Endometrial Specimens Research Articles

Immune Microenvironment in Microsatellite-Instable Endometrial Cancers: Hereditary or Sporadic Origin Matters

Purpose: Recent studies show that colorectal tumors with high microsatellite instability (MSI-H) have increased immunogenicity and response to immunotherapy compared with microsatellite-stable (MSS) tumors. It is not yet clear whether MSI-H endometrial cancer may also benefit from these therapies. It is also unknown whether immune response is equivalent in MSI-H endometrial cancer with sporadic or inherited Lynch syndrome origins.Experimental Design: Multiplexed fluorescent IHC was used to compare matched MSI-H (n = 60) and MSS (n = 96) endometrial cancer specimens by evaluating immune cell populations in tumor and stroma compartments. Sporadic MSI-H and Lynch syndrome-associated (LS) MSI-H endometrial cancers were also directly compared.Results: Increased immune cells were present in stroma of MSI-H endometrial cancer compared with MSS, including granzyme B+ cells, activated CTLs (CD8+granzyme B+), and PD-L1+ cells. Granzyme B+ cells and activated CTLs were also increased in the tumor compartment of MSI-H endometrial cancers. Comparing sporadic and LS MSI-H endometrial cancer showed distinct differences in immune cell populations, indicating that mechanisms underlying microsatellite instability alter immune response. Specifically, LS MSI-H endometrial cancer showed increased CD8+ cells and activated CTLs in stroma, with reduced macrophages in stroma and tumor compared with sporadic MSI-H. Sporadic MSI-H had increased PD-L1+ macrophages in stroma and tumor compared with LS MSI-H endometrial cancer.Conclusions: MSI-H endometrial cancer has increased immune cell infiltration compared with MSS endometrial cancer and the hereditary or sporadic origin of microsatellite instability impacts immune response. Clinical trials to determine the role of immunotherapy in patients with MSI-H endometrial cancer must evaluate Lynch syndrome-related and sporadic MSI-H tumors separately. Clin Cancer Res; 23(15); 4473-81. ©2017 AACR.

Relationship of endometrial thickness and endometrial cancer risk in postmenopausal women

Evaluation of the vaginal bleeding of postmenopausal women is crucial to diagnose endometrial lesions. Endometrial thickness measured by transvaginal ultrasonography provides an important reference for diagnosis. Currently, no specific cut-off value of endometrial thickness has been identified to predict the malignant risk for endometrial thickening and postmenopausal women without any symptoms. Most of resected endometrial specimens from postmenopausal women without any symptoms are diagnosed as benign or normal tissues. It seems that postmenopausal women without any symptoms have no need for general screen or intervention of endometrial cancer.

Uterine Glands Agenesia in the Mare

All mammalian uteri contain endometrial glands that synthesize and secrete a substance termed histotroph, which is essential for early pregnancy nutrition. Because of that function, endometrial glands play a crucial role as regulators of survival and development of conceptus. In all mammalian females as well as in woman, endometrial glands undergo extensive hyperplasia and hypertrophy during pregnancy as a response to increasing demands of the developing conceptus for uterine histotroph. The process of endometrial glands development is called adenogenesis. Inhibition of the development of these glands through gene mutation or epigenetic strategies results in infertility. Two cases of total intrauterine glands developmental agenesis are presented, which were diagnosed on the base of histopathological examination of endometrial biopsy specimens. Despite the fact that endometrial glands agenesis in mare is a rare condition, it is worth while to emphasize that the endometrial biopsy is currently the best diagnostic tool to recognize the above developmental disturbance. Clinicians should be aware that the mare with normal reproductive organs during macroscopic clinical investigation can present with uterine glands agenesis.

Seasonal or pathological findings? Morphofunctional characteristics of the equine endometrium during the autumn and spring transition.

