Efficacy of fertility-sparing treatment with LNG-IUS is associated with different ProMisE subtypes of endometrial carcinoma or atypical endometrial hyperplasia.

OBJECTIVE: To determine whether proactive molecular risk classifier for endometrial cancer (ProMisE) could be used to assess the prognosis of patients with atypical endometrial hyperplasia (AEH) or early-stage endometrial cancer (EC) treated with levonorgestrel-releasing intrauterine system (LNG-IUS). METHODS: A retrospective cohort study was conducted among 93 AEH or early-stage EC patients who received LNG-IUS to preserve fertility . By immunohistochemistry and gene sequencing, 4 subtypes of ProMisE were identified (p53 wild type [p53 wt], mismatch repair-deficient [MMRd], p53-abnormal, and POLE-mutated). The primary outcome was the time to complete response (CR) after LNG-IUS therapy. Secondary outcomes included the recurrence rate after CR and success rate of conception. RESULTS: Among the 93 patients, 15 (16.1%) were classified as MMRd, 6 (6.5%) as POLE-mutated, 5 (5.4%) as p53-abnormal, and 67 (72.0%) as p53 wt. Comparison of serum cancer antigen 125, family history of tumor, and positive rates of programmed cell death 1 ligand 1 protein and Ki67 protein in 4 groups showed statistically significant differences (p<0.05). Patients with the p53-abnormal subtype had the lowest overall CR rate (40%) and the highest recurrence rate (2/2). Patients with POLE-mutated subtype had the best prognosis, and all 6 patients achieved CR. When patients achieved complete remission, assisted reproductive technology was more likely to help them conceive than natural conception (p<0.05). CONCLUSION: Patients with early-stage EC or AEH who are more likely to benefit from fertility-sparing treatment can be identified using ProMisE classifier. Patients with POLE-mutated are suitable for fertility-sparing treatment with LNG-IUS.

DNA methylation of selected tumor suppressor genes in endometrial hyperplasia.

AIMS: To investigate DNA methylation of specific gene promoters in endometrial hyperplasia compared to normal endometrial tissue. MATERIALS AND METHODS: To search for epigenetic events, methylation-specific multiplex ligation-dependent probe amplification was employed to compare the methylation status of 64 tissue samples with atypical endometrial hyperplasia, 60 tissue samples with endometrial hyperplasia without atypia, and 40 control tissue samples with normal endometrium. RESULTS: Differences in DNA methylation among the groups were found in PTEN, CDH13, and MSH6 promoters (PTEN: atypical hyperplasia 32%, benign hyperplasia 6.8%, normal endometrium 10%; P=0.004; CDH13: atypical hyperplasia, 50%; benign hyperplasia, 43%; normal endometrium 8.1%; P=0.003; MSH6 atypical hyperplasia 84%, benign hyperplasia, 62%; normal endometrium, 52%; P=0.008.) Higher rates of CDH13 promoter methylation were identified in the groups with both forms of endometrial hyperplasia when compared to the control group (atypical hyperplasia, P=0.003, benign hyperplasia, P=0.0002). A higher rate of DNA methylation of the PTEN and MSH6 promoters was observed in samples with atypical endometrial hyperplasia than in samples with benign endometrial hyperplasia (PTEN: P=0.02; MSH6: P=0.01) and samples with normal endometrial tissue (PTEN, P=0.04; MSH6, P=0.006). CONCLUSION: DNA methylation of CDH13, PTEN, and MSH6 appear to be involved in the development of endometrial hyperplasia.

[Fertility-preserving treatment outcomes in endometrial cancer and atypical hyperplasia patients with different molecular profiles].

