Cells Of Endometrium Research Articles

P-343 Metformin reduces endometrial implants and improves fertility in a mouse model of endometriosis

Abstract Study question Could endometriosis-associated infertility be mitigated by metformin? Summary answer Metformin treatment restored fertility to control rates in endometriosis-induced mice that present lower fertility. Endometrial implants presented a reduction in size with metformin treatment. What is known already Endometriosis is an inflammatory disease characterized by estrogen-dependent ectopic growth of endometrium causing pain and infertility. Hormone-based medication prescribed need to be interrupted when women intend to conceive. Corroborating observations in women, endometriosis decreases oocyte quality and pregnancy success in rodents. Thus, animal models of endometriosis constitute a valuable tool to elucidate the pathophysiology of the disease and putative therapies. Besides lowering glycaemia, metformin down-regulates estrogen secretion, inflammation and oxidation. Metformin can be safely used before and during pregnancy, it is currently used in gestational diabetes. Metformin has shown to induce regression of endometrial implants in an endometriosis rat model. Study design, size, duration B6CBA/F1 female mice were randomly divided in groups and subjected to treatment: 1-Endometriosis(E) (n = 20); 2-Sham-operated(S) (n = 12); 3-Endometriosis with metformin(EM) (n = 20); 4-Sham-operated with metformin(SM) (n = 20). Endometriosis was surgically induced by heterologous transplantation of endometrium from one donor in receptors from the same strain mice. Implants were confirmed and monitored by ultrasound. 50mg/kg/day of metformin was orally administrated during 3 months to Groups 3 and 4. Half of mice in each group were mated to fertility study. Participants/materials, setting, methods Endometrial implants were monitored at 3 timepoints during the experiments. Fertility rates were assessed by the average number of fetuses in each group. Histological characterization of eutopic and ectopic endometrium was performed by H&E and Masson's trichrome staining. Immunofluorescence for PCNA and CYP17A1 proteins, markers of cell proliferation and estrogen secretion, respectively were performed. Western blotting quantification for these proteins is in course. Statistical analysis was carried out and significant differences were considered for p < 0.05. Main results and the role of chance A decrease of 30% of fertility rate was verified in mice with endometriosis (p = 0,01); treatment with metformin was able to revert this decrease (p = 0,04). Interestingly, no differences in fertility were found in sham-operated mice under metformin treatment relatively with those of group 2 (p = 0,16). No biometrical differences were found between mice with endometriosis receiving metformin and those that do not receive the drug. Implants size were reduced by metformin treatment. Histological similarities were observed between the ectopic tissue and uterus. It is possible to observe that the ectopic endometrium layer is thinner in the EM group compared with the E group. PCNA and CYP17a labeling was present in the same cells of eutopic and ectopic endometrium, with an apparent lower number in the ectopic tissue in the EM group. In addition, in untreated animals, the cavity of ectopic tissue has more flaking cells than in animals with metformin treatment. In sum, metformin seems to have a positive role in the control of cell proliferation and estrogen production of ectopic endometrium. We postulate that these findings owes to a metformin-mediated decrease of oxidative imbalance and inflammatory response, induction of regression of endometriomas and regulation of oestrogen secretion. Limitations, reasons for caution Extrapolation of data from animal models to human needs caution, considering that endometriosis pattern differs between species. Also, further investigation, focused in identification of molecular targets of metformin and molecular pathways activated in endometriosis, is needed and in course. Wider implications of the findings With these results, we suggest metformin as a novel and safe strategy to mitigate endometriosis-related oxidative stress and indeed could be used as a valid pharmacological approach to ameliorate endometriosis-associated infertility Trial registration number Not applicable

P-296 Relationship between the percentage of stem cells and implantation success in women with in-vitro fertilization