The deep anoestrous phase in winter is part of the anovulatory season in mares and is bordered by the autumn and spring transitional periods (ATP/STP). To define an annual time span for effective prognostic biopsy sampling, the aim of this study was to provide a morphofunctional characterization of the endometrium during ATP and STP. To outline both transitional periods, endometrial specimens were taken in September, October and November (n=76) as well as February, March and April (n=184) with the requirement of a detailed clinical documentation. Tissue samples were examined histologically with special emphasis on the functional endometrial morphology. Additionally, an immunohistochemical evaluation was performed on selected specimens regarding the expression of oestrogen receptor α, progesterone receptor and Ki67-antigen. An absent to low endometrial activity was ascertained in more than 60% of all specimens from late October onwards, whereas a comparably lacking or low activity in STP was observed until early April. Approximately 30% (ATP) to 22% (STP) of all samples exhibited a predominantly "irregular" endometrial differentiation. During the transitional periods, the clinically evaluated ovarian status (transrectal palpation, transrectal ultrasonography and/or serum progesterone and oestrogen analyses) and the endometrial functional morphology were in accordance with approximately 70% of all cases. The expression of steroid hormone receptors and Ki67-antigen was generally low. Given that endometrial maldifferentiations were frequently found during ATP and STP, its occurence might display a characteristic and physiological feature of the transitional periods. Regarding the functional endometrial morphology, a diagnostic biopsy sampling should therefore be performed between late April and before September.

The Use of Endometrial Cancer Patient–Derived Organoid Culture for Drug Sensitivity Testing Is Feasible

Patient-derived organoids (PDOs), used in multiple tumor types, have allowed evaluation of tumor characteristics from individual patients. This study aimed to assess the feasibility of applying PDO in vitro culture for endocrine-based and drug sensitivity testing in endometrial cancer. Endometrial cancer cells were enzymatically dissociated from tumors retrieved from fresh hysterectomy specimens and cultured within basement membrane extract in serum-free medium. An organoid growth assay was developed to assess the inhibitory effects of a variety of drugs including endocrine treatments. Organoid cultures were also prepared for histological and immunohistochemical comparison to the tumors of origin. Fifteen endometrial cancer specimens were successfully cultured as PDOs. Small spherical structures formed within 24 hours, and many continued to grow to larger, denser organoids, providing the basis for an organoid growth assay. The STAT3 transcription factor inhibitor, BBI608 (Napabucasin), strongly inhibited growth in almost all PDO cultures, suggesting that stemness programing is involved in organoid formation and/or growth. Inhibition by different growth factor receptor tyrosine kinase inhibitors was observed in several PDO specimens. Four cultures were inhibited by fulvestrant, implying the importance of estrogen-receptor signaling in some PDO cultures. Organoids closely resembled their tumors of origin in both histomorphology and immunohistochemical expression. The use of endometrial cancer PDO cultures for development of drug sensitivity testing for individual patient tumors is feasible. The potential value of the PDO model for clinical decision making will require clinical trial evaluation.

Abstract 4324: Estrogen promotes proliferation and apoptosis of endometrial carcinoma cell lines Ishikawa and HEC1A via endometrial stromal cells

Abstract Endometrial carcinoma is the leading malignancies of women in Western countries and the second leading cancer of female in China. As endometrial stromal cells (ESCs) are sharply decreased in endometrial carcinoma (EC) compared to normal endometrium, it is interested to explore the roll of ESCs in the tumorigenesis of endometrium. Unopposed estrogen is considered as the etiology of endometrioid carcinoma, but the underlying mechanism is needed to further explore. This study is to determine the biological effects of primary cultured ESCs on two endometrial cancer cell lines-Ishikawa and HEC1A. ESCs from normal, atypical hyperplastic and malignant endometrial specimens were collected, validated by immunohistochemistry and cultured in vitro, which were maintained in a medium containing 10 nM estradiol (E2) for three months to simulate the unopposed estrogen stimulation of EC patients. Microarray were performed to detect the alteration of gene expression, which were then confirmed by real time RT-PCR. Co-culture of ESC and EC cell lines were performed by Transwell plate. Cell proliferation was analyzed by CCK8 assay. Apoptosis was analyzed by flow cytometry based on PI/Anexin V staining. Our results showed E2 promoted the proliferation of three types of ESCs. E2 has no direct effect on cancer cell lines Ishikawa and HEC1A, but when they co-cultured with ESCs, which were stimulated by E2, the proliferation of cancer cells was promoted by all three type of ESCs. Moor interestedly, we found that the much obvious promoted effects were occurred by the ESCs that extracted from malignant samples. Estrogen could also reduce the apoptosis of both cancer cell lines when co-culturing with ESCs from normal specimen. Gene microarray and real time RT-PCR indicated that collagen 9 were up-regulated 2.3 times when ESCs cells treated by E2. In conclusion, Estrogen altered the microenvironment of stromal cells, which could lead to the proliferation of endometrial carcinoma cells via paracrine function. The alteration of collagen synthesis by ESCs might contribute to this communication between the epithelial and mesenchymal cells. Citation Format: Xiang Tao, Jian Wu, Yinhua Yu. Estrogen promotes proliferation and apoptosis of endometrial carcinoma cell lines Ishikawa and HEC1A via endometrial stromal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4324. doi:10.1158/1538-7445.AM2017-4324