Objective: To investigate the impact of molecular classification and key oncogenes on the oncologic outcomes in patients with endometrial carcinoma (EC) and atypical endometrial hyperplasia (AEH) receiving fertility-preserving treatment. Methods: Patients with EC and AEH undergoing progestin-based fertility-preserving treatment and receiving molecular classification as well as key oncogenes test at Obstetrics and Gynecology Hospital, Fudan University from January 2021 to March 2023 were reviewed. Hysteroscopic lesion resection and endometrial biopsy were performed before initiating hormone therapy and every 3 months during the treatment to evaluate the efficacy. The risk factors which had impact on the treatment outcomes in EC and AEH patients were further analyzed. Results: Of the 171 patients analyzed, the median age was 32 years, including 86 patients with EC and 85 patients with AEH. The distribution of molecular classification was as follows: 157 cases (91.8%) were classified as having no specific molecular profile (NSMP); 9 cases (5.3%), mismatch repair deficient (MMR-d); 3 cases (1.8%), POLE-mutated; 2 cases (1.2%), p53 abnormal. No difference was found in the cumulative 40-week complete response (CR) rate between the patients having NSMP or MMR-d (61.6% vs 60.0%; P=0.593), while the patients having MMR-d had increased risk than those having NSMP to have recurrence after CR (50.0% vs 14.4%; P=0.005). Multi-variant analysis showed PTEN gene multi-loci mutation (HR=0.413, 95%CI: 0.259-0.658; P<0.001) and PIK3CA gene mutation (HR=0.499, 95%CI: 0.310-0.804; P=0.004) were associated with a lower cumulative 40-week CR rate, and progestin-insensitivity (HR=3.825, 95%CI: 1.570-9.317; P=0.003) and MMR-d (HR=9.014, 95%CI: 1.734-46.873; P=0.009) were independent risk factors of recurrence in EC and AEH patients. Conclusions: No difference in cumulative 40-week CR rate is found in the patients having NSMP or MMR-d who received progestin-based fertility-preserving treatment, where the use of hysteroscopy during the treatment might be the reason, while those having MMR-d have a higher risk of recurrence after CR. Oncogene mutation of PTEN or PIK3CA gene might be associated with a lower response to progestin treatment. The molecular profiles help predict the fertility-preserving treatment outcomes in EC and AEH patients.

Exploring the role of macrophages in the progression from atypical hyperplasia to endometrial carcinoma through single-cell transcriptomics and bulk transcriptomics analysis.

Introduction: In this study, we aimed to identify key genes in endometrial cancer by conducting single-cell analysis of macrophages. Methods: We sourced clinical data from the TCGA database as well as supplementary datasets GSE201926 and GSE173682. Using bulk-seq data of atypical endometrial hyperplasia and endometrial cancer, we pinpointed key differentially expressed genes. Single-cell RNA sequencing was utilized for further gene expression analysis. Cluster analysis was conducted on TCGA tumor data, identifying two distinct subtypes. Statistical methods employed included LASSO regression for diagnostic modeling and various clustering algorithms for subtype identification. Results: We found that subtype B was closely related to cellular metabolism. A diagnostic model was established using LASSO regression and was based on the genes CDH18, H19, PAGE2B, PXDN, and THRB. This model effectively differentiated the prognosis of cervical cancer. We also constructed a prognosis model and a column chart based on these key genes. Discussion: Through CIBERSORT analysis, CDH18 and PAGE2B were found to be strongly associated with macrophage M0. We propose that these genes influence the transformation from atypical endometrial hyperplasia to endometrial cancer by affecting macrophage M0. In conclusion, these key genes may serve as therapeutic targets for endometrial cancer. A new endometrial cancer risk prognosis model and column chart have been constructed based on these genes, offering a reliable direction for future cervical cancer treatment.

Endometrial telomerase activity in women with either endometrial cancer or hyperplasia: A systematic review and meta-analysis.

AIM: We performed a systematic review and meta-analysis to assess whether endometrial telomerase activity is associated with endometrial cancer or hyperplasia. METHODS: PubMed, Web of Science, Embase, Scielo, LILAC, and CNKI databases were searched to obtain relevant literature for articles published through June 2022, following PRISMA guidelines and a registered PROSPERO protocol. We included observational studies reporting endometrial telomerase activity in patients with either endometrial cancer or hyperplasia compared with benign endometrial tissue (control women). The Newcastle-Ottawa Scale was used to evaluate the quality of studies. Data were expressed as the odds ratios (OR) and 95 % confidence intervals (CI). Random effects and inverse variance methods were used to meta-analyze associations. The I2 test was used to assess heterogeneity. RESULTS: There were significant associations between endometrial telomerase activity and either endometrial cancer (20 studies, OR = 10.65, 95 % CI 6.39, 17.75, p = 0.00001, I2 = 21 %) or endometrial hyperplasia (nine studies, OR = 3.62, 95 % CI 1.61, 8.13, p = 0.002, I2 = 36 %) compared to women without endometrial cancer and hyperplasia. There was not a significant difference in telomerase activity in women with endometrial cancer compared to those with endometrial hyperplasia (seven studies, OR = 1.03; 95 % CI 0.31, 3.37, p = 0.96, I2 = 49 %). In subgroup analyses, there were no significant differences in telomerase activity in patients with endometrial cancer by type of observational studies and by countries of the studies. CONCLUSION: Endometrial telomerase activity is higher in women with either endometrial cancer or endometrial hyperplasia compared to control women without those lesions.