Abstract Study question Is there a relationship between the percentage of stem cells in human endometrial stroma during the mid-luteal phase of the menstrual cycle and implantation success? Summary answer Women with successful implantation have significantly higher percentage of endometrial stromal CD117-positive stem cells during the mid-luteal phase of the menstrual cycle. What is known already Human endometrium as a highly regenerative dynamic tissue has a population of certain types of adult stem cells localized in the stroma and epithelium. These cells, including mesenchymal and hematopoietic stromal stem cells, participate in the immense cellular turnover in the human endometrium before and during the implantation process. General adult stem cell markers such as CD117 and NOTCH1 have been identified in human endometrium. However, there is a scarce data about the quantity of these endometrial stromal stem cells and their influence on the process of implantation. Study design, size, duration This is a cohort study of 109 women who had an endometrial biopsy during the mid-lutheal phase (LH + 7) in a natural cycle and positive or negative hCG test after in-vitro fertilization (IVF) with euploid embryos within 6 months of biopsy. We used immunohistochemical biomarkers NOTCH1 (E-AB-12815, Elabscience), CD117 (CD117/c-Kit/SCF-Receptor) (RB-9038-RQ, Epredia), to identify stem cells in endometrial stroma. The study was conducted between March 2020 and January 2022. Participants/materials, setting, methods The percentages of NOTCH1-positive and CD117-positive stem cells in the stroma were calculated after enumeration by two independent investigators in multiple endometrial sections. After embryo transfer women were divided into the following groups: unsuccessful embryo implantation (negative hCG test) ( n = 45) and successful implantation (positive hCG test) ( n = 64). The percentage of NOTCH1-positive and CD117-positive cells in the endometrial stroma were compared between these groups through Wilcoxon signed-rank test. Main results and the role of chance The percentage of CD117-positive stem cells in the endometrial stroma ranged between 0% and 0.21%, with a median of 0.02%. The percentage of NOTCH1-positive stem cells in the endometrial stroma ranged between 0.003% and 2.11%, with a median of 0.06%. There was no significant difference in female age (years), BMI (kg/m2) and blastocyst quality between the studied patient groups (P < 0.05). The quantity of NOTCH1-positive cells also did not differ significantly between the two groups (P = 0.18). However, the mean percentage of CD117-positive stromal cells was significantly higher in women with successful implantation compared to patients with unsuccessful implantation (0.04% vs. 0.02%, respectively, P = 0.004). The optimal successful implantation cut-off value for the percentage of CD117-positive stromal cells, was 0.018% (AUC 0.67, 95% CI 0.56-0.77). Therefore, a percentage of 0.018% of CD117-positive stem cells in the endometrial stroma was associated with 63.1% sensitivity and 61.4% specificity for predicting successful implantation after IVF. Limitations, reasons for caution The study cohort was limited in sample size. Not all confounding factors such as potential immunological and genetic reasons for unsuccessful implantation have been taken into account. In addition, the endometrial biopsy could also have an effect on endometrial receptivity and pregnancy outcome. Wider implications of the findings The results of this study revealed that the quantity of certain types of stem cells is associated with successful embryo implantation. These findings emphasize the essential impact of stem cells in human endometrium on the process of implantation. Trial registration number not applicable

Killer Timing: The Temporal Uterine Natural Killer Cell Differentiation Pathway and Implications for Female Reproductive Health.

Natural killer (NK) cells are the predominant maternal uterine immune cell component, and they densely populate uterine mucosa to promote key changes in the post-ovulatory endometrium and in early pregnancy. It is broadly accepted that (a) immature, inactive endometrial NK (eNK) cells in the pre-ovulatory endometrium become activated and transition into decidual NK (dNK) cells in the secretory stage, peri-implantation endometrium, and continue to mature into early pregnancy; and (b) that secretory-stage and early pregnancy dNK cells promote uterine vascular growth and mediate trophoblast invasion, but do not exert their killing function. However, this may be an overly simplistic view. Evidence of specific dNK functional killer roles, as well as opposing effects of dNK cells on the uterine vasculature before and after conception, indicates the presence of a transitory secretory-stage dNK cell (s-dNK) phenotype with a unique angiodevelopmental profile during the peri-implantation period, that is that is functionally distinct from the angiomodulatory dNK cells that promote vessel destabilisation and vascular cell apoptosis to facilitate uterine vascular changes in early pregnancy. It is possible that abnormal activation and differentiation into the proposed transitory s-dNK phenotype may have implications in uterine pathologies ranging from infertility to cancer, as well as downstream effects on dNK cell differentiation in early pregnancy. Further, dysregulated transition into the angiomodulatory dNK phenotype in early pregnancy will likely have potential repercussions for adverse pregnancy outcomes, since impaired dNK function is associated with several obstetric complications. A comprehensive understanding of the uterine NK cell temporal differentiation pathway may therefore have important translational potential due to likely NK phenotypic functional implications in a range of reproductive, obstetric, and gynaecological pathologies.

