Endometrial Tissue Samples Research Articles

Apolipoprotein E enhances migration of endometrial cancer cells byactivating the ERK/MMP9 signaling pathway

To study the role of apolipoprotein E (APOE) in regulating endometrial cancer metastasis and explore the signaling pathway in the regulatory mechanism. Human endometrial cancer cell line HEC-1B was transfected with a control siRNA (siCtrl) or a specific siRNA targeting APOE (siAPOE) or with either pEGFP-N1 plasmid or an APOEoverexpressing plasmid. The changes in migration, proliferation, apoptosis and cell cycle of the transfected cells were examined using wound healing assay, Transwell migration assay, MTT assay, flow cytometry, and Hoechst staining. The activity of the ERK/MMP9 signaling pathway in the transfected cells was assessed using RT-qPCR and Western blotting. The expression level of APOE in clinical specimens of endometrial cancer tissues were detected using immunohistochemistry and its correlation with differentiation of endometrial cancer tissues was analyzed. Wound healing assay and Transwell migration assay showed that compared with those in siCtrl group, HEC-1B cells transfected with siAPOE showed significantly reduced migration ability (P < 0.05), whereas APOE overexpression significantly promoted the migration of the cells (P < 0.05). Neither APOE knockdown nor overexpression produced significant effects on HEC-1B cell proliferation as shown by MTT assay and flow cytometry. Hoechst staining revealed that transfection with siAPOE did not significantly affect apoptosis of HEC-1B cells. APOE knockdown obviously reduced and APOE overexpression enhanced ERK phosphorylation and MMP9 expression in HEC-1B cells (P < 0.05). Treatment with U0126 partially reversed the effects of APOE overexpression on ERK phosphorylation, migration and MMP9 expression in HEC-1B cells (P < 0.05). APOE is highly expressed in clinical samples of endometrial cancer tissues as compared with the adjacent tissues. APOE is highly expressed in endometrial cancer tissues to promote cancer cell migration by enhancing ERK phosphorylation and MMP9 expression.

Revealing the uterine blood vessel network via virtual pathology.

The distribution of the blood vessel network at any point in time in any body tissue, may provide valuable information with regards to the tissue condition and its angiogenesis functionality. The blood vessel three-dimensional network of the endometrium goes through a process of change over a relatively short period of 4 weeks on average. It is well accepted that this angiogenesis is closely related to the success or failure of the implantation of the embryo Objective and rationale: Our study aims to present a method to follow the three-dimensional evolution of the superficial blood vessel distribution in the endometrium throughout the uterine cycle. This method utilizes differences in the observed broadband colors of the blood vessels in order to assess their depth coordinate below the endometrial tissue surface. We implemented the method using microscopic images of fresh, ex-vivo, endometrial samples of different cycle days to obtain the statistical evolution track of the superficial blood vessel population in both human and animal (swine) samples. In human samples we observed a systematic and consistent trend in the BV diameter distribution at different tissue depths. We demonstrate that the magnitude of this trend evolves throughout the course of the female cycle. This method has the potential to further our understanding of the mechanisms of angiogenesis in tissues other than the endometrium. We propose that this method may also contribute to more precise endometrial dating and may assist in more accurate determination of embryo transfer timing within IVF treatments.

Expression pattern of circadian rhythm-related genes and its potential relationship with miRNAs activity in endometrial cancer.

The circadian clock is an autonomous oscillator that controls key aspects of cell physiology, including metabolism, transcriptional state, and cell signaling. Disturbances of circadian rhythms lead to disruption of cell and tissue homeostasis, which promotes carcinogenesis. The aim of the study was to determine the expression of circadian rhythm-related genes in endometrial cancer and to select miRNAs involved in the regulation of their expression. 50 endometrial tissue samples were collected from patients who underwent hysterectomy: 40 diagnosed with endometrial cancer and 10 without cancer. Expression profile of circadian rhythm-related genes was evaluated using microarrays and validated with RT-qPCR. MicroRNA expression was assessed using microarrays. Then mirTAR tool was used to identify miRNAs involved in the expression regulation of circadian rhythm-related genes. CLOCK expression is disrupted in endometrial cancer, which may be due to miR-15b, miR-331-3p and miR-200a overexpression. Elevated NPAS2 and CSNK1D levels may be associated with miR-432 silencing. In addition, high miR-874 and miR-200a expression may be potentially responsible for the reduction of PER3 level. Change of CLOCK, CSNK1D, NPAS2 and PER3 expression may suggest that circadian rhythms are disrupted in endometrial cancer. A possible mechanism of the observed changes may be related to miRNAs activity.