New approaches to the estimation of endometrial dysfunction

Introduction. The application of modern methods for assessing the morphofunctional state of the endometrium to verify and study the expression of sex steroid hormones, proinflammatory markers and markers of angiogenesis using confocal laser scanning microscopy will allow an objective study of the role of studied markers in the pathogenesis of the endometrial dysfunction.
 The aim of the study was to evaluate the expression of ER and PR receptors in endometrium in patients with endometrial dysfunction.
 Material and methods. Endometrial biopsy specimens obtained with the aid of a pile-biopsy or a scraping from the uterine cavity are used to conduct the method of immunofluorescent confocal laser scanning microscopy. It is possible to use both cryostat material and paraffin blocks to provide the immunohistochemical analysis. Monoclonal antibodies to ER (1 : 60, Dako, Denmark) and PR (1 : 50, Dako, Denmark) are used as primary antibodies, antibodies conjugated with fluorochrome Alexa Fluor 647 (1 : 1000, Abcam, England) are used as secondary antibodies). Hoechst 33258 (Sigma, USA) is used for staining of cell nuclei.
 Results. A method of confocal laser scanning microscopy makes it possible to conduct qualitative and quantitative evaluation of the studied markers in different structures of the endometrium.

Evaluation of whole exome sequencing by targeted gene sequencing and Sanger sequencing

BackgroundTargeted gene sequencing (TGS) and whole exome sequencing (WES) are being used in clinical testing in laboratories. We compared the performances of TGS and WES using the same DNA samples. MethodsDNA was extracted from 10 endometrial tumor tissue specimens. Sequencing were performed with an Illumina HiSeq 2000. We randomly selected variants to confirm through Sanger sequencing or mutant-enriched PCR with Sanger sequencing. ResultsWe found that the variants identified in both TGS and WES were true positives (47/47), regardless of the sequencing depth. Most variants found in TGS only were true positives (34/40), and most of the variants found by WES only were false positives (8/18). From these results, we suggest that the sequencing depth may not play important role in the accuracy of NGS-based methods. After analysis, we found that WES had a sensitivity of 72.70%, specificity of 96.27%, precision of 99.44%, and accuracy of 75.03%. ConclusionsThe results of NGS-based methods must currently be validated, especially for important reported variants regardless of the methods used, and for the use of WES in cancers a higher false negative rate must be considered. More sensitive methods should be used to confirm the NGS results in uneven cancer tissues.

Evaluation of pattern of angiogenesis in various menstrual disorders in different district in Gujarat, India.

Backgound: Menstrual disturbance is one of the commonest gynecological problems. Majority of these women have dysfunctional uterine bleeding (DUB), the rest being associated with leiomyomas, carcinomas, adenomyosis, endometrial polyps, hyperplasia and oral contraceptive uses. Aims: To study of alteration in blood vessels morphology in various menstrual disorders have significant role in prognosis and in various anti-angiogenic therapies. Methods : A Cross-sectional study done at Anand, Surendranagar and Junagadh district from October 2011 to October 2016. 1867 endometrial specimens were studied to document the changes in blood vessels in various phases of menstrual cycle, menstrual disturbances and in unexplained infertility. 761 cases were taken as control and 1106 cases as study group which included cases of dysfunctional uterine bleeding (DUB), endometrial polyps, fibroids, adenomyosis, infertility, atrophic endometrium, endometrial hyperplasia, pill endometrium and carcinomas. Using light microscopy, the vascular morphology was studied. Statistical analyses were performed using the IBM SPSS (Statistical package for the Social Sciences v15.0) and Microsoft Excel 2007 software. Results: The blood vessels were concentrated more in basal layer in the proliferative phase and in functional layer in the secretory phase. Cases of complex hyperplasia and pill endometrium had significantly higher vessel concentration. Congestion and dilatation of blood vessels were significantly higher in cases of DUB. Conclusions: The present study showed a positive correlation between endometrial angiogenesis and menstrual disorders. The alteration in blood vessel morphology has significant role in prognosis and in various anti-angiogenic therapies. DOI: 10.21276/APALM.1180