Image processing and machine learning approaches for the automatic diagnosis of endometrial cancer

In many medical imaging based diagnosis, Deep Learning (DL) algorithms play an essential role. A DL algorithm can be used to identify abnormal and normal cells in the uterus's endometrium in order to discover Endometrial Cancer (EC) cells. EC is difficult to diagnose since it develops without causing any symptoms. DL algorithm can distinguish between normal, abnormal, and malignant cells, producing more accurate findings than screening by hand procedures such as liquid cytology and Pap smear test. For the accurate and easier detection of EC cells, DL employs multiple architectures. The findings of an analysis and survey of the many forms of DL architecture, as well as their accuracy and performance, are addressed in this work. Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) are the most modalities of advanced imaging used for the non-invasive diagnosis of EC. EC is considered as the fourth most prevalent malignancy in women and one of the common most gynaecological cancers. Diagnostic imaging in clinical evaluation with has not been proven yet to be precise enough to substitute surgical staging in determining the spread of cancer. It may allow for improved surgical process optimization and a more customised therapeutic plan.

Detection of stem cells in human endometrium: immune-histochemical study

In this work, we recruited CD105 and CD90 markers to identify the endometrial stem cells (EnSCs) in the human uterine endometrium. Fifty endometrial specimens have been extracted from twenty-five deceased women. All specimens have been embedded in paraffin block and prepared for immune-histochemical processing. The expression of CD105 and CD90 was counted per high power field (HPF). Paired sample t-test was used to compare the means between groups and age variables. The Statistical analysis was conducted using “SPSS16 software" The statistical significance was considered at less than 0.05. Results of paired sample t-test showed that the expression count (EC) at the Basalis layer (with CD105) was 5.26 points higher than the Functionalis layer (95% CI [3.43, 7.09]) and the correlation was strongly and positively related (r = 0.829, P < 0.001). The EC at Basalis layer (with CD90) was 4.96 points higher than Functionalis layer (95% CI [3.02, 6.89]) and the correlation was strongly and positively related (r = 0.746, P= < 0.001). The EC at Functionalis layer (with CD105) was 0.9 points higher than the Stroma layer (95% CI [2.27, 4.11]); however, the correlation was weak and positively related (r = 0.429, P = 0.032). The EC at Functionalis layer (with CD90) was 1.4 points higher than the Stroma layer (95% CI [2.1, 4.2]), but the correlation was moderately and positively related r = 0.547, P = 0.005). Findings of an independent-sample t-test showed that the EC of stem cells at the Functionalis, Basalis, and Stroma layers (with CD105 and CD90 markers) was more among patients of reproductive age (<50 years) than patients who were at non-reproductive age (50 years and above), a statistically significant difference [(m = 9.6, 95% CI (5.3, 14.6), t (19.630) = 4.413, P < 0.001)], [(m = 10.2, 95% CI (5.2, 15.2), t (20.714) = 4.202, P < 0.001)] and [(m = 8.4, 95% CI (3.5, 13.4), t (12.313) = 3.523, P = 0.002)], respectively. In conclusion, the EC of the stem cells in the endometrium decreases with age.

Unique expression patterns of the embryonal stem cell marker SOX2 and hormone receptors suggest the existence of a subpopulation of epithelial stem/progenitor cells in porcine and bovine endometrium.