Endometrial Heparin-Binding Epidermal Growth Factor Gene Expression and Hormone Level Changes in Implantation Window of Obese Women with Polycystic Ovarian Syndrome

Polycystic ovarian syndrome (PCOS) is a common endocrine disorder amongst reproductive-age women, and 61% to 76% of women with PCOS are obese. Obese women with PCOS are usually burdened with infertility problems due to implantation failure. Thus, progesterone treatment is usually used to improve implantation rates. Although Hb-EGF expression is actively involved in endometrial receptivity and implantation, the data on heparin-binding epidermal growth factor (Hb-EGF) expression following progesterone therapy in obese women with PCOS are still lacking. To investigate the changes in serum follicle-stimulating hormone (FSH), luteinising hormone (LH), dehydroepiandrosterone sulphate (DHEA), progesterone and oestradiol levels and Hb-EGF expression in obese women with PCOS during the implantation window following progesterone therapy. A total of 40 participants aged 18-40 years old were recruited following the provision of written consent. The participants were divided into the obese PCOS, normal-weight PCOS, obese fertile and normal-weight fertile groups. First blood collection was done before ovulation. Then, daily oral micronised progesterone (Utrogestan 200 mg) was given to the PCOS group for 10 days. The treatment was followed by a second blood collection and endometrial tissue sampling by using a Pipelle de Cornier catheter. In the fertile group, ovulation was confirmed by using ultrasound, and a second blood sample was collected on days 7 to 9 postovulation. The serum levels of FSH, LH, DHEA, progesterone and oestradiol were measured in all participants. Wilcoxon signed-rank test was used to compare FSH, LH, DHEA, progesterone and oestradiol levels during pre- and postovulation. Mann-Whitney test was performed to compare FSH, LH, DHEA, progesterone and oestradiol levels between two groups: (1) the PCOS group and the fertile group, (2) the obese PCOS group and the non-obese PCOS group and (3) the obese group and the non-obese fertile group. Serum FSH levels were lower in obese women in their follicular phase than in women with normal weight regardless of their PCOS status, whereas serum LH/FSH ratios and DHEA levels were higher in women with PCOS than in women without PCOS. However, endometrial Hb-EGF expression was lower in the obese PCOS group than in the normal-weight PCOS group. Different patterns of hormonal levels and Hb-EGF expression levels were seen between the studied groups. However, further in vitro and in vivo studies are needed to investigate the mechanism underlying the changes in FSH, LH/FSH ratio, DHEA and Hb-EGF expression in PCOS after progesterone treatment.

Metformin Inhibits the Estrogen-mediated Epithelial-Mesenchymal Transition of Ectopic Endometrial Stromal Cells in Endometriosis.

Endometriosis is an estrogen-dependent disease characterized by the ectopic implantation and growth of endometrial tissue outside the uterus. Endometrial stromal cells (ESCs) play a crucial role in the pathogenesis of endometriosis. Epithelial-mesenchymal transition (EMT) has recently been described in endometriosis and was induced by estrogen. Metformin has been shown to inhibit EMT in various diseases, but its role in endometriosis remains unclear. We collected endometrial tissue samples from patients with endometriosis and healthy controls and isolated primary ESCs. We performed gene expression analysis using the Gene Expression Omnibus (GEO) dataset and validated the results by immunohistochemistry in tissue samples. We also assessed the effects of metformin on the proliferation, migration and invasion of ectopic ESCs (EESCs) by Cell Counting Kit-8 and Transwell migration and invasion assays, respectively. We analyzed the protein expression of EMT-related markers (N-cadherin, vimentin, twist, and snail) and β-catenin by Western blotting and immunohistochemistry. We found that vimentin was highly expressed in ectopic endometrial tissues compared to normal endometrial tissues. Metformin treatment inhibited the proliferation, migration and invasion of EESCs in a dose-dependent manner. Metformin treatment also downregulated the expression of EMT-related markers and reduced the expression and nuclear translocation of β-catenin in EESCs. Our results suggest that metformin inhibits estrogen-induced EMT and regulates the expression of β-catenin in EESCs. This study provides new insights into the potential therapeutic role of metformin in endometriosis.

Патогенетические особенности сигнального пути прогестерона и экспрессия PR-B при глубоком инфильтративном эндометриозе

Objective. To study the expression of progesterone receptor isoform B (PR-B) in patients with deep infiltrating endometriosis (DIE). Patients and methods. This article presents a comparative analysis of PR-B expression in eutopic and ectopic endometrial tissue samples obtained from patients with DIE (study group, n = 50) and tubo-peritoneal factor infertility (comparison group, n = 9). PR-B expression was analyzed by immunohistochemistry. Results. In the group of patients with DIE, PR-B expression in ectopic endometrial glandular and stromal cells of endometriosis lesions in the peritoneum and colon was significantly higher compared to the capsule of ovarian endometriotic cyst (p &lt; 0.05). Analysis of PR-B expression in eutopic endometrium showed no significant differences between patients in the study group and comparison group (p &gt; 0.05). Stromal cell PR-B expression in patients with infertility in the study group was significantly lower compared to fertile patients with DIE (p = 0.04). Conclusion. No decrease in PR-B expression in the endometrium of patients with DIE was found, which may indicate a favorable response to postoperative hormonal therapy in this group of patients. At the same time, we observed a decrease in PR-B expression in eutopic endometrial stromal cells in patients with DIE and infertility compared to fertile patients, which confirms the role of PR in disrupting pregnancy onset in endometriosis. Search for prognostic markers is currently implemented to develop a personalized approach to the treatment of endometriosis and individual selection of hormone therapy for preventing primary and subsequent surgical interventions and reducing severe pain syndrome in endometriosis. Key words: progesterone, PR-B expression, deep endometriosis, pain syndrome, dienogest