Dysregulation of the ADAM17/Notch signalling pathways in endometriosis: from oxidative stress to fibrosis.

Is oxidative stress associated with the A disintegrin and metalloproteases (ADAM) metallopeptidase domain 17 (ADAM17)/Notch signalling pathway and fibrosis in the development of endometriosis? Oxidative stress is correlated with hyperactivation of the ADAM17/Notch signalling pathway and a consequent increase in fibrosis in patients with endometriosis. It is nowadays accepted that oxidative stress plays an important role in the onset and progression of endometriosis. Oxidative stress is able to induce the synthesis of some members of the 'ADAM' family, such as ADAM17. ADAM17/Notch signalling is dysregulated in other profibrotic and inflammatory diseases. This was a prospective laboratory study conducted in a tertiary-care university hospital between January 2011 and April 2013. We investigated non-pregnant, younger than 42-year-old patients (n = 202) during surgery for a benign gynaecological condition. After complete surgical exploration of the abdominopelvic cavity, 121 women with histologically proven endometriosis and 81 endometriosis-free control women were enrolled. Peritoneal fluid (PF) samples were obtained from all the study participants during surgery in order to detect advanced oxidation protein products (AOPPs) and metalloproteinase activity of ADAM17. Stromal cells from endometrial specimens (n = 8) were obtained from endometrium of control patients (Cs), and from eutopic (Es) and ectopic (Ps) endometrium of patients with deep infiltrating endometriosis (DIE) (n = 8). ADAM17, Notch and the fibrosis markers α-smooth muscle actin (α-SMA) and type-I collagen were assessed using immunoblotting in all the endometrial samples obtained. Additionally, fibrosis was assessed after using Notch cleavage inhibitors (DAPT and FLI-06). Notch and fibrosis were also evaluated after stimulation of stromal endometrial cells with ADAM17 purified protein, increasing concentrations of H2O2 and primary cell culture supernatants. Patients with DIE presented higher PF AOPP and ADAM17 protein levels than controls (P < 0.01 and P < 0.05, respectively). In addition, these two markers were positively correlated (r = 0.614; P < 0.001). At the cellular level, ADAM17 activity was increased in Es and Ps compared to Cs (P < 0.001 and P < 0.01, respectively). Furthermore, Ps presented hyperactivation of Notch signalling (P < 0.05) and augmentation of fibrosis markers (P = 0.009 for α-SMA and P = 0.015 for type-I collagen) compared to controls. The use of DAPT and FLI-06 reduced both fibrosis markers in Ps but not in Cs. Stimulation with ADAM17, H2O2 and Ps supernatant culture significantly increased Notch and fibrosis in both Ps and Cs. N/A. The control group consisted of women who underwent surgery for benign gynaecological conditions, which could lead to biases because some of these conditions may cause alterations in oxidative stress and the ADAM17/Notch pathways. The small sample size of endometrial biopsies used for each group of patients (n = 8) is a limitation of the study, and results should be interpreted with caution. We propose a novel pathway in endometriosis pathogenesis that correlates oxidative stress, hyperactivation of ADAM17/Notch signalling and a consequent increase in fibrosis. This study suggests that Notch signalling plays a key role in the fibrotic processes that take place in ectopic lesions of patients with DIE, as already observed in other pro-fibrotic diseases. This work was supported by grants from University Paris Descartes, INSERM and Fundación Alfonso Martín Escudero. The authors have no competing interests to declare.

The efficacy of the cyclin-dependent kinase 4/6 inhibitor in endometrial cancer.