BackgroundThere are currently insufficient data on the population of endometrial epithelial stem/progenitor cells in farm animals.ObjectivesWith the aim of identifying a potential population of epithelial stem/progenitor cells in the porcine and bovine endometrium, this study immunohistochemically examined the expression patterns of the oestrogen and progesterone receptors, as well as that of the embryonal stem cell marker SOX2.MethodsA total of 24 endometrial tissue samples obtained from cycling pigs (n = 12) and cows (n = 12) were included in our study. Each endometrium was divided into basal, middle and luminal portions. The percentage of marker‐positive cells and the intensity of the immunoreaction in each portion of the endometrium were determined.ResultsInverse expression patterns of SOX2 and progesterone receptors were found in both animal species throughout the oestrous cycle. Strong diffuse SOX2 expression was detected in the basal portions of the glands, while a significant decrease in positivity and a weak immunoreaction were found in the luminal two thirds of the glandular epithelium. Strong progesterone receptor expression was observed in at least 90% of glandular cells in the middle and luminal portions, whereas weak staining and significant decrease in positivity were detected in the basal portions of the glands. One oestrogen receptor expression pattern resembled that of progesterone receptors.ConclusionThe inverse expression patterns of SOX2 and hormone (especially progesterone) receptors suggest that endometrial epithelial stem/progenitor cells represent a subset of cells that reside in the basal portions of the endometrial glands in both the bovine and porcine endometrium.

Epigenetic Factors in Eutopic Endometrium in Women with Endometriosis and Infertility.

Eutopic endometrium in patients with endometriosis is characterized by aberrant expression of essential genes during the implantation window. It predisposes to disturbance of endometrial receptivity. The pathomechanism of implantation failures in women with endometriosis remains unclear. This paper aims to summarize the knowledge on epigenetic mechanisms in eutopic endometrium in the group of patients with both endometriosis and infertility. The impaired DNA methylation patterns of gene promoter regions in eutopic tissue was established. The global profile of histone acetylation and methylation and the analysis of selected histone modifications showed significant differences in the endometrium of women with endometriosis. Aberrant expression of the proposed candidate genes may promote an unfavorable embryonic implantation environment of the endometrium due to an immunological dysfunction, inflammatory reaction, and apoptotic response in women with endometriosis. The role of the newly discovered proteins regulating gene expression, i.e., TET proteins, in endometrial pathology is not yet completely known. The cells of the eutopic endometrium in women with endometriosis contain a stable, impaired methylation pattern and a histone code. Medication targeting critical genes responsible for the aberrant gene expression pattern in eutopic endometrium may help treat infertility in women with endometriosis.

Impaired Expression of Membrane Type-2 and Type-3 Matrix Metalloproteinases in Endometriosis but Not in Adenomyosis.

Matrix metalloproteinases (MMPs) play an important role in menstruation and endometriosis; however, the membrane-type matrix metalloproteinases (MT-MMPs) are not well studied in endometriosis and adenomyosis. We analyzed MT2-MMP (MMP15) and MT3-MMP (MMP16) in eutopic endometrium with and without endometriosis and with and without adenomyosis and ectopic endometrium of deep infiltrating endometriosis (DIE), peritoneal endometriosis (PE), and ovarian endometriosis (Ov) by immunohistochemistry. Preferential expression of both proteins was observed in the glandular and luminal epithelial cells of the eutopic endometrium of patients with and without endometriosis with a ~2.5-fold stronger expression of MT3-MMP compared to MT2-MMP. We did not observe any differences during menstrual cycling and in eutopic endometrium of patients with and without endometriosis. Similarly, eutopic endometrium and adenomyotic tissue with and without endometriosis showed similar protein levels of MT2-MMP and MT3-MMP. In contrast, MT2-MMP and MT3-MMP protein was decreased in ectopic compared to eutopic endometrium and adenomyosis. The similar expression of MT2-MMP and MT3-MMP in eutopic endometrium in patients with and without endometriosis in contrast to the impaired expression in ectopic endometrium suggests that alterations occur after and not before endometrial implantation possibly by distinct interactions with the different environments. The differential protein expression of MT2/3-MMP in adenomyosis compared to endometriosis might suggest a different pathogenesis pathway for the two diseases.