ARID1A Regulates Progesterone Receptor Expression in Early Endometrial Endometrioid Carcinoma Pathogenesis.

Loss of progesterone receptor (PR) expression is an established risk factor for unresponsiveness to progesterone therapy in patients with endometrial atypical hyperplasia and endometrioid carcinoma. ARID1A is one of the most commonly mutated genes in endometrioid carcinomas, and the loss of its expression is associated with tumor progression. In this study, we investigated the roles of ARID1A deficiency in PR expression in human and murine endometrial epithelial neoplasia. An analysis of genome-wide chromatin immunoprecipitation sequencing in isogenic ARID1A-/- and ARID1A+/+ human endometrial epithelial cells revealed that ARID1A-/- cells showed significantly reduced chromatin immunoprecipitation sequencing signals for ARID1A, BRG1, and H3K27AC in the PgR enhancer region. We then performed immunohistochemistry to correlate the protein expression levels of ARID1A, estrogen receptor, and PR in 50 human samples of endometrial atypical hyperplasia and 75 human samples of endometrial carcinomas. The expression levels of PR but not were significantly lower in ARID1A-deficient low-grade endometrial carcinomas and atypical hyperplasia (P = .0002). When Pten and Pten/Arid1a conditional knockout murine models were used, Pten-/-;Arid1a-/- mice exhibited significantly decreased epithelial PR expression in endometrial carcinomas (P = .003) and atypical hyperplasia (P < .0001) compared with that in the same tissues from Pten-/-;Arid1a+/+ mice. Our data suggest that the loss of ARID1A expression, as occurs in ARID1A-mutated endometrioid carcinomas, decreases PgR transcription by modulating the PgR enhancer region during early tumor development.

Potential serum metabolites and long-chain noncoding RNA biomarkers for endometrial cancer tissue.

BACKGROUND: Endometrial carcinoma (EC) is one of the most common tumors in the female reproductive system. There are nearly 200 000 new cases every year. It is the third most common gynecological malignant tumor leading to female death. The incidence rate is closely related to lifestyle, and the incidence rate varies in different regions. The incidence rate of EC is ranking the first in the female reproductive system cancer just second only to breast, lung, and colorectal cancer in North America and Europe and the incidence rate of EC is only second, followed by breast cancer and cervical cancer in China. PURPOSE: The potential metabolic markers of endometrial cancer were screened by liquid chromatograph mass spectrometer (LC-MS), and the tissues of patients with hysteromyoma and endometrial cancer were sequenced to explore the relationship between the disease and change in the content of long-chain noncoding RNA (lncRNA). METHODS: Serum and tissue samples were collected from patients with endometrial dysplasia, endometrial cancer stage I, and endometrial cancer stage III. The metabolites in all serum samples were extracted, and the metabolites in all samples were detected by LC-MS/MS technology. The Pareto-scaling method was used for normalization, and the MetaboAnalyst 4.0 software was used for different analyses. The T test between groups showed that p ≤ 0.05 was regarded as the metabolite with a difference. Further, the function of differential metabolites was determined by metabolite function enrichment and co-expression analysis. Meanwhile, the differentially expressed lncRNA was detected by Illumina second-generation high-throughput sequencing technology, and the expression was analyzed by DEGseq software. Different lncRNA were screened according to p < 0.05. LncRNA with significant differences were screened by p < 0.01, q < 0.001, fold change ≥2, and false discovery rate (FDR) ≤0.001. RESULTS: Through synthesis of T test, cluster heatmap, and ROC curve analysis, five biomarkers with potential diagnostic ability were obtained, including 2,3-Pyridinedicarboxylic acid (area under the curve (AUC) = 0.69), Hematommic acid, ethyl ester (AUC = 0.69), Maltitol (AUC = 0.69), 13(S)-HODE (AUC = 0.88), and D-Mannitol (AUC = 0.69) had potential diagnostic ability between EC phase I versus EC phase III. At the same time, lncRNA sequencing results showed that when endometrial atypical hyperplasia continued to change, including LINC00511, PVT1, and IQCH-AS1 (downregulated), and only changed significantly in the endometrial dysplasia group, including MALAT1, CARMN (downregulated) and LINC00648, BISPR, LINC01534, and LINC00930 (upregulated). Moreover, both differential metabolites and differential lncRNA were annotated to the lipid metabolism pathway, suggesting that this pathway played an important role in the occurrence and development of endometrial carcinoma. CONCLUSIONS: It can combine the results of metabolomics and lncRNA sequencing to assist in the early diagnosis of endometrial precancerous lesions and endometrial cancer patients, to enhance the sensitivity and specificity of diagnosis, which has a certain clinical application prospect.

