High Stromal Senescence During the Window of Implantation Is Linked to Plasma Cell Presence and Cluster Formation in the Endometrium

Timing of endometrial biopsy: Are we one step closer to the definition of chronic endometritis?

INTRAUTERINE PATHOLOGY IS ASSOCIATED WITH HIGHER INCIDENCE OF ENDOMETRIAL PLASMA CELL INFILTRATE

Should patients be screened for chronic endometritis before assisted reproductive technology?

Is chronic endometritis a causative factor for repeated implantation failure in IVF-ET?

Profiling the gene signature of endometrial receptivity: clinical results

CHRONIC ENDOMETRITIS: WHAT IS THE PREVALENCE AMONG HEALTHY CONTROLS?

The menstrual cycle phase impacts the detection of plasma cells and the diagnosis of chronic endometritis in endometrial biopsy specimens

Study question This study aimed to systematically understand the endometrial leukocyte types, inflammatory environment, and impaired receptivity at single-cell resolution. Summary answer These results provide new insights into the endometrial immune microenvironment and impaired endometrial receptivity in infertile women with minimal/mild endometriosis. What is known already Endometriosis is a common inflammatory disorder in women of reproductive age due to an abnormal endometrial immune environment and is associated with infertility. Study design, size, duration We profiled single-cell RNA transcriptomes of 138,057 endometrial cells from endometriosis patients (n = 6) and control (n = 7), respectively, using 10x Genomics platform. Participants/materials, setting, methods We profiled single-cell RNA transcriptomes of 138,057 endometrial cells from endometriosis patients (n = 6) and control (n = 7), respectively, using 10x Genomics platform. Main results and the role of chance We found that one cluster of epithelial cells that expressed PAEP and CXCL14 was mostly from the control during the window of implantation (WOI). This epithelial cell type is absent in the eutopic endometrium during the secretory phase. The proportion of endometrial immune cells decreased in the secretory phase in the control group, whereas the cycle variation of total immune cells, NK cells, and T cells was absent in endometriosis. Endometrial immune cells secreted more IL-10 in the secretory phase than in the proliferative phase in the control group; the opposite trend was observed in endometriosis. Proinflammatory cytokines levels in the endometrial immune cells were higher in endometriosis than in the control group. Trajectory analysis revealed that the secretory phase epithelial cells decreased in endometriosis. Ligand–receptor analysis revealed that 11 ligand-receptor pairs were upregulated between endometrial immune and epithelial cells during WOI. Limitations, reasons for caution Our study had some limitations, such as the small sample size and lack of validation experiments. Further studies are needed to demonstrated the conclusions of our bioinformatic analysis, like in situ validation of receptor-ligand pairs analysis. Therefore, more evidence is needed to clearly state the pathogenesis of endometriosis-associated infertility. Wider implications of the findings This database will provide an essential resource for understanding the eutopic endometrial inflammatory environment and defective endometrial receptivity in patients with endometriosis-associated infertility. Trial registration number 82071625

Endometriosis is a common inflammatory disorder in women of reproductive age due to an abnormal endometrial immune environment and is associated with infertility. This study aimed to systematically understand the endometrial leukocyte types, inflammatory environment, and impaired receptivity at single-cell resolution. We profiled single-cell RNA transcriptomes of 138 057 endometrial cells from endometriosis patients (n = 6) and control (n = 7), respectively, using 10x Genomics platform. We found that one cluster of epithelial cells that expressed PAEP and CXCL14 was mostly from the control during the window of implantation (WOI). This epithelial cell type is absent in the eutopic endometrium during the secretory phase. The proportion of endometrial immune cells decreased in the secretory phase in the control group, whereas the cycle variation of total immune cells, NK cells, and T cells was absent in endometriosis. Endometrial immune cells secreted more IL-10 in the secretory phase than in the proliferative phase in the control group; the opposite trend was observed in endometriosis. Proinflammatory cytokines levels in the endometrial immune cells were higher in endometriosis than in the control group. Trajectory analysis revealed that the secretory phase epithelial cells decreased in endometriosis. Ligand-receptor analysis revealed that 11 ligand-receptor pairs were upregulated between endometrial immune and epithelial cells during WOI. These results provide new insights into the endometrial immune microenvironment and impaired endometrial receptivity in infertile women with minimal/mild endometriosis.

