Biopsy vitrification: New tool for endometrial tissue cryopreservation for research applications.

Patient-derived endometrial biopsies serve as a crucial source for molecular studies, highlighting the necessity for tissue cryopreservation methods that preserve cell viability and tissue morphology with minimal to no impact. The passive slow freezing (PSF) protocol has demonstrated efficacy for cryopreserving endometrial biopsies, allowing for the subsequent isolation of viable epithelial and stromal cells. Vitrification (VT) enables the avoidance of ice crystal formation and could therefore potentially prevent mechanical injury to tissues. In this study, PSF and VT techniques were applied to endometrial biopsies, and the effects of cryopreservation on tissue samples were evaluated using traditional histology. In addition, transmission electron microscopy (TEM), gene expression profiling analyses, the viability of endometrial cells, and the ability to form epithelial organoids were compared between PSF and VT endometrial biopsies in a subset of samples. The histology and TEM studies demonstrated relatively mild cellular and sub-cellular damage in both cryopreservation protocols which did not affect tissue functionality and the formation of the organoids. Additionally, the cryopreservation methodology did not affect the gene expression profile of the 68 endometrial-receptivity associated genes studied. In conclusion, our findings indicate that although current cryopreservation methodologies need further improvements, they still allow us to achieve acceptable cell viability and functionality, showing promising potential for facilitating the utilization of cryopreserved endometrial tissue samples for research purposes.

Endometrial Proliferative Phase-Centered View of Transcriptome Dynamics across the Menstrual Cycle.

The endometrium, the inner mucosal lining of the uterus, undergoes complex molecular and cellular changes across the menstrual cycle in preparation for embryo implantation. Transcriptome-wide analyses have mainly been utilized to study endometrial receptivity, the prerequisite for successful implantation, with most studies, so far, comparing the endometrial transcriptomes between (i) secretory and proliferative endometrium or (ii) mid-secretory and early secretory endometrium. In the current study, we provide a complete transcriptome description of the endometrium across the entire menstrual cycle and, for the first time, comprehensively characterize the proliferative phase of the endometrium. Our temporal transcriptome analysis includes five time points including the mid-proliferative, late proliferative (peri-ovulatory phase), early secretory, mid-secretory, and late secretory phases. Thus, we unveil exhaustively the transitions between the consecutive proliferative and secretory phases, highlighting their unique gene expression profiles and possible distinct biological functions. The transcriptome analysis reveals many differentially expressed genes (DEGs) across the menstrual cycle, most of which are phase-specific. As an example of coordinated gene activity, the expression profile of histone-encoding genes within the HIST cluster on chromosome 6 shows an increase in cluster activity during the late proliferative and a decline during the mid-secretory phase. Moreover, numerous DEGs are shared among all phases. In conclusion, in the current study, we delineate the endometrial proliferative phase-centered view of transcriptome dynamics across the menstrual cycle. Our data analysis highlights significant transcriptomic and functional changes occurring during the late proliferative phase-an essential transition point from the proliferative phase to the secretory phase. Future studies should explore how the biology of the late proliferative phase endometrium impacts the achievement of mid-secretory endometrial receptivity or contributes to molecular aberrations leading to embryo implantation failure.

The expression pattern of endometrial receptivity genes is desynchronized between endometrium and matched endometriomas.

RESEARCH QUESTION: Are paired samples of endometrium and ovarian endometriomas synchronous with each other throughout the menstrual cycle? DESIGN: The expression levels of 57 endometrial receptivity-associated genes were determined from matched endometrial and endometrioma samples (n=31) collected from women with endometriosis throughout the menstrual cycle. RESULTS: The expression profile of endometrial receptivity genes divided endometrial samples according to their menstrual cycle phase. Endometrioma samples grouped together irrespective of the menstrual cycle phase and formed a cluster distinct from endometrial samples. Pairwise comparison showed 21, 16, 33 and 23 differentially expressed genes (adjusted P < 0.001-0.05) between the lesions and endometria collected in the proliferative, early-secretory, mid-secretory and late-secretory menstrual cycle phases, respectively, confirming the distinct expression profiles of endometrium and endometrioma. CONCLUSIONS: No menstrual cycle synchronicity was found between matched eutopic and ectopic endometrium, suggesting that the concept of cycling endometrial tissue inside the endometrioma should be revised.

Differences in microbial profile of endometrial fluid and tissue samples in women with in vitro fertilization failure are driven by Lactobacillus abundance.

