protein 1 (IGF2BP1) via immunohistochemistry. TGFBR2 and IGF2BP1 were positively stained in 81% and 62.5% of leiomyoma tissues but not in adjacent myometrium. Both were more frequently positive in patients with ≥ 6 cm leiomyoma and mass effect. The mean expression levels of miR-181a-5p, 127-3p, 28-3p, 30b-5p and let-7c-5p were higher in cases with TGFBR2 and IGF2BP1 positive leiomyoma. We observed several miRs were overexpressed in leiomyoma tissues, and these results provide insight into the role of miRs in the development and progression of leiomyoma and underscore the need to validate their utility as diagnostic or therapeutic targets.