This aim of this study was to determine the association between uterine natural killer (uNK) cell density and chronic endometritis (CE). Endometrial biopsies from 135 women with recurrent miscarriage were obtained precisely 7 days after luteinizing hormone surge in natural cycles. Endometrial sections were immunostained for CD56 for uNK cells and CD138 for plasma cells, respectively. Uterine NK cell counting was performed according to a standardized protocol and results were expressed as percentage of CD56+ cells/ total stromal cells. High uNK cell density was defined as >4.5% and CE was diagnosed when the plasma cell density > 5.15 cells/ 10 mm2 . The uNK cells density in women with CE (median, 5.1%; range, 3.4-8.8%) was significantly (P < 0.05) higher than that of those without CE (median, 3.8%; range, 1.2%-7.3%). The prevalence of high uNK cell density in women with CE (11/29, 37.9%) was significantly (P < 0.05) higher than that of women without CE (8/106, 7.5%). To conclude, there was a significant association between high uNK cell density and CE. In women with high uNK cell density, plasma cell should be examined to determine if the underlying cause is associated with CE.

Study question What is the occurrence of Chronic Endometritis (CE) during the mid-luteal phase in patients with Recurrent Implantation Failure (RIF) and fertile controls? Summary answer Higher occurrence of CD138-cells and high %CD56+-cells, relating to CE, is seen in RIF-patients compared to controls. What is known already A key issue for the success of Medical Assisted Reproduction (MAR) is the receptivity of the endometrium to achieve implantation of the embryo. In an estimated 4-10% of MAR-patients embryo implantation repeatedly fails, even after multiple high-quality embryo transfers, a condition termed RIF. RIF has a multifactorial pathogenesis including aberrant activation of immune cells altering implantation. Endometrial immune factors and diagnosing CE have received considerable attention to address recurrent failures. Presence of CD138 plasma cells on immunohistochemistry has been the main criteria for diagnosis of CE and high uterine natural killer (uNK) cell numbers have been considered indicative of CE. Study design, size, duration A cohort study was conducted among RIF-patients and a control group. Recruitment occurred between 2019 and 2024 in a university hospital outpatient RIF-clinic. RIF was defined as consecutive implantation failure of three high quality embryos, based on the Gardner grading scale, or ten embryos without quality criterion. Women referred because of severe male factor, bilateral tubal factor or preimplantation genetic testing (PGT) for hereditary disorders without concurrent subfertility were recruited as control group. Participants/materials, setting, methods Clinical characteristics and endometrial biopsies were collected 5-8 days after the LH-peak in natural cycles. Endometrial biopsies were stained by immunohistochemistry and analysed through a custom built AI-program that distinguished the stained cells and the unstained cells. Data are expressed as presence of CD138 and %CD56 + (uNK) cells/total stromal cells. Means were compared by the independent t-test and categorical variables by the chi-square test with SPSS 28.0. A p-value of &amp;lt;0.05 was considered statistically significant. Main results and the role of chance Clinical characteristics showed that women with RIF (n = 32) are on average older (33 vs 30 years, p &amp;lt; 0.001) they give birth less often (nullipara 69% vs 52%, p = 0.174) and they endure miscarriages more often (53% vs 21%, p = 0.009) in comparison to control subjects (n = 29). There were no significant differences in BMI, ethnicity and smoking habits. The staining showed a significant difference in the presence of CD138 cells between RIF-patients and control subjects (63% vs 21%, p &amp;lt; 0.001). Contrarily, no significant differences in the percentage of CD56+ cells / total stromal cells (RIF: 17,81%, control: 13,14% and p = 0.184) and CD56+ mean number of cell clusters (RIF: 0.75, control: 1.46 and p = 0.183) were found. However, looking at the occurrence of CD56+ above 10%, which has been suggested as a marker for CE in previous studies, there was a higher occurrence in RIF-patients compared to the control subjects (69% vs 34%, p = 0.007). Limitations, reasons for caution The absence of a standardized immune cell quantification method in the endometrium is seen as a barrier to determine reference intervals for endometrial immune cells and to investigate the functions of these cells in the window of implantation which limits the interpretation of our results. Wider implications of the findings This study suggests a role for CE in the pathology of RIF due to the higher occurrence of CD138 cells among RIF-patients in comparison to control subjects. Antibiotics can be considered for the treatment of CE as described in other studies. Trial registration number Yes