INTRODUCTION: The endometrial microbiota has been linked to several gynecological disorders, including infertility. It has been shown that the microbial profile of endometrium could have a role in fertilization and pregnancy outcomes. In this study we aim to assess the microbial community of endometrial tissue (ET) and endometrial fluid (EF) samples in women receiving in vitro fertilization (IVF) treatment. We also search for possible associations between chronic endometritis (CE) and endometrial microbiota. MATERIAL AND METHODS: This was a cohort study involving 25 women aged between 28 and 42 years with both primary and secondary infertility and with at least one IVF failure. The ET and EF sample collection was carried out between September 2016 and November 2018. Each of the participants provided two types of samples-tissue and fluid samples (50 samples in total). A 16S rRNA sequencing was performed on both of the sample types for microbial profile evaluation. CE was diagnosed based on a CD138 immunohistochemistry where CE diagnosis was confirmed in the presence of one or more plasma cells. Microbial profiles of women with and without CE were compared in both sample types separately. RESULTS: We report no differences in the microbial composition and alpha diversity (pObserved  = 0.07, pShannon  = 0.65, pInverse Simpson  = 0.59) between the EF and ET samples of IVF patients. We show that the abundance of the genus Lactobacillus influences the variation in microbial beta diversity between and fluid samples (r2  = 0.34; false discovery rate [FDR] <9.9 × 10-5 ). We report that 32% (8/25) of the participants had differences in Lactobacillus dominance in the paired samples and these samples also present a different microbial diversity (pShannon  = 0.06, FDRweighted UniFrac  = 0.01). These results suggest that the microbial differences between ET and fluid samples are driven by the abundance of genus Lactobacillus. The microbiome of CE and without CE (ie non-CE) women in our sample set of IVF patients was similar. CONCLUSIONS: Our findings show that genus Lactobacillus dominance is an important factor influencing the microbial composition of ET and fluid samples.

Syndecan-1 modulates the invasive potential of endometrioma via TGF-ß signalling in a subgroup of women with endometriosis.

STUDY QUESTION: What is the physiological role of transforming growth factor-beta (TGF-ß1) and syndecans (SDC1, SDC4) in endometriotic cells in women with endometriosis? SUMMARY ANSWER: We observed an abnormal, pro-invasive phenotype in a subgroup of samples with ovarian endometriosis, which was reversed by combining gene silencing of SDC1 with the TGF-ß1 treatment. WHAT IS KNOWN ALREADY: Women with endometriosis express high levels of TGF-ß1 and the proteoglycan co-receptors SDC1 and SDC4 within endometriotic cysts. However, how SDC1 and SDC4 expression is regulated by TGF-ß1 and the physiological significance of the high expression in endometriotic cysts remains unknown as does the potential role in disease severity. STUDY DESIGN, SIZE, DURATION: We utilized a pre-validated panel of stem- and cancer cell-associated markers on endometriotic tissue (n = 15) to stratify subgroups of women with endometriosis. Furthermore, CD90+CD73+CD105+ (SC+) endometriotic stromal cells from these patient subgroups were explored for their invasive behaviour in vitro by transient gene inhibition of SDC1 or SDC4, both in the presence or absence of TGF-ß1 treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometriotic cyst biopsies (n = 15) were obtained from women diagnosed with ovarian endometriosis (ASRM Stage III-IV). Gene expression variability was assessed on tissue samples by applying gene clustering tools for the dataset generated from the pre-validated panel of markers. Three-dimensional (3D) spheroids from endometriotic SC+ were treated in vitro with increasing doses of TGF-ß1 or the TGFBRI/II inhibitor Ly2109761 and assessed for SDC1, SDC4 expression and in vitro 3D-spheroid invasion. Transcriptomic signatures from the invaded 3D spheroids were evaluated upon combining transient gene silencing of SDC1 or SDC4, both in presence or absence of TGF-ß1 treatment. Furthermore, nanoscale changes on the surface of endometriotic cells were analysed after treatment with TGF-ß1 or TGFBRI/II inhibitor using atomic force microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: Gene clustering analysis revealed that endometriotic tissues displayed variability in their gene expression patterns; a small subgroup of samples (2/15, Endo-hi) exhibited high levels of SDC1, SDC4 and molecules involved in TGF-ß signalling (TGF-ß1, ESR1, CTNNB1, SNAI1, BMI1). The remaining endometriotic samples (Endo-lo) showed a uniform, low gene expression profile. Three-dimensional spheroids derived from Endo-hi SC+ but not Endo-lo SC+ samples showed an aberrant expression of SDC1 and exhibited enhanced 3D-spheroid invasion in vitro, upon rhTGF-ß1 treatment. However, this abnormal, pro-invasive response of Endo-hi SC+ was reversed upon gene silencing of SDC1 with the TGF-ß1 treatment. Interestingly, transcriptomic signatures of 3D spheroids silenced for SDC1 and consecutively treated with TGF-ß1, showed a down-regulation of cancer-associated pathways such as WNT and GPCR signalling. LARGE SCALE DATA: Transcriptomic data were deposited in NCBI's Gene Expression Omnibus (GEO) and could be retrieved using GEO series accession number: GSE135122. LIMITATIONS, REASONS FOR CAUTION: It is estimated that about 2.5% of endometriosis patients have a potential risk for developing ovarian cancer later in life. It is possible that the pro-oncogenic molecular changes observed in this cohort of endometriotic samples may not correlate with clinical occurrence of ovarian cancer later in life, thus a validation will be required. WIDER IMPLICATIONS OF THE FINDINGS: This study emphasizes the importance of interactions between syndecans and TGF-ß1 in the pathophysiology of endometriosis. We believe that this knowledge could be important in order to better understand endometriosis-associated complications such as ovarian cancer or infertility. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Cancerfonden (CAN 2016/696), Radiumhemmets Forskningsfonder (Project no. 154143 and 184033), EU MSCA-RISE-2015 project MOMENDO (691058), Estonian Ministry of Education and Research (IUT34-16), Enterprise Estonia (EU48695) and Karolinska Institute. Authors do not have any conflict of interest.