Immunohistochemical analysis of selected stem cell markers including stage-specific mouse embryonic antigen 1 (SSEA-1) in porcine and bovine endometrium throughout the oestrous cycle

There has been a lack of research regarding endometrial stem cells in farm animals, and particularly, regarding epithelial stem/progenitor cells. We aimed to identify potential stem cell subpopulations in porcine and bovine endometrium by measuring the expression of selected stem cell markers (OCT3/4, CD44, SSEA-1, SOX10, CD73, and CD117) during the follicular and luteal phases of the oestrous cycle. We studied 28 endometrial tissue samples collected from 14 healthy, cycling pigs and cows. The endometrial mucosa of each sample was divided into basal, middle, and luminal portions, and the immunohistochemical staining intensity and percentages of cells that were marker-positive were recorded for each portion of the endometrium. Expression of OCT-3/4 was identified in the glands and stroma, and that of SOX10 and CD117 was identified in stroma of both porcine and bovine endometrium. In porcine endometrium, CD44 was only expressed in the glands, whereas SSEA-1 was expressed in the glands and stroma. In bovine endometrium, CD73 was only expressed in the glands. Differences in the expression of most of these markers were found between phases of the oestrous cycle and/or portions of the endometrial mucosa. Our data indicate the existence of both epithelial and mesenchymal stem cell subpopulations in the endometrium of pigs and cows during their oestrous cycles. The expression pattern of the stem cell marker SSEA-1 in porcine endometrium suggests a possible alternative location of the epithelial stem/progenitor cell population in the middle portion of the endometrial glands.

Evaluation of genes and molecular pathway related pathogenesis of endometriosis: A bioinformatics approach

ObjectiveAlthough recent studies have focused on the role of genes in the pathogenesis of endometriosis, the investigation of genes and molecular pathways involved in the pathogenesis of endometriosis with a bioinformatics approach has not been addressed yet. Hence, we aimed to investigate this issue in our study. Materials and methodsThe gene expression datasets GSE7305 and GSE124010 were obtained from the GEO database, consisting of endometriosis and normal endometrial tissue samples. Differential expression analysis was performed using the R software and LIMMA package, applying cutoff criteria of p-value <0.05 and |logFC| > 1 to identify differentially expressed miRNAs (DEmiRs) and genes (DEGs). The MultiMiR package was used to predict target genes for DEmiRs, considering the overlapping predictions from multiple databases. Venn diagrams were used to establish miRNA-target relationships and identify interactions with DEGs. Functional enrichment analyses were conducted using the Enrichr analysis tool to predict the molecular function, biological processes, cellular components, and pathways associated with the target genes. Protein-protein interaction networks were constructed using the STRING database and visualized in Cytoscape, and hub genes were identified based on their centrality measures. Key hub genes were selected by considering those shared among all three centrality measures. The expression levels of hub genes were validated in endometriosis samples using ROC curves to assess diagnostic accuracy. The AUC was calculated to compare the diagnostic value of the hub genes, using GraphPad Prism version 9.1.0. ResultThe analysis identified six hub genes (CCNA2, ESR1, AURKA, MKI67, and VEGFA) in the protein-protein interaction network. Among the miRNAs, hsa-miR-29a-3p showed the highest number of interactions with these hub genes, making it the key miRNA in the study. These findings suggest the potential involvement of hsa-miR-29a-3p in regulating the expression of the identified hub genes in endometriosis. ConclusionIdentifying pathways related to the expression of hub genes can be useful in designing appropriate treatment strategies. On the other hand, examining the pathways activated by hub genes can be effective in preventing of endometriosis progression.