BackgroundPD-0332991, the selective cyclin-dependent kinase 4/6 inhibitor palbociclib, causes cell cycle arrest by inhibiting phosphorylation of retinoblastoma (Rb) protein. The aim of this study was to evaluate the therapeutic potential of PD-0332991 in endometrial cancer.Methods and findingsFour human endometrial cancer cell lines, ECC, HEC1A, HEC108 and TEN, were treated with PD-0332991 and their function was evaluated. In vivo, the therapeutic efficacy was evaluated in a model of subcutaneous endometrial cancer. An immunohistochemical analysis was performed in 337 endometrial cancer specimens. A proliferation assay revealed that 2 of the 4 cell lines that expressed Rb were sensitive to PD-0332991 with an IC50 of 0.65 μM (HEC1A) and 0.58 μM (HEC108), respectively. Both cell lines had G0/G1 cell cycle arrest after treatment with PD-0332991 according to flow cytometry. In vivo, PD-0332991 had antitumoral efficacy with a reduction in the activity of Ki67 and phosphorylation of Rb. Immunohistochemical analyses revealed that the positive rate of Rb was 67.7%, however, there was no significant relationship between the expression levels of Rb and the tumor grade.ConclusionsPD-0332991 had therapeutic potential against endometrial cancer cell lines expressing Rb protein. Our immunohistochemical analysis revealed that approximately 70% of patients with endometrial cancer might have therapeutic indications for PD-0332991. Of note, the tumor grade had no impact on the indications for treatment.

ВАЖЛИВІСТЬ ВИЗНАЧЕННЯ БІОМОЛЕКУЛЯРНИХ МАРКЕРІВ У ПАТОГЕНЕЗІ РОЗВИТКУ ГІПЕРПЛАСТИЧНИХ ПРОЦЕСІВ ЕНДОМЕТРІЯ У ПЕРИМЕНОПАУЗАЛЬНОМУ ПЕРІОДІ

Мета дослідження – вивчити біомолекулярні маркери при гіперплазії ендометрія (ГПЕ) в жінок у періоді перименопаузи і відповідність ступеня їх активності морфологічним змінам слизової оболонки матки.Матеріали та методи. Досліджені 70 жінок з ГПЕ, що склали основну групу, з яких у 38 (54,5 %) хворих виявлені морфологічні ознаки простої форми типової гіперплазії ендометрія (ПГЕ), у 32 (45,5 %) – складної гіперплазії (СГЕ). 20 жінок склали контрольну групу. У 50 пацієнток з різними типами ГПЕ були проведені імуногістохімічні дослідження з визначенням маркера апоптозу – білка p53, маркерів проліферативної активності – протеїну Ki-67, білка p27 і рецепторів стероїдних гормонів – естрогену і прогестерону. Результати дослідження та їх обговорення. Показники експресії проонкогенного білка Ki-67 свідчать про високу проліферативну активність клітин ендометрія в жінок 2 групи із СГЕ (р&lt;0,05), в той час як при ПГЕ цей показник був значно меншим та практично не відрізнявся від контрольної групи. Експресія антионкогенів білка р53 у залозах і клітинах строми при СГЕ була значно нижче показників у біоптатах ендометрія у пацієнток з ПГЕ. Аналіз вмісту білків, що регулюють клітинний цикл, вказує на зниження вмісту інгібітору циклу р27. При ГПЕ відбувалося достовірне підвищення маркера проліферації Ki-67: при ПГЕ з 4,3 до 9,2 % (клітини епітелію), при СГЕ в біоптатах ендометрія з 11,1 до 23,4 %, а також зниження маркера апоптозу р53 з 10,7 до 4,3 % та з 13,4 до 5,8 % відповідно (р&lt;0,05). Також спостерігалась різна імуногістохімічна реакція естрогенових та прогестеронових рецепторів залежно від типу ГПЕ. Був запропонований алгоритм обстеження пацієнток із гіперпластичними процесами в перименопаузі, в основі якого лежить верифікація морфологічного діагнозу з використанням імуногістохімічних критеріїв.Висновок. Диференційований підхід до діагностики ГПЕ оснований на імуногістохімічному визначенні біомолекулярних маркерів (Ki-67, р27, р53), що дозволяє не тільки прогнозувати перебіг гіперпластичного процесу, а й оцінити ефективність проведеного лікування.