MUC20 expression marks the receptive phase of the human endometrium.

RESEARCH QUESTION: How does mucin MUC20 expression change during the menstrual cycle in different cell types of human endometrium? DESIGN: Study involved examination of MUC20 expression in two previously published RNA-seq datasets in whole endometrial tissue (n = 10), sorted endometrial epithelial (n = 44) or stromal (n = 42) cell samples. RNA-Seq results were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in whole tissue (n = 10), sorted epithelial (n = 17) and stromal (n = 17) cell samples. MUC20 protein localization and expression were analysed in human endometrium by immunohistochemical analysis of intact endometrial tissue (n = 6) and also Western blot of cultured stromal and epithelial cells (n = 2). RESULTS: MUC20 is differentially expressed in the endometrium between the pre-receptive and receptive phases. We show that MUC20 is predominantly expressed by epithelial cells of the receptive endometrium, both at the mRNA (RNA-Seq, P = 0.005; qRT-PCR, P = 0.039) and protein levels (Western blot; immunohistochemistry, P = 0.029). CONCLUSION: Our results indicate MUC20 as a novel marker of mid-secretory endometrial biology. We propose a model of MUC20 function in the hepatocyte growth factor (HGF)-activated mesenchymal-epithelial transition (MET) receptor signalling specifically in the receptive phase. Further investigations should reveal the precise function of MUC20 in human endometrium and the possible connection between MUC20 and HGF-activated MET receptor signalling. MUC20 could potentially be included in the list of endometrial receptivity markers after further clinical validation.

DNA methylation alterations-potential cause of endometriosis pathogenesis or a reflection of tissue heterogeneity?

Alterations in the DNA methylation pattern of endometriotic lesions and endometrium of endometriosis patients have been proposed as one potential factor accompanying the endometriosis development. Although many differentially methylated genes have been associated with the pathogenesis of this disease, the overlap between the results of different studies has remained small. Among other potential confounders, the impact of tissue heterogeneity on the outcome of DNA methylation studies should be considered, as tissues are mixtures of different cell types with their own specific DNA methylation signatures. This review focuses on the results of DNA methylation studies in endometriosis from the cellular heterogeneity perspective. We consider both the studies using highly heterogeneous whole-lesion biopsies and endometrial tissue, as well as pure cell fractions isolated from lesions and endometrium to understand the potential impact of the cellular composition to the results of endometriosis DNA methylation studies. Also, future perspectives on how to diminish the impact of tissue heterogeneity in similar studies are provided.

Aberrant expression of genes associated with stemness and cancer in endometria and endometrioma in a subset of women with endometriosis.