Chronic endometritis identified by plasma cells can often be diagnosed in patients with recurrent implantation failure.

Study question Does a standardized histopathological classification system lead to a robust interobserver agreement to diagnose chronic endometritis (CE) and can an accurate computation algorithm be developed? Summary answer The proposed scoring system has a particularly good to excellent interobserver agreement among pathologists. A machine-learning algorithm outperformed pathologists in the clinically most relevant 2-tier-system. What is known already Plasma cell (PC) presence in the stromal endometrium has been proposed as a marker for CE, yet no standardized classification system that considers the amount or distribution of PCs exists. Also, interobserver agreement has been insufficiently reported in the previous publications on the topic. Without a robust histopathological score, the potential clinical impact of CE on ART treatment outcomes cannot be investigated. This issue is strongly reflected in the heterogeneity of the current diagnostic and therapeutic approaches, specifically for patients experiencing repeated implantation failure (RIF) or recurrent pregnancy loss (RPL). Study design, size, duration A 5-tier classification system based on CD138-positive PCs (Roche B-A38/automated Ventana system) was established: class0 (no PCs), classIPC (isolated PCs), class1 (³1 cluster of 5-19 PCs/0.25mm2), class2 (³1 cluster of 20-49 PCs/0.25mm2), class3 (³1 cluster of 50 or more PCs/0.25mm2). A test set of 78 endometrial biopsies (collected over two years) was selected by a certified pathologist who sorted &amp;gt;1000 biopsies to ensure the full spectrum of PC counts/distribution was accounted for. Participants/materials, setting, methods The 3DHistech-platform digitized the slides (pixel size 0.2738x0.2738mm) and a scoring session was set up in the Pathomation software suite. Six pathologists scored independently after training. Overall agreement was expressed by Fleiss’ Kappa values. Pairwise interobserver agreement was calculated using Cohen’s Kappa statistics. A supervised machine-learning algorithm was developed in QuPath(V0.4.2) and considered a seventh observer in the interobserver agreement calculations. Main results and the role of chance The CD138-based semiquantitative scoring system showed particularly good to excellent interobserver agreement among all observers. Overall agreement values were 0.722 for the 5-tier system and 0.858 for the 2-tier system (0+IPC versus 1 + 2+3, which was considered the clinically most relevant classification, i.e. CE absent versus diagnosed). Pairwise interobserver agreement was 0.871-0.951. Intra-class correlations varied from 0.727 to 0.839 in the 5-tier system and 0.893 to 0.965 in the 2-tier system. All findings were statistically significant (p &amp;lt; 0.001). The supervised machine-learning algorithm was able to compete with human pathologist observers and outperformed them in the 2-tier system. The proposed classification system provides a framework for assessing the presence and severity of endometrial PC infiltration and can guide further research. Limitations, reasons for caution Inconsistent staining intensity, non-specific epithelial staining and background interference are common problems that might limit interlaboratory use of the algorithm. Furthermore, the algorithm has not yet been associated to patient/clinical parameters. Accurate analysis of an endometrial biopsy might still be insufficient to provide a formal diagnosis of CE. Wider implications of the findings To unravel the association between PCs in endometrial biopsies (as a proxy for the diagnosis of CE) and the reproductive outcomes following ART, a standardized methodology is of crucial importance for future studies. Higher accuracy, efficieny and automation are advantages when using computer-aided diagnostics for CE. Trial registration number BUN 143202042810

P-389: Immunohistochemistry confirms the presence of chronic endometritis in patients with recurrent implantation failure

It’s time to pay attention to the endometrium