Decreased CD44v3 expression impairs endometrial stromal cell proliferation and decidualization in women with recurrent implantation failure

BackgroundThe precise pathogenesis of poor endometrial receptivity in recurrent implantation failure (RIF) remains unclear. This study was aimed at exploring the effects of different CD44 isoforms in the mid-secretory phase endometrium on endometrial receptivity in women with RIF.MethodsMid-secretory phase endometrial tissue samples were obtained from the following two groups of women who had undergone IVF: (a) 24 patients with RIF and (b) 18 patients with infertility due to tubal obstruction, who had achieved a successful clinical pregnancy after the first embryo transfer in IVF (control group). Identification of differentially expressed CD44 isoforms in endometrial tissues was assessed using immunohistochemistry, qPCR, and western blotting. Effects of overexpression and knockdown of CD44v3 on proliferation and decidualization of immortalized human endometrial stromal cells (T-HESCs) and primary HESCs were investigated by qPCR and western blot analysis. A heterologous coculture system of embryo implantation was constructed to mimic the process of trophoblast invasion during implantation.ResultsThe expression of CD44v3 was significantly higher in the mid-secretory phase of endometrial stromal cells than in the proliferation phase, but was notably lower in RIF patients. Knockdown of CD44v3 significantly downregulated cell proliferation both in T-HESCs and HESCs. The expression of decidualization markers, prolactin (PRL) and insulin like growth factor binding protein-1 (IGFBP1), was notably decreased following the knockdown of CD44v3, whereas the expression of both PRL and IGFBP1 increased after its overexpression in HESCs. Furthermore, the CD44v3-knockdown HESCs displayed significant deficiency in supporting trophoblast outgrowth in a coculture system of embryo implantation; however, overexpression of CD44v3 in HESCs promoted trophoblast outgrowth.ConclusionThe reduced expression of CD44v3 suppresses the proliferation and decidualization of HESCs, which might play a pivotal role in poor endometrial receptivity in women with RIF.

Point-of-Care Diagnosis of Endometrial Cancer Using the Surgical Intelligent Knife (iKnife)—A Prospective Pilot Study of Diagnostic Accuracy

Introduction: Delays in the diagnosis and treatment of endometrial cancer negatively impact patient survival. The aim of this study was to establish whether rapid evaporative ionisation mass spectrometry using the iKnife can accurately distinguish between normal and malignant endometrial biopsy tissue samples in real time, enabling point-of-care (POC) diagnoses. Methods: Pipelle biopsy samples were obtained from consecutive women needing biopsies for clinical reasons. A Waters G2-XS Xevo Q-Tof mass spectrometer was used in conjunction with a modified handheld diathermy (collectively called the 'iKnife'). Each tissue sample was processed with diathermy, and the resultant surgical aerosol containing ionic lipid species was then analysed, producing spectra. Principal component analyses and linear discriminant analyses were performed to determine variance in spectral signatures. Leave-one-patient-out cross-validation was used to test the diagnostic accuracy. Results: One hundred and fifty patients provided Pipelle biopsy samples (85 normal, 59 malignant, 4 hyperplasia and 2 insufficient), yielding 453 spectra. The iKnife differentiated between normal and malignant endometrial tissues on the basis of differential phospholipid spectra. Cross-validation revealed a diagnostic accuracy of 89% with sensitivity, specificity, positive predictive value and negative predictive value of 85%, 93%, 94% and 85%, respectively. Conclusions: This study is the first to use the iKnife to identify cancer in endometrial Pipelle biopsy samples. These results are highly encouraging and suggest that the iKnife could be used in the clinic to provide a POC diagnosis.

Endometrial blood vessel morphometry in patients presenting with abnormal uterine bleeding.

: Abnormal uterine bleeding (AUB) is one of the most common problems encountered in gynecological practice. Various benign and malignant disorders of the endometrial tissue show vascular changes such as congestion, dilatation, and vessel wall irregularities. To evaluate the vascular morphometry of the endometrial tissue in AUB. : A descriptive cross-sectional study of the endometrial tissue in patients presented with AUB was undertaken for vascular morphometric analysis. Histopathological processing of the endometrial tissue samples was done as per the standard format, and the slides were evaluated for vascular morphometry. Out of 150 cases of endometrial tissue in patients presented with AUB, 80 cases were reported as proliferative phase, 41 as secretory phase, 15 as disordered proliferative endometrium, 6 as atrophic phase endometrium, and 4 each of endometrial hyperplasia without atypia and endometrial carcinoma. An average number of endometrial blood vessels and large-sized blood vessels were more in endometrial carcinoma and endometrial hyperplasia without atypia as compared to proliferative phase, secretory phase, atrophic endometrium, and disordered proliferative endometrium. Vessel shape irregularities and vascular congestion were observed in all the cases of atrophic endometrium, endometrial carcinoma, and endometrial hyperplasia without atypia. Endometrial carcinoma showed severe dilatation of the endometrial blood vessels. Vascular morphometry changes were noted in endometrial hyperplasia, endometrial carcinoma, disordered proliferative endometrium, and atrophic phase endometrium. These findings suggest that studies or trials related to anti-angiogenic therapy may help to plan anti-angiogenic therapy in patients with AUB.