STUDY QUESTION: Is there molecular evidence for a link between endometriosis and endometriosis-associated ovarian cancers (EAOC)? STUDY ANSWER: We identified aberrant gene expression signatures associated with malignant transformation in a small subgroup of women with ovarian endometriosis. WHAT IS KNOWN ALREADY: Epidemiological studies have shown an increased risk of EAOC in women with ovarian endometriosis. However, the cellular and molecular changes leading to EAOC are largely unexplored. STUDY DESIGN, SIZE, DURATION: CD73+CD90+CD105+ multipotent stem cells/progenitors (SC cohort) were isolated from endometrium (n = 18) and endometrioma (n = 11) of endometriosis patients as well as from the endometrium of healthy women (n = 14). Extensive phenotypic and functional analyses were performed in vitro on expanded multipotent stem cells/progenitors to confirm their altered characteristics. Aberrant gene signatures were also validated in paired-endometrium and -endometrioma tissue samples from another cohort (Tissue cohort, n = 19) of endometriosis patients. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Paired-endometrial and -endometriotic biopsies were obtained from women with endometriosis (ASRM stage III-IV) undergoing laparoscopic surgery. Control endometria were obtained from healthy volunteers. Isolated CD73+CD90+CD105+ SC were evaluated for the presence of known endometrial surface markers, colony forming efficiency, multi-lineage differentiation, cell cycle distribution and 3D-spheroid formation capacity. Targeted RT-PCR arrays, along with hierarchical and multivariate clustering tools, were used to determine both intergroup and intragroup gene expression variability for stem cell and cancer-associated markers, in both SC+ and tissue cohorts. MAIN RESULTS AND THE ROLE OF CHANCE: Isolated and expanded SC+ from both control and patient groups showed significantly higher surface expression of W5C5+, clonal expansion and 3D-spheroid formation capacity (P < 0.05) compared with SC-. The SC+ cells also undergo mesenchymal lineage differentiation, unlike SC-. Gene expression from paired-endometriosis samples showed significant downregulation of PTEN, ARID1A and TNFα (P < 0.05) in endometrioma compared with paired-endometrium SC+ samples. Hierarchical and multivariate clustering from both SC+ and tissue cohorts together identified 4 out of 30 endometrioma samples with aberrant expression of stem cell and cancer-associated genes, such as KIT, HIF2α and E-cadherin, altered expression ratio of ER-ß/ER-α and downregulation of tumour suppressor genes (PTEN and ARID1A). Thus, we speculate that above changes may be potentially relevant to the development of EAOC. LARGE-SCALE DATA: N/A. LIMITATIONS, REASON FOR CAUTION: As the reported frequency of EAOC is very low, we did not have access to those samples in our study. Moreover, by adopting a targeted gene array approach, we might have missed several other potentially-relevant genes associated with EAOC pathogenesis. The above panel of markers should be further validated in archived tissue samples from women with endometriosis who later in life developed EAOC. WIDER IMPLICATIONS OF THE FINDINGS: Knowledge gained from this study, with further confirmation on EAOC cases, may help in developing screening methods to identify women with increased risk of EAOC. STUDY FUNDING/COMPETING INTEREST(S): The study is funded by the Swedish Research Council (2012-2844), a joint grant from Stockholm County and Karolinska Institutet (ALF), RGD network at Karolinska Institutet, Karolinska Institutet for doctoral education (KID), Estonian Ministry of Education and Research (IUT34-16), Enterprise Estonia (EU48695), Horizon 2020 innovation program (WIDENLIFE, 692065), European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways funding (IAPP, SARM, EU324509) and MSCA-RISE-2015 project MOMENDO (691058). All authors have no competing interest.

Differentially-Expressed miRNAs in Ectopic Stromal Cells Contribute to Endometriosis Development: The Plausible Role of miR-139-5p and miR-375.

microRNA (miRNA) expression level alterations between endometrial tissue and endometriotic lesions indicate their involvement in endometriosis pathogenesis. However, as both endometrium and endometriotic lesions consist of different cell types in various proportions, it is not clear which cells contribute to variability in miRNA levels and the overall knowledge about cell-type specific miRNA expression in ectopic cells is scarce. Therefore, we utilized fluorescence-activated cell sorting to isolate endometrial stromal cells from paired endometrial and endometrioma biopsies and combined it with high-throughput sequencing to determine miRNA alterations in endometriotic stroma. The analysis revealed 149 abnormally expressed miRNAs in endometriotic lesions, including extensive upregulation of miR-139-5p and downregulation of miR-375 compared to eutopic cells. miRNA transfection experiments in the endometrial stromal cell line ST-T1b showed that the overexpression of miR-139-5p resulted in the downregulation of homeobox A9 (HOXA9) and HOXA10 expression, whereas the endothelin 1 (EDN1) gene was regulated by miR-375. The results of this study provide further insights into the complex molecular mechanisms involved in endometriosis pathogenesis and demonstrate the necessity for cell-type-specific analysis of ectopic tissues to understand the interactions between different cell populations in disease onset and progression.

Challenges in endometriosis miRNA studies - From tissue heterogeneity to disease specific miRNAs.

In order to uncover miRNA changes in endometriosis pathogenesis, both endometriotic lesions and endometrial biopsies, as well as stromal and epithelial cells isolated from these tissues have been investigated and a large number of dysregulated miRNAs have been reported. However, the concordance between the result of different studies has remained small. One potential explanation for limited overlap between the proposed disease-related miRNAs could be the heterogeneity in tissue composition, as some studies have compared highly heterogeneous whole-lesion biopsies with endometrial tissue, some have compared the endometrium from patients and controls, and some have used pure cell fractions isolated from lesions and endometrium. This review focuses on the results of published miRNA studies in endometriosis to reveal the potential impact of tissue heterogeneity on the discovery of disease-specific miRNA alterations in endometriosis. Additionally, functional studies that explore the roles of endometriosis-involved miRNAs are discussed.