Endometrial hyperplasia with loss of APC in a novel population of Lyz2-expressing mouse endometrial epithelial cells.

Loss of heterozygosity and promoter hypermethylation of APC is frequently observed in human endometrial cancer, which is the most common gynecological cancer in the USA, but its carcinogenic driver status in the endometrial epithelium has not been confirmed. We have identified a novel population of progenitor endometrial epithelial cells (EECs) in mice that express lysozyme M (LysM) and give rise to approximately 15% of all EECs in adult mice. LysM is a glycoside hydrolase that is encoded by Lyz2 and functions to protect cells from bacteria as part of the innate immune system. Its expression has been shown in a subset of hematopoietic stem cells and in specialized lung and small intestinal epithelial cells. Conditional deletion of Apc in LysM + EECs results in significantly more epithelial cells compared to wild-type mice. At 5 months of age, the ApccKO mice have enlarged uterine horns with pathology that is consistent with endometrial hyperplasia with cystic endometrial glands, non-villous luminal papillae and nuclear atypia. Nuclear accumulation of ß-catenin and ERα, both of which are known to induce endometrial hyperplasia, was observed in the EECs of the ApccKO mice. These results confirm that loss of APC in EECs can result in a phenotype similar to endometrial hyperplasia.

Application of molecular classification to guiding fertility-sparing therapy for patients with endometrial cancer or endometrial intraepithelial neoplasia.

BACKGROUND: The aim of this study was to evaluate whether molecular classification was associated with treatment response in women with endometrial endometrioid carcinoma (EEC) or Endometrial atypical hyperplasia/endometrial intraepithelial neoplasia (EAH/EIN) treated with progestin. METHODS: A retrospective analysis of 59 patients with EEC or EAH/EIN who received fertility-sparing therapy between 2013 and 2021 was performed. For each patient, medical records and pathological reports were reviewed. The treatment efficacy and tumor prognosis were evaluated. Immunohistochemistry analysis for p53 and MSH2, MSH6, PSM2, MLH1 were performed. Molecular classification was analyzed using a 11-gene panel based on next generation sequencing technology. RESULTS: 23 of 39 patients with EEC received complete response (CR) after fertility-sparing treatment which was significantly lower than the EAH/EIN group (58.97 % vs 80.0 %, P < 0.05). Molecular classification via the Cancer Genome Atlas (TCGA) algorithm was successfully applied to 59 cases. The distribution of specimens into the four molecular classes was as follows: 83.05 % (49/59) CNL(copy number-low)，6.78 % (4/59) MSI-H (microsatellite instability -high), 5.08 %(3/59) POLE-mutated and 5.08 % (3/59) CNH(copy number-high). MSI and TP53 sequencing results were concordant with immunohistochemistry analyses of MMR and p53 protein. The patients with CNH and MSI-H subtypes showed worse prognosis than those with POLE-mutated and CNL subtypes. CONCLUSIONS: Molecular classification of EAH/EIN prior to management with progestin treatment was feasible and may predict patients at risk of progression.

[Significance of molecular classification in fertility-sparing treatment of endometrial carcinoma and atypical endometrial hyperplasia].