NR3C1 and glucocorticoid-regulatory genes mRNA and protein expression in the endometrium and ampulla during the bovine estrous cycle

The bovine reproductive tract exhibits changes during the estrous cycle modulated by the interplay of steroid hormones. Glucocorticoids can be detrimental when stress-induced but are relevant at baseline levels for appropriate reproductive function. Here, an analysis of quantitative real-time PCR was performed to study the bovine glucocorticoid-related baseline gene transcription in endometrial and ampullar tissue samples derived from three time points of the estrous cycle, stage I (Days 1–4), stage III (Days 11–17) and stage IV (Days 18–20). Our results revealed expression differences during stages, as expression observed in the ampulla was higher during the post-ovulatory phase (stage I), including the glucocorticoid receptor NR3C1, and some of its regulators, involved in glucocorticoid availability (HSD11B1 and HSD11B2) and transcriptional actions (FKBP4 and FKBP5). In contrast, in the endometrium, higher expression of the steroid receptors was observed during the late luteal phase (stage III), including ESR1, ESR2, PGRMC1 and PGRMC2, and HSD11B1 expression decreased, while HSD11B2 increased. Moreover, at protein level, FKBP4 was higher expressed during the late luteal phase, and NR3C1 during the pre-ovulatory phase (stage IV). These results suggest that tight regulation of the glucocorticoid activity is promoted in the ampulla, when reproductive events are taking place, including oocyte maturation. Moreover, most expression changes in the endometrium were observed during the late luteal phase, and may be related to the embryonic maternal recognition. In conclusion, the glucocorticoid regulation changes across the estrous cycle and may be playing a role on the reproductive events occurring in the bovine ampulla and endometrium.

Histopathological evaluation of endometrial curettings in women with abnormal uterine bleeding: A retrospective analysis

Endometrial pathologies have been a predominate problem faced by women globally.Among all the endometrial pathologies, abnormal uterine bleeding is first in the list among the developing countries. To analyse the percentage of endometrial pathology detected in women who were diagnosed from abnormal uterine bleeding in our tertiary health care centre.A retrospective study of 158 women patients who were diagnosed with abnormal uterine bleeding and their endometrial tissue sampling were evaluated for endometrial pathology. The study data were collected from February 2017 till January 2019 based on Inclusion criteria and Exclusion criteria.According to the inclusion criteria, the 152 cases medical data which includes clinical evaluations along with endometrial histopathology evaluation were analysed. According to our analysis, the histopathological examination reports revealed most of the cases were diagnosed with benign endometrial pathology (34.72%) and abnormal endometrial pathology (9.09%). The 9.09% includes 7.44% presented with endometrial hyperplasia and 1.65% presented with endometrial cancer.Histopathological study of endometrial curettage tissue is the approved method for detecting any endometrial pathology. This is an onerous task for pathologists as not to miss any important findings. According to our analysis the commonest incidences of endometrial pathology were normal endometrium 56.19% of cases and benign pathology 34.72% and the less incidences of preneoplastic lesions 7.44% and endometrial carcinoma 1.65%.

Human endometrial cell-type-specific RNA sequencing provides new insights into the embryo-endometrium interplay.