High-throughput mRNA sequencing of stromal cells from endometriomas and endometrium.

The aetiology of endometriosis is still unclear and to find mechanisms behind the disease development, it is important to study each cell type from endometrium and ectopic lesions independently. The objective of this study was to uncover complete mRNA profiles in uncultured stromal cells from paired samples of endometriomas and eutopic endometrium. High-throughput mRNA sequencing revealed over 1300 dysregulated genes in stromal cells from ectopic lesions, including several novel genes in the context of endometriosis. Functional annotation analysis of differentially expressed genes highlighted pathways related to cell adhesion, extracellular matrix-receptor interaction and complement and coagulation cascade. Most importantly, we found a simultaneous upregulation of complement system components and inhibitors, indicating major imbalances in complement regulation in ectopic stromal cells. We also performed in vitro experiments to evaluate the effect of endometriosis patients' peritoneal fluid (PF) on complement system gene expression levels, but no significant impact of PF on C3, CD55 and CFH levels was observed. In conclusion, the use of isolated stromal cells enables to determine gene expression levels without the background interference of other cell types. In the future, a new standard design studying all cell types from endometriotic lesions separately should be applied to reveal novel mechanisms behind endometriosis pathogenesis.

Pregnancy rate in endometriosis patients according to the severity of the disease after using a combined approach of laparoscopy, GnRH agonist treatment and in vitro fertilization.

AIM: To evaluate the effects of combined treatment approaches on endometriosis-associated infertility in different stages of endometriosis using laparoscopy, gonadotropin-releasing hormone (GnRH) agonist (GnRHa) therapy and in vitro fertilization (IVF). METHODS: This retrospective study was carried out on 179 women with surgically confirmed endometriosis. Patients were divided into subgroups: group 1 (stage I-II, n = 121) and group 2 (stage III-IV, n = 58). Patients eligible for IVF, who were found to have adenomyosis or moderate to severe endometriosis, were also given postoperative GnRHa. Pregnancy and delivery rates were cumulatively calculated during 5 years according to the severity of the disease. RESULTS: The overall pregnancy, delivery and miscarriage rates were 66.5, 56.4 and 15.1%, respectively, for all patients following spontaneous and assisted conception. There were no significant differences in reproductive outcomes between the study groups. The pregnancy and delivery rates were also comparable within group 1 between the patients with and without GnRHa treatment. CONCLUSION: Pregnancy and delivery rates at different stages of endometriosis were not affected by the different approaches used for infertility treatment, with >60 and >50% of patients having conceived and delivered a baby, respectively, in both groups. The usefulness of GnRHa treatment for endometriosis patients with minimal to mild forms is questionable and deserves further studies.

Dynamic changes in AI-based analysis of endometrial cellular composition: Analysis of PCOS and RIF endometrium.

Background: The human endometrium undergoes a monthly cycle of tissue growth and degeneration. During the mid-secretory phase, the endometrium establishes an optimal niche for embryo implantation by regulating cellular composition (e.g., epithelial and stromal cells) and differentiation. Impaired endometrial development observed in conditions such as polycystic ovary syndrome (PCOS) and recurrent implantation failure (RIF) contributes to infertility. Surprisingly, despite the importance of the endometrial lining properly developing prior to pregnancy, precise measures of endometrial cellular composition in these two infertility-associated conditions are entirely lacking. Additionally, current methods for measuring the epithelial and stromal area have limitations, including intra- and inter-observer variability and efficiency. Methods: We utilized a deep-learning artificial intelligence (AI) model, created on a cloud-based platform and developed in our previous study. The AI model underwent training to segment both areas populated by epithelial and stromal endometrial cells. During the training step, a total of 28.36 mm2 areas were annotated, comprising 2.56 mm2 of epithelium and 24.87 mm2 of stroma. Two experienced pathologists validated the performance of the AI model. 73 endometrial samples from healthy control women were included in the sample set to establish cycle phase-dependent dynamics of the endometrial epithelial-to-stroma ratio from the proliferative (PE) to secretory (SE) phases. In addition, 91 samples from PCOS cases, accounting for the presence or absence of ovulation and representing all menstrual cycle phases, and 29 samples from RIF patients on day 5 after progesterone administration in the hormone replacement treatment cycle were also included and analyzed in terms of cellular composition. Results: Our AI model exhibited reliable and reproducible performance in delineating epithelial and stromal compartments, achieving an accuracy of 92.40% and 99.23%, respectively. Moreover, the performance of the AI model was comparable to the pathologists' assessment, with F1 scores exceeding 82% for the epithelium and >96% for the stroma. Next, we compared the endometrial epithelial-to-stromal ratio during the menstrual cycle in women with PCOS and in relation to endometrial receptivity status in RIF patients. The ovulatory PCOS endometrium exhibited epithelial cell proportions similar to those of control and healthy women's samples in every cycle phase, from the PE to the late SE, correlating with progesterone levels (control SE, r2 = 0.64, FDR < 0.001; PCOS SE, r2 = 0.52, FDR < 0.001). The mid-SE endometrium showed the highest epithelial percentage compared to both the early and late SE endometrium in both healthy women and PCOS patients. Anovulatory PCOS cases showed epithelial cellular fractions comparable to those of PCOS cases in the PE (Anovulatory, 14.54%; PCOS PE, 15.56%, p = 1.00). We did not observe significant differences in the epithelial-to-stroma ratio in the hormone-induced endometrium in RIF patients with different receptivity statuses. Conclusion: The AI model rapidly and accurately identifies endometrial histology features by calculating areas occupied by epithelial and stromal cells. The AI model demonstrates changes in epithelial cellular proportions according to the menstrual cycle phase and reveals no changes in epithelial cellular proportions based on PCOS and RIF conditions. In conclusion, the AI model can potentially improve endometrial histology assessment by accelerating the analysis of the cellular composition of the tissue and by ensuring maximal objectivity for research and clinical purposes.