Correlation between three-dimensional power Doppler and morphometric measurement of endometrial vascularity at the time of embryo implantation in women with unexplained recurrent miscarriage

Power Doppler in combination with three-dimensional (3D-PD) ultrasonography has been used as a noninvasive tool to evaluate the vascularity. However, it is unclear whether 3D-PD can accurately reflect endometrial vascularization and replace the invasive endometrial biopsy. This study aims to investigate the correlation between 3D-PD and micro vessel morphometric measurement of endometrial vascularity. Twenty-five women with unexplained recurrent miscarriage were recruited for 3D-PD and endometrial biopsy on precisely day LH + 7. Immunohistochemistry using vWF was employed to identify micro vessels in endometrial biopsy specimens followed by the use of morphometric technique to measure the mean vessel diameter and volume fractions. The vascularization index (VI), flow index (FI) and vascularization flow index (VFI) assessed by 3D-PD were calculated for both the endometrial and sub-endometrial regions. There were no significant correlations between any of the ultrasonographic measurements (endometrial thickness, endometrial volume, endometrial VI/FI/VFI, sub-endometrial volume, sub-endometrial VI/FI/VFI) and morphometric features (number of micro vessel, mean diameter of micro vessel and volume fraction measurement of vessel). This study indicates that endometrial vascularity assessed by 3D-PD could not be used to reflect changes in micro vessels of the endometrium at the time of embryo implantation in women with unexplained recurrent miscarriage.

Prevalence of female genital tuberculosis, its risk factors and associated clinical features among the women of Andaman Islands, India: a community-based study

ObjectiveThere is scarcity of information on the prevalence of female genital tuberculosis (FGTB) in the community. The present study was carried out to estimate the prevalence of FGTB, its risk factors and associated clinical features. Study designCommunity-based cross-sectional survey. MethodsThis study was carried during October 2011 and May 2014 in the Andaman Islands. A total of 13,300 women aged 20–59 years were primarily screened using a structured questionnaire. About 721 (5.4%) were found initially eligible for screening for genital tuberculosis by clinical examination and specimen collection for laboratory tests but only 460 (63.8%) expressed their willingness. Endometrial specimens were collected from 405 (88%) subjects. The association of the potential risk factors with genital tuberculosis was tested by Chi-squared test. A similar analysis was performed to identify clinical features associated with genital tuberculosis. ResultsThe estimated prevalence of FGTB was 45.1 cases per 100,000 women (95% confidence interval [CI]: 16.6–98.1). Infertility and oligomenorrhoea were identified as clinical features associated with FGTB. Past history of tuberculosis and history of close contact with tuberculosis cases were identified as risk factors. ConclusionsThis study shows the prevalence of FGTB among the female population of the Andaman Islands. Though the estimated prevalence was close to the expected prevalence, but as only 63.8% of the eligible women could be adequately screened, a much higher prevalence of FGTB could not be ruled out. Infertility, oligomenorrhoea, past history of tuberculosis and contact with tuberculosis case were identified as factors associated with genital tuberculosis.

Liquid-Based Endometrial Cytology Using SurePath™ Is Not Inferior to Suction Endometrial Tissue Biopsy in Clinical Performance for Detecting Endometrial Cancer Including Atypical Endometrial Hyperplasia

Objective: We evaluated the clinical performance of liquid-based endometrial cytology (SurePath™) for detecting endometrial malignancies by comparison with the performance of suction endometrial tissue biopsy. Study Design: From November 2011 to May 2013, we consecutively collected 1,118 liquid-based endometrial cytology specimens and 674 suction endometrial tissue biopsy specimens. Results: The rate of nonpositive final histology in nonpositive liquid-based endometrial cytology (98.2%) was higher than the rate of nonpositive final histology in nonpositive suction endometrial tissue biopsy (97.0%). None of the clinical performance values of liquid-based endometrial cytology for detecting the endometrial malignancies were statistically inferior to those of the suction endometrial tissue biopsy. When the positivity threshold was more than “atypical endometrial cells of undetermined significance,” the rate of positive liquid-based endometrial cytology from cases with a positive final histology (84.5%) was higher than the rate of positive suction endometrial tissue biopsy from cases with a positive final histology (69.8%). However, there were still no significant differences among all the performance values. Conclusions: Our liquid-based endometrial cytology would be more appropriate in various clinical situations as the initial detection tool for endometrial malignancies, rather than suction endometrial tissue biopsy. In addition, it could be used in screening for endometrial malignancies on a broader scale.