Which genes regulate receptivity in the epithelial and stromal cellular compartments of the human endometrium, and which molecules are interacting in the implantation process between the blastocyst and the endometrial cells? A set of receptivity-specific genes in the endometrial epithelial and stromal cells was identified, and the role of galectins (LGALS1 and LGALS3), integrin β1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in embryo-endometrium dialogue among many other protein-protein interactions were highlighted. The molecular dialogue taking place between the human embryo and the endometrium is poorly understood due to ethical and technical reasons, leaving human embryo implantation mostly uncharted. Paired pre-receptive and receptive phase endometrial tissue samples from 16 healthy women were used for RNA sequencing. Trophectoderm RNA sequences were from blastocysts. Cell-type-specific RNA-seq analysis of freshly isolated endometrial epithelial and stromal cells using fluorescence-activated cell sorting (FACS) from 16 paired pre-receptive and receptive tissue samples was performed. Endometrial transcriptome data were further combined in silico with trophectodermal gene expression data from 466 single cells originating from 17 blastocysts to characterize the first steps of embryo implantation. We constructed a protein-protein interaction network between endometrial epithelial and embryonal trophectodermal cells, and between endometrial stromal and trophectodermal cells, thereby focusing on the very first phases of embryo implantation, and highlighting the molecules likely to be involved in the embryo apposition, attachment and invasion. In total, 499 epithelial and 581 stromal genes were up-regulated in the receptive phase endometria when compared to pre-receptive samples. The constructed protein-protein interactions identified a complex network of 558 prioritized protein-protein interactions between trophectodermal, epithelial and stromal cells, which were grouped into clusters based on the function of the involved molecules. The role of galectins (LGALS1 and LGALS3), integrin β1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in the embryo implantation process were highlighted. RNA-seq data are available at www.ncbi.nlm.nih.gov/geo under accession number GSE97929. Providing a static snap-shot of a dynamic process and the nature of prediction analysis is limited to the known interactions available in databases. Furthermore, the cell sorting technique used separated enriched epithelial cells and stromal cells but did not separate luminal from glandular epithelium. Also, the use of biopsies taken from non-pregnant women and using spare IVF embryos (due to ethical considerations) might miss some of the critical interactions characteristic of natural conception only. The findings of our study provide new insights into the molecular embryo-endometrium interplay in the first steps of implantation process in humans. Knowledge about the endometrial cell-type-specific molecules that coordinate successful implantation is vital for understanding human reproduction and the underlying causes of implantation failure and infertility. Our study results provide a useful resource for future reproductive research, allowing the exploration of unknown mechanisms of implantation. We envision that those studies will help to improve the understanding of the complex embryo implantation process, and hopefully generate new prognostic and diagnostic biomarkers and therapeutic approaches to target both infertility and fertility, in the form of new contraceptives. This research was funded by the Estonian Research Council (grant PRG1076); Horizon 2020 innovation grant (ERIN, grant no. EU952516); Enterprise Estonia (grant EU48695); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, grant SARM, EU324509); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER) (grants RYC-2016-21199, ENDORE SAF2017-87526-R, and Endo-Map PID2021-127280OB-100); Programa Operativo FEDER Andalucía (B-CTS-500-UGR18; A-CTS-614-UGR20), Junta de Andalucía (PAIDI P20_00158); Margarita Salas program for the Requalification of the Spanish University system (UJAR01MS); the Knut and Alice Wallenberg Foundation (KAW 2015.0096); Swedish Research Council (2012-2844); and Sigrid Jusélius Foundation; Academy of Finland. A.S.-L. is funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-085440). K.G.-D. has received consulting fees and/or honoraria from RemovAid AS, Norway Bayer, MSD, Gedeon Richter, Mithra, Exeltis, MedinCell, Natural cycles, Exelgyn, Vifor, Organon, Campus Pharma and HRA-Pharma and NIH support to the institution; D.B. is an employee of IGENOMIX. The rest of the authors declare no conflict of interest.

The amount of preoperative endometrial tissue surface in relation to final endometrial cancer classification

ObjectiveTo evaluate whether the amount of preoperative endometrial tissue surface is related to the degree of concordance with final low- and high-grade endometrial cancer (EC). In addition, to determine whether discordance is influenced by sampling method and impacts outcome. MethodsA retrospective cohort study within the European Network for Individualized Treatment of Endometrial Cancer (ENITEC). Surface of preoperative endometrial tissue samples was digitally calculated using ImageJ. Tumor samples were classified into low-grade (grade 1–2 endometrioid EC (EEC)) and high-grade (grade 3 EEC + non-endometroid EC). ResultsThe study cohort included 573 tumor samples. Overall concordance between pre- and postoperative diagnosis was 60.0%, and 88.8% when classified into low- and high-grade EC. Upgrading (preoperative low-grade, postoperative high-grade EC) was found in 7.8% and downgrading (preoperative high-grade, postoperative low-grade EC) in 26.7%. The median endometrial tissue surface was significantly lower in concordant diagnoses when compared to discordant diagnoses, respectively 18.7 mm2 and 23.5 mm2 (P = 0.022). Sampling method did not influence the concordance in tumor classification. Patients with preoperative high-grade and postoperative low-grade showed significant lower DSS compared to patients with concordant low-grade EC (P = 0.039). ConclusionThe amount of preoperative endometrial tissue surface was inversely related to the degree of concordance with final tumor low- and high-grade. Obtaining higher amount of preoperative endometrial tissue surface does not increase the concordance between pre- and postoperative low- and high-grade diagnosis in EC. Awareness of clinically relevant down- and upgrading is crucial to reduce subsequent over- or undertreatment with impact on outcome.

Comprehensive Analysis of circRNA-Mediated ceRNA Regulatory Networks in relation to Recurrent Implantation Failure.

Recurrent implantation failure (RIF) is attributed to endometrial receptivity dysfunction with many unanswered questions. Up to now, there is no explanation for RIF, and therapeutic strategies are usually limited to supportive care. In this study, we differentially analyzed the raw data deposited in three eligible microarray datasets, GSE111974, GSE121219, and GSE147442 to screen DE-mRNAs, DE-miRNAs, and DE-circRNAs, respectively. The value of log2-fold change |log2FC| ≥ 1 and the adjusted p value < 0.05 were considered differentially expressed between RIF and fertile control. We found 350 DE-mRNAs, 43 DE-miRNAs, and 1968 DE-circRNAs between RIF and fertile control. The PPI network identified 6 hub genes with degree ≥10, KDR, AGT, POSTN, TOP2A, RRM2, and PTGS2, in RIF. KDR, AGT, POSTN, TOP2A, and RRM2 were downregulated in endometrial tissue samples of RIF compared with those of fertile control, while PTGS2 was upregulated in endometrial tissue samples of RIF compared with those of fertile control. According to the ceRNA hypothesis, 15 groups of ceRNA network based on 10 circRNAs, hsa_circ_001572, hsa_circ_001884, hsa_circ_001375, hsa_circ_001449, hsa_circ_000029, hsa_circ_001168, hsa_circ_000210, hsa_circ_001484, hsa_circ_001698, and hsa_circ_000089 were constructed in RIF. In conclusion, the present study examined the possible role of circRNAs and their related ceRNA network involved in the pathogenesis of RIF.