Carboxypeptidase Inhibitor LXN Expression in Endometrial Tissue Is Menstrual Cycle Phase-Dependent and Is Upregulated in Endometriotic Lesions.

Endometriosis is a chronic hormone-dependent disease characterized by the spread of endometrial cells outside the uterus, which form endometriotic lesions and disrupt the functions of the affected organs. The etiopathogenesis of endometriosis is still unclear, and thus it is important to examine the genes that may contribute to the establishment of endometriotic lesions. The aim of this study was to investigate the expression of new potential candidate gene latexin (LXN), an inhibitor of carboxypeptidases, in endometrium and endometriotic lesions to elucidate its possible role in endometriosis development. LXN expression in tissues was assessed using quantitative reverse transcription PCR (qRT-PCR) analysis and immunohistochemical staining (IHC). The functions of LXN were examined using Transwell and MTT assays. qRT-PCR analysis revealed that LXN expression in endometrium was menstrual cycle-dependent, being lowest in the early-secretory phase and highest in the late-secretory phase and was significantly upregulated in endometriotic lesions. IHC confirmed LXN expression in endometrial stromal cells, and in vitro assays demonstrated that knockdown of LXN effectively reduced the migratory capacity of endometrial stromal cells while promoting cell viability. In conclusion, our results showed that LXN can be involved in the pathogenesis of endometriosis by regulating the proliferation and migration activity of endometriotic stromal cells.

AI-algorithm training and validation for identification of endometrial CD138+ cells in infertility-associated conditions; polycystic ovary syndrome (PCOS) and recurrent implantation failure (RIF).

Background: Endometrial CD138+ plasma cells serve as a diagnostic biomarker for endometrial inflammation, and their elevated occurrence correlates positively with adverse pregnancy outcomes. Infertility-related conditions like polycystic ovary syndrome (PCOS) and recurrent implantation failure (RIF) are closely associated with systemic and local chronic inflammatory status, wherein endometrial CD138+ plasma cell accumulation could also contribute to endometrial pathology. Current methods for quantifying CD138+ cells typically involve laborious and time-consuming microscopic assessments of only a few random areas from a slide. These methods have limitations in accurately representing the entire slide and are susceptible to significant biases arising from intra- and interobserver variations. Implementing artificial intelligence (AI) for CD138+ cell identification could enhance the accuracy, reproducibility, and reliability of analysis. Methods: Here, an AI algorithm was developed to identify CD138+ plasma cells within endometrial tissue. The AI model comprised two layers of convolutional neural networks (CNNs). CNN1 was trained to segment epithelium and stroma across 28,363 mm2 (2.56 mm2 of epithelium and 24.87 mm2 of stroma), while CNN2 was trained to distinguish stromal cells based on CD138 staining, encompassing 7345 cells in the object layers (6942 CD138- cells and 403 CD138+ cells). The training and performance of the AI model were validated by three experienced pathologists. We collected 193 endometrial tissues from healthy controls (n = 73), women with PCOS (n = 91), and RIF patients (n = 29) and compared the CD138+ cell percentages based on cycle phases, ovulation status, and endometrial receptivity utilizing the AI model. Results: The AI algorithm consistently and reliably distinguished CD138- and CD138+ cells, with total error rates of 6.32% and 3.23%, respectively. During the training validation, there was a complete agreement between the decisions made by the pathologists and the AI algorithm, while the performance validation demonstrated excellent accuracy between the AI and human evaluation methods (intraclass correlation; 0.76, 95% confidence intervals; 0.36-0.93, p = 0.002) and a positive correlation (Spearman's rank correlation coefficient: 0.79, p < 0.01). In the AI analysis, the AI model revealed higher CD138+ cell percentages in the proliferative phase (PE) endometrium compared to the secretory phase or anovulatory PCOS endometrium, irrespective of PCOS diagnosis. Interestingly, CD138+ percentages differed according to PCOS phenotype in the PE (p = 0.03). On the other hand, the receptivity status had no impact on the cell percentages in RIF samples. Conclusion: Our findings emphasize the potential and accuracy of the AI algorithm in detecting endometrial CD138+ plasma cells, offering distinct advantages over manual inspection, such as rapid analysis of whole slide images, reduction of intra- and interobserver variations, sparing the valuable time of trained specialists, and consistent productivity. This supports the application of AI technology to help clinical decision-making, for example, in understanding endometrial cycle phase-related dynamics, as well as different reproductive disorders.