Oncogenic histone methyltransferase EZH2: A novel prognostic marker with therapeutic potential in endometrial cancer.

The histone methyltransferase EZH2, a key epigenetic modifier, is known to be associated with human tumorigenesis. However, the physiological importance of EZH2 and its clinical relevance in endometrial cancer remain unclear. Hence, in the present study, we investigated the expression and function of EZH2 in endometrial cancer. In a quantitative real-time PCR analysis of 11 endometrial cancer cell lines and 52 clinical endometrial cancer specimens, EZH2 was significantly overexpressed in cancer cells and tissues compared to that in corresponding normal control cells and tissues. Kaplan-Meier survival analysis using data of the TCGA RNA-seq database and tissue microarrays (TMAs) indicated that EZH2 overexpression is associated with endometrial cancer prognosis. In addition, knockdown of EZH2 using specific siRNAs resulted in growth suppression and apoptosis induction of endometrial cancer cells, accompanied by attenuation of H3K27 trimethylation. Consistent with these results, treatment with GSK126, a specific EZH2 inhibitor, suppressed endometrial cancer cell growth and decreased the number of cancer cell colonies. Furthermore, GSK126 showed additive effects with doxorubicin or cisplatin, which are conventional drugs for treatment of endometrial cancer. Further studies should explore the therapeutic potential of inhibiting EZH2 in patients with endometrial cancer.

Endometrial MicroRNA Signature during the Window of Implantation Changed in Patients with Repeated Implantation Failure.

Background:At present, a diagnostic tool with high specificity for impaired endometrial receptivity, which may lead to implantation failure, remains to be developed. We aimed to assess the different endometrial microRNA (miRNA) signatures for impaired endometrial receptivity by microarray analysis.Methods:A total of 12 repeated implantation failure (RIF) patients and 10 infertile patients, who conceived and delivered after one embryo transfer attempt, were recruited as RIF and control groups, respectively. Endometrial specimens from the window of implantation (WOI) were collected from these two groups. MiRNA microarray was conducted on seven and five samples from the RIF and control groups, respectively. Comparative, functional, and network analyses were performed for the microarray results. Quantitative real-time polymerase chain reaction (PCR) was performed on other samples to validate the expression of specific miRNAs.Results:Compared with those in the control group, the expression levels of 105 miRNAs in the RIF group were found to be significantly up- or down-regulated (at least 2-fold) by microarray analysis. The most relevant miRNA functional sets of these dysregulated miRNAs were miR-30 family, human embryonic stem cell regulation, epithelial-mesenchymal transition, and miRNA tumor suppressors by tool for annotations of microRNA analysis. Network regulatory analysis found 176 miRNA-mRNA interactions, and the top 3 core miRNAs were has-miR-4668-5p, has-miR-429, and has-miR-5088. Expression levels of the 18 selected miRNAs in new samples by real-time PCR were found to be regulated with the same trend, as the result of microarray analysis.Conclusions:There is a significant different expression of certain miRNAs in the WOI endometrium for RIF patients. These miRNAs may contribute to impaired endometrial receptivity.