Towards less invasive molecular diagnostics for endometrial cancer: massively parallel sequencing of endometrial lavage specimens in women attending for an office hysteroscopy.

We aimed to detect endometrial cancer (EC)-associated mutations in endometrial lavage specimens collected in an office setting and to compare the detected mutations with those identified in tissue samples. Participants included 16 women attending for an office hysteroscopy because of suspected EC between July 2020 and October 2021. Massively parallel sequencing was conducted using the targeted 72 cancer-associated genes. Endometrial lavage specimens, endometrial tissue samples, and blood samples were simultaneously sequenced to establish the concordance of genetic alterations. In this study, the vast majority of EC-associated mutations identified in lavage samples (R2 = 0.948) were identical to those detected in endometrial tissues. Of the 13 patients with EC, 12 (92.3%) had at least one mutation identified in endometrial lavage samples. Notably, no mutations in lavage samples were identified in the two patients with a previous history of EC but no actual endometrial lesions, supporting a high negative predictive value of the test. A patient previously diagnosed with EC and with current evidence of atypical hyperplasia showed persisting PTEN, PIK3R1, and KRAS mutations in her endometrial lavage specimen. PTEN was the most commonly mutated gene, followed by PIK3R1, ARID1A, PIK3CA, CTNNB1, and KRAS. In conclusions, our study provides pilot evidence on the actionability of uterine lavage samples sequencing to detect EC-associated mutations in women with suspected endometrial lesions. In a precision medicine framework, the high mutational concordance between uterine lavage samples and tissue specimens may help inform less invasive diagnostic protocols and the need for ongoing surveillance in patients with EC who wished for fertility-preserving treatment. KEY MESSAGES: • Sequencing of uterine lavage samples collected by office hysteroscopy is feasible. •Most EC mutations identified in lavage were identical to endometrial tissues. •Sequencing of uterine lavage samples may help inform diagnostic protocols for EC. •This approach can be used for recurrence surveillance in patients with EC.

The incidence of endometrial carcinoma in patients with atypical endometrial hyperplasia versus atypical endometrial polyp (438)