Endometriotic lesions exhibit distinct metabolic signature compared to paired eutopic endometrium at the single-cell level.

Current therapeutics of endometriosis focus on hormonal disruption of endometriotic lesions (ectopic endometrium, EcE). Recent findings show higher glycolysis utilization in EcE, suggesting non-hormonal strategy for disease treatment that addresses cellular metabolism. Identifying metabolically altered cell types in EcE is important for targeted metabolic drug therapy without affecting eutopic endometrium (EuE). Here, using single-cell RNA-sequencing, we examine twelve metabolic pathways in paired samples of EuE and EcE from women with confirmed endometriosis. We detect nine major cell types in both EuE and EcE. Metabolic pathways are most differentially regulated in perivascular, stromal, and endothelial cells, with the highest changes in AMPK signaling, HIF-1 signaling, glutathione metabolism, oxidative phosphorylation, and glycolysis. We identify transcriptomic co-activation of glycolytic and oxidative metabolism in perivascular and stromal cells of EcE, indicating a critical role of metabolic reprogramming in maintaining endometriotic lesion growth. Perivascular cells, involved in endometrial stroma repair and angiogenesis, may be potential targets for non-hormonal treatment of endometriosis.

Aging promotes accumulation of senescent and multiciliated cells in human endometrial epithelium.

STUDY QUESTION: What changes occur in the endometrium during aging, and do they impact fertility? SUMMARY ANSWER: Both the transcriptome and cellular composition of endometrial samples from women of advanced maternal age (AMA) are significantly different from that of samples from young women, suggesting specific changes in epithelial cells that may affect endometrial receptivity. WHAT IS KNOWN ALREADY: Aging is associated with the accumulation of senescent cells in aging tissues. Reproductive aging is mostly attributed to the decline in ovarian reserve and oocyte quality, whereas the endometrium is a unique complex tissue that is monthly renewed under hormonal regulation. Several clinical studies have reported lower implantation and pregnancy rates in oocyte recipients of AMA during IVF. Molecular studies have indicated the presence of specific mutations within the epithelial cells of AMA endometrium, along with altered gene expression of bulk endometrial tissue. STUDY DESIGN SIZE DURATION: Endometrial transcriptome profiling was performed for 44 women undergoing HRT during the assessment of endometrial receptivity before IVF. Patients younger than 28 years were considered as the young maternal age (YMA) group (age 23-27 years) and women older than 45 years were considered as the AMA group (age 47-50 years). Endometrial biopsies were obtained on Day 5 of progesterone treatment and RNA was extracted. All endometrial samples were evaluated as being receptive based on the expression of 68 common endometrial receptivity markers. Endometrial samples from another 24 women classified into four age groups (YMA, intermediate age group 1 (IMA1, age 29-35), intermediate age group 2 (IMA2, age 36-44), and AMA) were obtained in the mid-secretory stage of a natural cycle (NC) and used for validation studies across the reproductive lifespan. PARTICIPANTS/MATERIALS SETTING METHODS: A total of 24 HRT samples (12 YMA and 12 AMA) were subject to RNA sequencing (RNA-seq) and differential gene expression analysis, 20 samples (10 YMA and 10 AMA) were used for qPCR validation, and 24 NC samples (6 YMA, 6 IMA1, 6 IMA2 and 6AMA) were used for RNA-seq validation of AMA genes across the woman's reproductive lifespan. Immunohistochemistry (IHC) was used to confirm some expression changes at the protein level. Computational deconvolution using six endometrial cell type-specific transcriptomic profiles was conducted to compare the cellular composition between the groups. MAIN RESULTS AND THE ROLE OF CHANCE: Comparisons between YMA and AMA samples identified a lower proportion of receptive endometria in the AMA group (P = 0.007). Gene expression profiling identified 491 differentially expressed age-sensitive genes (P adj < 0.05) that revealed the effects of age on endometrial epithelial growth and receptivity, likely contributing to decreased reproductive performance. Our results indicate that changes in the expression of the cellular senescence marker p16INK4a and genes associated with metabolism, inflammation, and hormone response are involved in endometrial aging. Importantly, we demonstrate that the proportion of multi-ciliated cells, as discovered based on RNA-seq data deconvolution and tissue IHC results, is affected by endometrial aging, and propose a putative onset of age-related changes. Furthermore, we propose that aging has an impact on the transcriptomic profile of endometrial tissue in the context of endometrial receptivity. LARGE SCALE DATA: The raw sequencing data reported in this article are deposited at the Gene Expression Omnibus under accession code GSE236128. LIMITATIONS REASONS FOR CAUTION: This retrospective study identified changes in the endometrium of patients undergoing hormonal replacement and validated these changes using samples obtained during a NC. However, future studies must clarify the importance of these findings on the clinical outcomes of assisted reproduction. WIDER IMPLICATIONS OF THE FINDINGS: The findings reported in this study have important implications for devising future strategies aimed at improving fertility management in women of advanced reproductive age. STUDY FUNDING/COMPETING INTERESTS: This research was funded by the Estonian Research Council (grant no. PRG1076), Horizon 2020 innovation grant (ERIN, grant no. EU952516), Enterprise Estonia (grant no. EU48695), MSCA-RISE-2020 project TRENDO (grant no. 101008193), EU 874867 project HUTER, the Horizon Europe NESTOR grant (grant no. 101120075) of the European Commission, the EVA specialty program (grant no. KP111513) of the Maastricht University Medical Center (MUMC+), MICIU/AEI/10.13039/501100011033 and FEDER, EU projects Endo-Map (grant no. PID2021-12728OB-100), ROSY (grant no. CNS2022-135999), and the National Science Fund of Bulgaria (grant no. KII-06 H31/2). The authors declare no competing interests.