Objective: To investigate the molecular classification of endometrial carcinoma (EC) and atypical endometrial hyperplasia (AEH) treated with fertility-sparing therapy, and to analyze its relationship with clinicopathological factors and treatment efficacy. Methods: A total of 46 EC and AEH patients who received fertility-sparing therapy and molecular classification tested by next generation sequencing in Peking University People's Hospital from June 2020 to December 2021, were retrospectively collected. The relationships between molecular classification and clinicopathological factors and treatment outcomes were analyzed. Results: (1) Of the 46 patients, including 40 EC and 6 AEH patients, 32 cases (71%, 32/45) had complete response (CR) after treatment, with median CR time of 8 months, 6 cases (13%, 6/45) had partial response, and 8 cases (25%, 8/32) had recurrence. (2) The cases were distributed as no specific molecular profile (NSMP) 34 cases (74%, 34/46) subtype mainly, high microsatellite instability (MSI-H) 7 cases (15%, 7/46), POLE ultra-mutated 3 cases (7%, 3/46), and copy number high (CNH) 2 cases (4%, 2/46). Patients with CNH had the hightest serum cancer antigen 125 (CA125) level [(34.3±35.2) kU/L]. MSI-H subtype had more family history of tumors (6/7), more with loss of mismatch repair (MMR) protein expression by immunohistochemical (7/7), and higher nuclear antigen associated with cell proliferation (Ki-67) expression level (3/3). (3) Patients in MSI-H subgroup had the lowest CR rate at 6 months (0/6; P=0.019), and survival analysis showed that they were less likely to achieve CR than those with NSMP subtype (P=0.022). Subgroup analysis of patients with NSMP showed that age ≥30 years related with longer treatment time to CR (P=0.010). In addition, CR was obtained after treatment in 2/3 POLE ultra-mutated cases and 2/2 CNH, respectively. Conclusions: Molecular classification relates with the treatment response in patients with EC and AEH treated with fertility-sparing therapy. Patients with MSI-H subtype have poor treatment efficacy, and patients with NSMP need to be further studied and predict treatment benefit. However, there are few cases in POLE ultra-mutated and CNH subtypes, which need further clinical research.

Brusatol sensitizes endometrial hyperplasia and cancer to progestin by suppressing NRF2-TET1-AKR1C1-mediated progestin metabolism.

Progestin resistance is the main obstacle for the conservative therapy to maintain fertility in women with endometrial cancer. Brusatol was identified as an inhibitor of the NRF2 pathway; however, its impact on progestin resistance and the underlying mechanism remains unclear. Here, we found that brusatol sensitized endometrial cancer to progestin by suppressing NRF2-TET1-AKR1C1-mediated progestin metabolism. Brusatol transcriptionally suppressed AKR1C1 via modifying the hydroxymethylation status in its promoter region through TET1 inhibition. Suppression of AKR1C1 by brusatol resulted in decreased progesterone catabolism and maintained potent progesterone to inhibit endometrial cancer growth. This inhibition pattern has also been found in the established xenograft mouse and organoid models. Aberrant overexpression of AKR1C1 was found in paired endometrial hyperplasia and cancer samples from the same individuals with progestin resistance, whereas attenuated or loss of AKR1C1 was observed in post-treatment samples with well progestin response as compared with paired pre-treatment tissues. Our findings suggest that AKR1C1 expression pattern may serve as an important biomarker of progestin resistance in endometrial cancer.

Loss of HOXA10 causes endometrial hyperplasia progressing to endometrial cancer.

Endometrial cancer is the fourth most common malignancy in women and the precursor lesion is endometrial hyperplasia. HOXA10 is a transcription factor that plays key roles in endometrial functions such as the endowment of receptivity, embryo implantation, and trophoblast invasion. Herein, using testicular transgenesis, we developed transgenic mice that expressed a shRNA against HOXA10 and there was a nearly 70% reduction in the expression of HOXA10 in these animals. We observed that downregulation of HOXA10 led to the development of endometrial hyperplasia in the young animals (3 months), and as they aged (>1 year), most animals developed well-differentiated endometrial adenocarcinoma. In the endometrium of animals with reduced HOXA10, there was increased proliferation and elevated levels of ERα and ERß. In parallel, there was increased expression of Wnt4 and ß-Catenin, SOX9, and YAP1. We propose that chronic reduction in HOXA10 expression disrupts multiple pathways in the uterus that aids in the development of endometrial hyperplasia which progresses to endometrial cancer with age.

Role of Nucleolin in Endometrial Precancerous Hyperplasia and Carcinogenesis: Ex Vivo and In Silico Study.