Objectives: The objective of the present study was to compare the incidence of endometrial carcinoma following hysterectomy in patients with presurgical diagnoses of atypical endometrial hyperplasia (non-polypoid) versus atypical endometrial hyperplasia confined to an endometrial polyp. Methods: Medical records of women who underwent staging surgery, including hysterectomy, bilateral salpingo-oophorectomy, and sentinel lymph node dissection, for atypical endometrial hyperplasia or atypical endometrial polyp between 2016 and 2020 were reviewed. Results: Seventy-nine women who underwent surgery were included. Of those, 46 women were diagnosed with an atypical endometrial polyp that was resected via hysteroscopy (endometrial polyp group), and 33 women had non-polypoid atypical endometrial hyperplasia following endometrial tissue sampling (non-polypoid group). The mean age at diagnosis was 60.2 ± 9.9 years in the endometrial polyp group compared to 61.7 ± 12 years in the non-polypoid group (p=0.5). Most women (81.8%) in the non-polypoid group presented with post-menopausal bleeding, whereas 41.3% of the women in the endometrial polyp group were asymptomatic (p=0.001). Pathology results from the hysterectomy specimens revealed concurrent endometrial carcinoma in 23.9% of women in the endometrial polyp group compared to 51.5% of women in the non-polypoid group (p=0.001). Ninety-one percent of cancers were grade 1 in the endometrial polyp group compared to 53% of cancers in the non-polypoid group. Grade 2 was found in 9% of cancers in the endometrial polyp group compared to 35% of women in the non-polypoid group (p=0.04). Most women were diagnosed at stage IA, 91% in the endometrial polyp group compared to 82% in the non-polypoid group (p=0.6). In both groups, none of the sentinel lymph nodes harvested during surgery were involved by cancer. Conclusions: Concurrent cancer is less frequent with atypical endometrial polyp as compared to atypical endometrial hyperplasia. Still, the high incidence of endometrial carcinoma in both groups supports the current advice to perform a hysterectomy and bilateral salpingo- oophorectomy for peri and post-menopausal women. Our data do not support performing sentinel lymph node dissection for atypical endometrial hyperplasia. Objectives: The objective of the present study was to compare the incidence of endometrial carcinoma following hysterectomy in patients with presurgical diagnoses of atypical endometrial hyperplasia (non-polypoid) versus atypical endometrial hyperplasia confined to an endometrial polyp. Methods: Medical records of women who underwent staging surgery, including hysterectomy, bilateral salpingo-oophorectomy, and sentinel lymph node dissection, for atypical endometrial hyperplasia or atypical endometrial polyp between 2016 and 2020 were reviewed. Results: Seventy-nine women who underwent surgery were included. Of those, 46 women were diagnosed with an atypical endometrial polyp that was resected via hysteroscopy (endometrial polyp group), and 33 women had non-polypoid atypical endometrial hyperplasia following endometrial tissue sampling (non-polypoid group). The mean age at diagnosis was 60.2 ± 9.9 years in the endometrial polyp group compared to 61.7 ± 12 years in the non-polypoid group (p=0.5). Most women (81.8%) in the non-polypoid group presented with post-menopausal bleeding, whereas 41.3% of the women in the endometrial polyp group were asymptomatic (p=0.001). Pathology results from the hysterectomy specimens revealed concurrent endometrial carcinoma in 23.9% of women in the endometrial polyp group compared to 51.5% of women in the non-polypoid group (p=0.001). Ninety-one percent of cancers were grade 1 in the endometrial polyp group compared to 53% of cancers in the non-polypoid group. Grade 2 was found in 9% of cancers in the endometrial polyp group compared to 35% of women in the non-polypoid group (p=0.04). Most women were diagnosed at stage IA, 91% in the endometrial polyp group compared to 82% in the non-polypoid group (p=0.6). In both groups, none of the sentinel lymph nodes harvested during surgery were involved by cancer. Conclusions: Concurrent cancer is less frequent with atypical endometrial polyp as compared to atypical endometrial hyperplasia. Still, the high incidence of endometrial carcinoma in both groups supports the current advice to perform a hysterectomy and bilateral salpingo- oophorectomy for peri and post-menopausal women. Our data do not support performing sentinel lymph node dissection for atypical endometrial hyperplasia.

BHLHE22 Expression Is Associated with a Proinflammatory Immune Microenvironment and Confers a Favorable Prognosis in Endometrial Cancer.

Endometrial cancer (EC) rates are rising annually. Additional prediction markers need to be evaluated because only 10–20% of EC cases show an objective response to immune-checkpoint inhibitors (ICIs). Our previous methylomic study found that BHLHE22 is hypermethylated in EC tissues and can be detected using a Pap-smear sample. BHLHE22, a basic helix loop helix transcription factor family member, is known as a transcriptional repressor and is involved in cell differentiation. However, the role of BHLHE22 in EC remains poorly understood. Herein, we analyzed BHLHE22 expression in 54 paired cancer and normal endometrial tissue samples, and confirmed with databases (TCGA, GTEx, and human protein atlas). We found that BHLHE22 protein expression was significantly downregulated in EC compared with normal endometrium. High BHLHE22 expression was associated with microsatellite-instable subtype, endometrioid type, grade, and age. It showed a significant favorable survival. BHLHE22 overexpression inhibited the proliferation and migration of EC cells. Functional enrichment analysis showed that BHLHE22 was significantly associated with immune-related pathways. Furthermore, BHLHE22 was positively correlated with proinflammatory leukocyte infiltration and expression of chemokine genes in EC. In conclusion, BHLHE22 regulates immune-related pathways and modulates the immune microenvironment of EC.

Evaluation of the relationship between miR-337-3p and RAP1A gene in endometriosis

Background: Endometriosis, a prevalent multifactorial condition, has a different effect on mental and physical health in women. MicroRNAs have been reported as a main epigenetic factor in endometriosis pathogenesis. However, the role of miR-337-3p and its direct target gene, RAP1A, in endometriosis tissues have not been investigated. Objective: The aim of this study was to evaluate the expression level of miR-337-3p and RAP1A gene in endometriosis tissues and normal endometrium tissues. Materials and methods: We measured the expression levels of miR-337-3p and RAP1A gene by quantitative polymerase chain reaction (qRT-PCR) in 15 eutopic and ectopic tissue samples of superficial peritoneal lesions from women with endometriosis and 15 normal endometrial tissue samples from women without any symptom of endometriosis. Results: The results showed the expression level of RAP1A gene significantly increased in endometriosis tissue samples (both of ectopic and eutopic tissues), while miR-337-3p expression level decreased significantly in these tissues compared to the normal endometrium. Conclusion: In this study, we observed an inverse relationship between miR-337-3p and RAP1A gene expression in endometriosis. Dysregulation of these genes can also be interpreted as their role in the pathogenesis and progression of endometriosis.