HLA Class Ib-receptor interactions during embryo implantation and early pregnancy.

Although the immune system intuitively must have an important role in embryo implantation and in the achievement of a pregnancy, the molecular details have for long been controversial. The role of the human leukocyte antigen (HLA) system has been debated. The unique HLA expression profile of the HLA Class Ia molecule HLA-C and the HLA Class Ib molecules HLA-E, HLA-F and HLA-G at the feto-maternal interface is now recognized. However, HLA Class Ib molecules may also have a role in embryo implantation and pregnancy success. The aim of this review was to evaluate the literature and recent discoveries on the role of the non-polymorphic HLA Class Ib molecules with a focus on HLA-F and HLA-G molecules at the time of implantation, including the interaction with uterine immune cells through the specific receptors immunoglobulin-like transcript 2 (ILT2), ILT4 and a number of killer cell immunoglobulin-like receptors (KIRs), and the importance of HLA-F and HLA-G genetic variation that influences fertility and time-to-pregnancy. Drawing on recent advances in basic and clinical studies, we performed a narrative review of the scientific literature to provide a timely update on the role of HLA Class Ib in embryo implantation, fertility and infertility. Pertinent studies were searched in PubMed/Medline using relevant key words. Both HLA-F and HLA-G interact with inhibitory or activating ILT2 or ILT4 receptors and KIRs on uterine immune cells, especially uterine natural killer (NK) cells that are highly abundant in the mid-secretory endometrium and in early pregnancy. The binding of HLA-G to ILT2 stimulates the secretion of growth-promoting factors from decidual NK cells. However, functional aspects of a HLA-F-receptor interaction remain to be clarified. Recent studies indicate that HLA-F and HLA-G are expressed in mid-secretory endometrium and HLA-G is expressed in the blastocyst. HLA-F fluctuates during the menstrual cycle with high levels during the implantation window. The level of HLA-F protein expression correlates with the number of CD56-positive NK cells in the mid-secretory endometrium. HLA-F and HLA-G gene polymorphisms, including a single nucleotide polymorphism (SNP) in a progesterone-responsive element, are associated with time-to-pregnancy. Depending on the SNP genotype, the effect of progesterone varies resulting in differences in HLA-F expression and thereby the interaction with receptors on the uterine NK cells. Studies suggest that the expression of HLA-G and HLA-F, both by the embryonic-derived trophoblast cells and by cells in the endometrium and decidua, and the interaction between HLA-G and HLA-F with specific receptors on uterine immune cells, stimulate and facilitate embryo implantation and placentation by secretion of growth factors, cytokines and angiogenic factors. A detailed understanding of the molecular mechanisms controlling the expression of HLA-F and HLA-G periconceptionally and in early pregnancy may improve the success of ART and holds promise for further insight into pathophysiological aspects of certain pregnancy complications.

Estrogenic effects of the seeds and stem bark extracts of Ricinodendron heudelotii in adult ovariectomized Wistar albino rats

Background: Infertility is a major public health problem in Africa. Most herbal remedies used for the treatment of women infertility are known to be estrogenic. Objective: The aim of this study was to investigate the estrogenic effects of the aqueous extracts of Ricinodendron heudelotii seeds and stem bark in ovariectomized rats. Methods: Adult ovariectomized rats were divided in groups of five animals and treated orally for seven consecutive days with both the seeds extract (SERH) and the bark extract (BERH) at 100 and 300 mg/kg and with Ethinylestradiol (0.02 mg/kg). The control group received distilled water. After treatments, vaginal smears were performed from the rats. Then the uterus, cervix and adrenal glands were weighted and the uterus stored in formalin 10% for histological analysis. Results: Ethinylestradiol as well as SERH at 100 mg/kg induced vaginal cornification and highly significant increase of the relative weight of the uterus and cervix in comparison to controls. SERH at 300 mg/kg and BERH at 100 and 300 mg/kg also induced significantly an increase of the relative weight of these organs. In addition, the adrenal glands relative weight was significantly augmented by SERH and BERH treatment. The histological examination of uterus showed the proliferation of the endometrium cells and development of numerous uterine glands in rats treated with Ethinylestradiol and the extracts at 100 mg/kg. Conclusion: R. heudelotii was found to show estrogenic effects on vagina, uterus, cervix and adrenal glands and the seeds extract exhibited greater estrogenic activities than the bark extract.