Chronic endometritis modifies decidualization in human endometrial stromal cells

BackgroundChronic endometritis (CE) is a continuous inflammation of uterine endometrium, and it is usually symptomless. As CE has been thought not to affect the reproductive status and general health of affected women, its significance has not been explored. However, recent studies have shown that CE is related with repeated implantation failures after in vitro fertilization-embryo transfer, unexplained infertility, and recurrent miscarriages. As decidua differentiates to support the implantation process and maintains the pregnancy, we hypothesized that CE may influence the process of decidualization.MethodsSeventeen patients were employed in the experiment involving culture of endometrial stromal cells (ESCs). After obtaining endometrial samples, ESCs were harvested and cultured for 13 days. The concentrations in culture media and the protein expressions in ESCs of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two well known decidualization markers used in a large number of in vitro models, were analyzed by ELISA and Western blotting, respectively, and the cell numbers were also counted. The mRNA levels of PRL and IGFBP-1 were tested by quantitative real time polymerase chain reaction (RT-PCR). Since sex hormone induce proliferation and differentiation to decidua via binding to the sex hormone receptors (ERα, ERβ, PRA, and PRB), their expression was assessed in another 17 patients’ paraffin-embedded endometrial tissue specimens by immunohistochemistry and semi-quantified by H-score.ResultsIncreased cell numbers and reduced secretion of PRL and IGFBP-1 were detected by ELISA in the ESCs of CE patients after culture for 13 days compared with non-CE patients. The decreased protein expression of IGFBP-1 in ESCs of CE patients was detected by Western blotting. The decreased expression of PRL mRNA and IGFBP-1 mRNA were detected by RT-PCR. Increased expressions of ERα, ERβ, PRA, and PRB were observed in the stromal cells of CE patients in comparison to non-CE patients, whereas increased expressions of ERα and ERβ were detected in the glandular cells of CE.ConclusionOur data suggests that CE modifies decidualization of human ESC through untuning the function of sex steroid hormone receptor.

Ki-67 in endometrial cancer: scoring optimization and prognostic relevance for window studies

Ki-67, a marker of cellular proliferation, is increasingly being used in pre-surgical window studies in endometrial cancer as a primary outcome measure. Unlike in breast cancer, however, there are no guidelines standardizing its measurement and its clinical relevance as a response biomarker is undetermined. It is, therefore, imperative that Ki-67 scoring protocols are optimized and its association with patient survival rigorously evaluated, in order to be able to clinically interpret the results of these studies. Using the International Ki-67 in Breast Cancer Working Group guidelines as a basis, whole slide, hot spot and invasive edge scoring protocols were evaluated using endometrial biopsies and hysterectomy specimens from 179 women. Whole sections and tissue microarrays, manual and semi-automated scoring using Definiens Developer software were additionally compared. Ki-67 scores were related to clinicopathological variables and cancer-specific survival in uni- and multivariate analysis. Against criteria of time efficiency, intra- and inter-observer variability and consistency, semi-automated hot spot scoring was the preferred method. Ki-67 scores positively correlated with grade, stage and depth of myometrial invasion (P-values all <0.03). By univariate analysis, higher Ki-67 scores were associated with a significant reduction in cancer-specific survival (P≤0.05); however, this effect was substantially attenuated in the multivariate model. In conclusion, hot spot scoring of whole sections using Definiens is an optimal method to quantify Ki-67 in endometrial cancer window study specimens. Measured this way, it is a clinically relevant marker, though further work is required to determine whether reductions in Ki-67 in neoadjuvant intervention studies translate into improved patient outcome.

Spectrum of Histopathological Findings in Postmenopausal Bleeding.

To determine the frequencies of histopathological findings in endometrial and endocervical biopsy samples with clinical history of Postmenopausal Bleeding (PMB). Descriptive cross-sectional study. Section of Histopathology, Department of Pathology and Laboratory Medicine, The Aga Khan University Hospital, Karachi, from February 2012 to January 2013. A total of 157 consecutive endometrial and endocervical biopsy specimens with history of postmenopausal bleeding were included. After microscopic examination, frequencies of histological findings in different age groups were generated. Chi-square and independent sample t-tests were applied to see whether the difference was significant which was set at p < 0.05. One hundred and twenty-one (77.1%) specimens showed benign pathologies while 36 (22.9%) were malignant. Endometrial polyp was seen in 67 (42.7%) cases followed by endometrial carcinomas in 25 (15.9%), endometrial hyperplasia in 21 (13.4%), cervical carcinoma in 12 (7.6%) and cervical polyps in 9 (5.7%) cases. A highly significant increase in the percentage of malignant and pre-malignant lesions was seen with increasing age group (p < 0.001). Mean age of patients with type-2 endometrial carcinoma was higher than type-1 endometrial carcinoma but statistical significance was not observed (70.2 ±6.5 vs. 61.8 ±9.1 years respectively, p=0.069). Although benign pathologies were more common in postmenopausal bleeding but the collective proportion of endometrial and cervical malignancies and pre-malignant conditions was quite high. Therefore, PMB should be urgently evaluated for cause and early commencement of treatment.