Endometrial cancer (EC) is the most common gynaecological malignancy. Nucleolin (NCL) is involved in rDNA transcription, cell proliferation, and apoptosis, with high expression associated with worse overall survival (OS) in other adenocarcinomas. Our aims were to assess NCL gene and protein expression and explore the differential expression of NCL-associated genes (NAGs) in endometrial carcinogenesis. Endometrial samples were obtained from 157 women to include healthy, hyperplastic (EH), EC, and metastatic groups. RT-qPCR and immunohistochemistry were employed to assess NCL gene and protein levels. In silico analysis of NAGs in TCGA and GEO datasets was performed, with the prognostic value determined via Human Protein Atlas. NCL mRNA level of EC was lower than in healthy post-menopausal endometrium (p < 0.01). EH samples had lower NCL immuno-expression scores than healthy pre-menopausal (p < 0.001), benign post-menopausal (p < 0.01), and EC (p < 0.0001) samples. Metastatic lesions demonstrated higher NCL quick scores than primary tissue (p = 0.04). Higher NCL Immuno quick scores carried a worse OS in high-grade EC (p = 0.01). Interrogating Uterine Corpus Endometrial Carcinoma (TCGA-UCEC) and Uterine Carcinosarcoma (TCGA-UCS) cohorts revealed NCL to be the most highly upregulated gene in carcinosarcoma, with S100A11, LMNB2, RERG, E2F1 and CCNA2 representing key dysregulated NAGs in EC. Since NCL is implicated in transforming hyperplastic glands into cancer, with further involvement in metastasis, it is suggested to be a promising target for better-informed diagnosis, risk stratification, and management of EC.

YTHDF2 is a novel diagnostic marker of endometrial adenocarcinoma and endometrial atypical hyperplasia/ intraepithelial neoplasia.

Numerous studies show that some biomarkers are aberrantly expressed in endometrial endometrioid adenocarcinoma (EMAC) and endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (EAH/EIN) compared to endometrial benign lesions. Because of low sensitivity and/or specificity, the utility of these markers to distinguish EMAC and EAH/EIN from benign endometrial lesions is limited. YTH domain family 2 (YTHDF2) is a functional N6-methyladenosine (m6A)-specific reader protein that mainly regulates mRNA stability. Aberrant YTHDF2 expression has been reported in many cancers and plays important functions in tumorigenesis and cancer progression. However, its expression in endometrial benign and malignant lesions has not been investigated. We evaluated YTHDF2 mRNA and protein expression in EMAC and normal endometrium using the UALCAN database and validated the bioinformatic results in EMAC cells using qRT-PCR, Western blot, and immunohistochemical (IHC) staining. We found that YTHDF2 was weakly expressed in normal endometrium, benign endometrial lesions, endometrial hyperplasia without atypia, and adenomyosis. In contrast, YTHDF2 was upregulated in EAH/EIN and EMAC. These results indicate that YTHDF2 immunostaining may be a useful tool to distinguish EAH/EIN from EHWA. Finally, YTHDF2 expression can accurately assess the depth of myometrial invasion (DMI) in EMAC when EMAC coexists with adenomyosis.

Role of metformin in functional endometrial hyperplasia and polycystic ovary syndrome involves the regulation of MEG3/miR­223/GLUT4 and SNHG20/miR­4486/GLUT4 signaling.

Metformin (MET) can effectively treat endometrial hyperplasia (EH), and the expression of glucose transporter type 4 insulin­responsive (GLUT4) is closely associated with the development of EH. The present study aimed to verify the effect of MET in functional EH and polycystic ovary syndrome (PCOS). H&E staining was performed to analyze the severity of EH, and immunohistochemistry was performed to evaluate the expression of GLUT4 in the endometrium of PCOS rats. Reverse transcription­quantitative PCR was used to calculate the expression of long non­coding (lnc)RNA­maternally expressed gene 3 (MEG3), lncRNA­small nucleolar RNA host gene 20 (SNHG20), GLUT4 mRNA, microRNA (miR)­223 and miR­4486. Sequence analysis and luciferase assays were performed to explore the regulatory relationship among certain lncRNAs, miRNAs and target genes. EH in PCOS rats was efficiently inhibited by MET administration. The increased expression of GLUT4 in PCOS rats was attenuated by MET treatment. Moreover, the expression levels of lncRNA­MEG3 and lncRNA­SNHG20 were significantly inhibited in the endometrium of PCOS rats. MET treatment also showed remarkable efficiency in restoring the expression of lncRNA­MEG3 and lncRNA­SNHG20. Meanwhile, the expression levels of miR­223 and miR­4486 were notably elevated in the endometrium of PCOS rats, while MET treatment reduced the expression of miR­223 and miR­4486 in PCOS rats. Furthermore, a luciferase assay confirmed the inhibitory relationship between miR­223 and lncRNA­MEG3/GLUT4 expression, as well as between miR­4486 and lncRNA­SNHG20/GLUT4 expression. GLUT4 knockdown restored the decreased viability of HCC­94 cells induced by overexpression of lncRNA­MEG3. To conclude, MET exhibited a therapeutic effect in the treatment of EH by modulating the lncRNA­MEG3/miR­223/GLUT4 and lncRNA­SNHG20/miR­4486/GLUT4 signaling pathways. This work provides mechanistic insight into the development of EH.