Down-regulation of PBK inhibits proliferation of human endometrial stromal cells in thin endometrium

BackgroundThin endometrium (TE) is a challenging clinical issue in the reproductive medicine characterized by inadequate endometrial thickness, poor response to estrogen and no effective treatments currently. At present, the precise pathogenesis of thin endometria remains to be elucidated. We aimed to explore the related molecular mechanism of TE by comparing the transcriptome profiles of late-proliferative phase endometria between TE and matched controls.MethodsWe performed a bulk RNA-Seq (RNA-sequencing) of endometrial tissues in the late-proliferative phase in 7 TE and 7 matched controls for the first time. Differential gene expression analysis, gene ontology enrichment analysis and protein-protein interactions (PPIs) network analysis were performed. Immunohistochemistry was used for molecular expression and localization in endometria. Human endometrial stromal cells (HESCs) were isolated and cultured for verifying the functions of hub gene.ResultsIntegrative data mining of our RNA-seq data in endometria revealed that most genes related to cell division and cell cycle were significantly inhibited, while inflammation activation, immune response and reactive oxygen species associated genes were upregulated in TE. PBK was identified as a hub of PPIs network, and its expression level was decreased by 2.43-fold in endometria of TE patients, particularly reduced in the stromal cells, which was paralleled by the decreased expression of Ki67. In vitro experiments showed that the depletion of PBK reduced the proliferation of HESCs by 50% and increased the apoptosis of HESCs by 1 time, meanwhile PBK expression was inhibited by oxidative stress (reduced by 76.2%), hypoxia (reduced by 51.9%) and inflammatory factors (reduced by approximately 50%). These results suggested that the insufficient expression of PBK was involved in the poor endometrial thickness in TE.ConclusionsThe endometrial transcriptome in late-proliferative phase showed suppressed cell proliferation in women with thin endometria and decreased expression of PBK in human endometrial stromal cells (HESCs), to which inflammation and reactive oxygen species contributed.

CD200S-positive granulated lymphoid cells in endometrium appear to be CD56-positive uterine NK cells

The eutopic secretory phase endometrium in endometriosis overproduces and releases a soluble immunosuppressive CD200 molecule (CD200L) and is populated by stromal cells that contain a truncated CD200 (CD200S) that promotes a proinflammatory environment. The CD200S+ cell population persists when pregnancy occurs and are abundant in the early pregnancy decidua of women with missed abortion. In the present study, CD200S+, CD56+, and CD68+ cells were enumerated in formalin-fixed paraffin-embedded tissue sections from women with endometriosis and non-endometriosis controls. CD200S+ cells were more numerous than CD68+ macrophages and were similar in number and location to CD56bright endometrial NK cells. In some endometria, there was an additional population of CD200S- CD56+ NK cells. In ectopic endometrial peritoneal deposits and in ectopic myometrial deposits (adenomyosis), CD200S+ cells were less frequent, consistent with the known paucity of CD56+ NK cells in sites of ectopic deposits. CD200S+ cell frequency was greater in stroma surrounding the smaller ectopic cystic deposits. Dual immunofluorescent antibody staining confirmed CD200S+ cells were CD56+ NK cells. CD200S+ NK cell frequency may be greater in endometriosis patients’ endometrium and may affect embryo survival in early pregnancy. In our opinion, regulation of alternative splicing that results in CD200S rather than CD200L may provide new diagnostic and therapeutic options for women with endometriosis.