Endometrial receptivity in women of advanced age: an underrated factor in infertility.

BACKGROUND: Modern lifestyle has led to an increase in the age at conception. Advanced age is one of the critical risk factors for female-related infertility. It is well known that maternal age positively correlates with the deterioration of oocyte quality and chromosomal abnormalities in oocytes and embryos. The effect of age on endometrial function may be an equally important factor influencing implantation rate, pregnancy rate, and overall female fertility. However, there are only a few published studies on this topic, suggesting that this area has been under-explored. Improving our knowledge of endometrial aging from the biological (cellular, molecular, histological) and clinical perspectives would broaden our understanding of the risks of age-related female infertility. OBJECTIVE AND RATIONALE: The objective of this narrative review is to critically evaluate the existing literature on endometrial aging with a focus on synthesizing the evidence for the impact of endometrial aging on conception and pregnancy success. This would provide insights into existing gaps in the clinical application of research findings and promote the development of treatment options in this field. SEARCH METHODS: The review was prepared using PubMed (Medline) until February 2023 with the keywords such as 'endometrial aging', 'receptivity', 'decidualization', 'hormone', 'senescence', 'cellular', 'molecular', 'methylation', 'biological age', 'epigenetic', 'oocyte recipient', 'oocyte donation', 'embryo transfer', and 'pregnancy rate'. Articles in a language other than English were excluded. OUTCOMES: In the aging endometrium, alterations occur at the molecular, cellular, and histological levels suggesting that aging has a negative effect on endometrial biology and may impair endometrial receptivity. Additionally, advanced age influences cellular senescence, which plays an important role during the initial phase of implantation and is a major obstacle in the development of suitable senolytic agents for endometrial aging. Aging is also accountable for chronic conditions associated with inflammaging, which eventually can lead to increased pro-inflammation and tissue fibrosis. Furthermore, advanced age influences epigenetic regulation in the endometrium, thus altering the relation between its epigenetic and chronological age. The studies in oocyte donation cycles to determine the effect of age on endometrial receptivity with respect to the rates of implantation, clinical pregnancy, miscarriage, and live birth have revealed contradictory inferences indicating the need for future research on the mechanisms and corresponding causal effects of women's age on endometrial receptivity. WIDER IMPLICATIONS: Increasing age can be accountable for female infertility and IVF failures. Based on the complied observations and synthesized conclusions in this review, advanced age has been shown to have a negative impact on endometrial functioning. This information can provide recommendations for future research focusing on molecular mechanisms of age-related cellular senescence, cellular composition, and transcriptomic changes in relation to endometrial aging. Additionally, further prospective research is needed to explore newly emerging therapeutic options, such as the senolytic agents that can target endometrial aging without affecting decidualization. Moreover, clinical trial protocols, focusing on oocyte donation cycles, would be beneficial in understanding the direct clinical implications of endometrial aging on pregnancy outcomes.