Canine cystic endometrial hyperplasia and pyometra may downregulate neuropeptide phoenixin and GPR173 receptor expression.

The most common uterine diseases affecting bitches are cystic endometrial hyperplasia (CEH) and pyometra. The neuropeptide phoenixin (PNX) and its receptor (GPR173) are potential key factors involved in the proliferative and inflammatory regulation of the reproductive system in females. This study aimed to evaluate the expression of PNX and GPR173 by qPCR, western blot and immunofluorescence assays in the endometrium of bitches suffering from CEH or pyometra compared to clinically healthy females. Additionally, PNX and progesterone (P4) plasma concentrations were analysed. The results showed a significantly lower expression levels of PNX and GPR173 (mRNA and protein production) in bitches with the CEH or pyometra groups compared to healthy animals. Immunofluorescence staining examination also confirmed a lower concentration of PNX and GPR173 signals in bitches with pathological uteri. Moreover, a lower concentration of PNX blood levels in bitches suffering from pyometra was observed. The PNX concentration was negatively correlated with P4 but only in healthy bitches. These results illustrate that the development of canine uterine disorders may cause a lower expression of PNX and its receptor GPR173.

PTEN mutations as predictive marker for the high-grade endometrial cancer development in slovak women.

Endometrial carcinoma (ECa) is one of the most common neoplasia of the female genital tract. The phosphatase and tensin (PTEN) homolog is the most frequently mutated tumor suppressor gene in endometrial carcinoma. PTEN encodes a phosphatase, a key regulatory enzyme involved in a signal transduction pathway that regulates cell growth, migration and apoptosis. The study evaluates an association between the morphological appearance of endometrial hyperplasia and ECa, and the presence of PTEN variations, PTEN protein´s level and intracellular localization. A total of 67 archived formalin-fixed and paraffin-embedded human biopsy tissue specimens with normal proliferative and secretory endometrium, endometrial hyperplasia without atypia and endometrial atypical hyperplasia, endometrioid the grade G1 and G3 and serous subtype of ECa were evaluated by sequencing for the presence of mutations in coding regions of PTEN gene of endometrial epithelial cells. The PTEN gene expression and intercellular localization of PTEN protein were evaluated immunohistochemically by immunoreactive score (IRS). PTEN mutation spectrum in endometrial carcinoma was identified for Slovak population. 28 non-silent mutations were identified in PTEN, twelve of them were novel, not annotated in Catalogue of Somatic Mutations in Cancer. Higher frequency of PTEN mutations was observed in serous carcinoma compared to global average. No correlation was observed between samples´ IRS, PTEN cellular localization and identified mutations. PTEN sequencing can be beneficial for patients considering prognosis of disease and sensitivity to treatment.

Co-existing TP53 and ARID1A mutations promote aggressive endometrial tumorigenesis.

TP53 and ARID1A are frequently mutated across cancer but rarely in the same primary tumor. Endometrial cancer has the highest TP53-ARID1A mutual exclusivity rate. However, the functional relationship between TP53 and ARID1A mutations in the endometrium has not been elucidated. We used genetically engineered mice and in vivo genomic approaches to discern both unique and overlapping roles of TP53 and ARID1A in the endometrium. TP53 loss with oncogenic PIK3CAH1047R in the endometrial epithelium results in features of endometrial hyperplasia, adenocarcinoma, and intraepithelial carcinoma. Mutant endometrial epithelial cells were transcriptome profiled and compared to control cells and ARID1A/PIK3CA mutant endometrium. In the context of either TP53 or ARID1A loss, PIK3CA mutant endometrium exhibited inflammatory pathway activation, but other gene expression programs differed based on TP53 or ARID1A status, such as epithelial-to-mesenchymal transition. Gene expression patterns observed in the genetic mouse models are reflective of human tumors with each respective genetic alteration. Consistent with TP53-ARID1A mutual exclusivity, the p53 pathway is activated following ARID1A loss in the endometrial epithelium, where ARID1A normally directly represses p53 pathway genes in vivo, including the stress-inducible transcription factor, ATF3. However, co-existing TP53-ARID1A mutations led to invasive adenocarcinoma associated with mutant ARID1A-driven ATF3 induction, reduced apoptosis, TP63+ squamous differentiation and invasion. These data suggest TP53 and ARID1A mutations drive shared and distinct tumorigenic programs in the endometrium and promote invasive endometrial cancer when existing simultaneously. Hence, TP53 and ARID1A mutations may co-occur in a subset of aggressive or metastatic endometrial cancers, with ARID1A loss promoting squamous differentiation and the acquisition of invasive properties.