APOBEC mediated mutagenesis drives genomic heterogeneity in endometriosis.

Endometriosis is a benign gynecologic condition, acting as a precursor of certain histological subtypes of ovarian cancers. The epithelial cells of endometriotic tissues and normal uterine endometrium accumulated somatic mutations in cancer-associated genes such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and Kirsten rat sarcoma (KRAS) proto-oncogene. To determine the genomic characteristic of endometriotic epithelial cells and normal uterine endometrium and to identify the predominant mutational process acting on them, we studied the somatic mutation profiles obtained from whole exome sequencing of 14 endometriotic epithelium and 11 normal uterine endometrium tissues and classified them into mutational signatures. We observed that single base substitutions 2/13 (SBS), attributed to Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit (APOBEC) induced mutagenesis, were significant in endometriotic tissues, but not in the normal uterine endometrium. Additionally, the larger number and wider allele frequency distribution of APOBEC signature mutations, compared to cancer-associated driver mutations in endometriotic epithelium suggested APOBEC mutagenesis as an important source of mutational burden and heterogeneity in endometriosis. Further, the relative risk of enriched APOBEC signature mutations was higher in endometriosis patients who were carriers of APOBEC3A/3B germline deletion, a common polymorphism in East Asians which involves the complete loss of APOBEC3B coding region. Our results illustrate the significance of APOBEC induced mutagenesis in driving the genomic heterogeneity of endometriosis.

Macrophages and Natural Killer Cells Characteristics in Variously Colored Endometriotic Lesions: A Cross-Sectional Analytic Study.

Background:Dysregulation of the immune response contribute to a significant role in endometriosis. This research examined macrophages and natural killer (NK) cells numbers in endometriotic lesions and their association with the different lesion colors: red, black, and white. To investigate the amount of the CD68 and CD56 in eutopic endometrium and different type of the endometriotic lesions.Materials and Methods:A cross-sectional analytic study was conducted. Women suspected endometriosis requiring laparoscopic surgery between July 2016 and January 2017 were recruited. Their lesions were classified as red, black, or white and these lesions were excised by standard laparoscopic surgery. Twenty-four endometriotic lesions from each color group were obtained from 45 women who met the inclusion criteria. One type of lesion was collected from 25 women. Two different lesion types and three-color lesion types were collected from the same women in 13 and 7 subjects, respectively. Immunohistochemistry staining with anti-human mouse cluster of differentiation (CD) 68 monoclonal antibody for macrophages and mouse anti-human CD56 monoclonal antibody for NK cells were performed.Results:The number of CD68 macrophages in red lesions was higher than in black and white lesions [median (25th-75th percentile); 10 (5-19.4), 0 (0-6.9), 0 (0-2.5) cells per mm2, respectively, adjusted P=0.001 for red vs. black lesions and red vs. white lesions, and adjusted P=1.000 for black and white lesions]. The number of CD56 NK cells was not significantly different among red, black, and white lesions [median (25th-75th percentile; 5 (2-16.5), 3.8 (0-14.4), 1.3 (0-6.9) respectively, adjusted P=1.000 for red vs. black lesions and black vs. white lesions, and adjusted P=0.617 for red vs. white lesions].Conclusion:The dynamic changes in the immune cells in ectopic endometrium were specific to the macrophages but not to the NK cells, as demonstrated by the highest number of CD68 macrophages in the red lesion, the earliest established ectopic endometrium. NK cells in endometriosis may have a role in the uterus.

The role of endometrial B cells in normal endometrium and benign female reproductive pathologies: a systematic review.