Loss of ten-eleven translocation 1 (TET1) expression as a diagnostic and prognostic biomarker of endometrial carcinoma.

Endometrial carcinoma (EC) is the most common gynecological cancer. However, there is currently no routinely used biomarker for differential diagnosis of malignant and premalignant endometrial lesions. Ten-eleven translocation (TET) proteins, especially TET1, were found to play a significant role in DNA demethylation, via conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). TET1, 5-mC, and 5-hmC expression profiles in endometrial carcinogenesis are currently unclear. We conducted a hospital-based retrospective review of the immunohistochemical expression of TET1, 5-mC, and 5-hmC in 181 endometrial samples. A "high" TET1 and 5-hmC expression score was observed in all cases of normal endometrium (100.0% and 100.0%, respectively) and in most samples of endometrial hyperplasia without atypia (90.9% and 78.8%, respectively) and atypical hyperplasia (90.6% and 93.8%, respectively), but a "high" score was found in only less than half of the EC samples (48.8% and 46.5%, respectively). The TET1 and 5-hmC expression scores were significantly higher in normal endometrium and premalignant endometrial lesions than in ECs (p < 0.001). A "high" 5-mC expression score was observed more frequently for ECs (81.4%) than for normal endometrium (40.0%), endometrial hyperplasia without atypia (51.5%), and atypical hyperplasia (53.1%) (p < 0.001). We also found that TET1 mRNA expression was lower in ECs compared to normal tissues (p = 0.0037). TET1 immunohistochemistry (IHC) scores were highly proportional to the TET1 mRNA levels and we summarize that the TET1 IHC scoring can be used for biomarker determinations. Most importantly, a higher TET1 score in EC cases was associated with a good overall survival (OS) rate, with a hazard ratio (HR) of 0.31 for death (95% confidence interval: 0.11-0.84). Our findings suggest that TET1, 5-mC, and 5-hmC expression is a potential histopathology biomarker for the differential diagnosis of malignant and premalignant endometrial lesions. TET1 is also a potential prognostic marker for EC.

Atypical Endometrial Hyperplasia, Low-grade: "Much ADO About Nothing".

Atypical endometrial hyperplasia (AEH) is considered a precursor of endometrioid carcinoma. The 2020 World Health Organization (WHO) classification divides endometrial hyperplasia into 2 categories: hyperplasia without atypia and atypical hyperplasia/endometrioid intraepithelial neoplasia (EIN); however, this classification does not consider the degree of nuclear atypia. We graded nuclear atypia for estimating the risk of finding carcinoma at hysterectomy. Also, we investigated genes involved in endometrial carcinogenesis including mismatch repair (MMR) genes and ARID1A, PIK3CA, PTEN, KRAS, and CTNNB1. We reviewed 79 biopsies of AEH from 79 patients who underwent hysterectomy within a 1-year interval. Intraobserver and interobserver agreement of grading nuclear atypia and the relationship between the grade of nuclear atypia at biopsy and the findings at hysterectomy were evaluated. Immunohistochemistry for MMR status was performed in all cases and targeted sequencing in 11. Using low-grade versus high-grade nuclear atypia, κ values ranged from 0.74 to 0.91 (89% to 96%) and from 0.72 to 0.81 (87% to 91%) for the intraobserver and the interobserver agreement, respectively. The degree of nuclear atypia at biopsy was highly predictive of the findings at hysterectomy (P=1.6×10-15). Of 53 patients with low-grade AEH, none had carcinoma at hysterectomy, whereas 6 (6/26; 23%) with high-grade AEH in the biopsy also had high-grade AEH in the uterus and 16 (16/26; 61%) had FIGO grade 1 carcinoma. MMR deficiency was found in 3 of the 79 patients. None of the genes showed a mutational load significantly associated with the degree of nuclear atypia. In summary, our data show high reproducibility within and between observers for the diagnosis of low-grade and high-grade AEH. Most cases of AEH had low-grade nuclear atypia and neither high-grade AEH nor carcinoma was encountered in the corresponding hysterectomy specimens.