STUDY QUESTIONWhat are the similarities and differences in endometrial B cells in the normal human endometrium and benign reproductive pathologies?SUMMARY ANSWEREndometrial B cells typically constitute <5% of total endometrial CD45+ lymphocytes, and no more than 2% of total cells in the normal endometrium, and while their relative abundance and phenotypes vary in benign gynaecological conditions, current evidence is inconsistent.WHAT IS KNOWN ALREADYB cells are vitally important in the mucosal immune environment and have been extensively characterized in secondary lymphoid organs and tertiary lymphoid structures (TLSs), with the associated microenvironment germinal centre. However, in the endometrium, B cells are largely overlooked, despite the crucial link between autoimmunity and reproductive pathologies and the fact that B cells are present in normal endometrium and benign female reproductive pathologies, scattered or in the form of lymphoid aggregates (LAs). A comprehensive summary of current data investigating B cells will facilitate our understanding of endometrial B cells in the endometrial mucosal immune environment.STUDY DESIGN, SIZE, DURATIONThis systematic review retrieved relevant studies from four databases (MEDLINE, EMBASE, Web of Science Core Collection and CINAHL) from database inception until November 2021.PARTICIPANTS/MATERIALS, SETTING, METHODSThe search strategy combined the use of subject headings and relevant text words related to ‘endometrium’, ‘B cells’ and B-cell derivatives, such as ‘antibody’ and ‘immunoglobulin’. Non-benign diseases were excluded using cancer-related free-text terms, and searches were limited to the English language and human subjects. Only peer-reviewed research papers were included. Each paper was graded as ‘Good’, ‘Fair’ or ‘Poor’ quality based on the NEWCASTLE-OTTAWA quality assessment scale. Only ‘Good’ quality papers were included.MAIN RESULTS AND THE ROLE OF CHANCETwenty-seven studies met the selection criteria and were included in this review: 10 cross-sectional studies investigated B cells in the normal endometrium; and 17 case–control studies compared the characteristics of endometrial B cells in control and benign female reproductive pathologies including endometritis, endometriosis, infertility, abnormal uterine bleeding, endometrial polyps and uterine fibroids. In all studies, B cells were present in the endometrium, scattered or in the form of LAs. CD20+ B cells were more abundant in patients with endometritis, but the data were inconsistent as to whether B-cell numbers were increased in endometriosis and patients with reproductive pathologies.LIMITATIONS, REASONS FOR CAUTIONAlthough only ‘good’ quality papers were included in this systematic review, there were variations in patients’ age, diagnostic criteria for different diseases and sample collection time among included studies. Additionally, a large number of the included studies only used immunohistochemistry as the identification method for endometrial B cells, which may fail to provide an accurate representation of the numbers of endometrial B cells.WIDER IMPLICATIONS OF THE FINDINGSHistological studies found that endometrial B cells are either scattered or surrounded by T cells in LAs: the latter structure seems to be under hormonal control throughout the menstrual cycle and resembles TLSs that have been observed in other tissues. Further characterization of endometrial B cells and LAs could offer insights to endometrial B-cell function, particularly in the context of autoimmune-associated pathologies, such as endometriosis. Additionally, clinicians should be aware of the limited value of diagnosing plasma cell infiltration using only CD138.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by Finox Biotech. The authors have no conflicts of interest to declare.PROSPERO REGISTRATION NUMBERThis systematic review was registered in PROSPERO in January 2020 (PROSPERO ID: CRD42020152915).

Podocalyxin promotes an impermeable epithelium and inhibits pro-implantation factors to negatively regulate endometrial receptivity

Embryo implantation is a key step in establishing pregnancy and a major limiting factor in IVF. Implantation requires a receptive endometrium but the mechanisms governing receptivity are not well understood. We have recently discovered that podocalyxin (PCX or PODXL) is a key negative regulator of human endometrial receptivity. PCX is expressed in all endometrial epithelial cells in the non-receptive endometrium but selectively down-regulated in the luminal epithelium at receptivity. We have further demonstrated that this down-regulation is essential for implantation because PCX inhibits embryo attachment and penetration. However, how PCX confers this role is unknown. In this study, through RNAseq analysis of Ishikawa cell line stably overexpressing PCX, we discovered that PCX suppresses expression of genes controlling cell adhesion and communication, but increases those governing epithelial barrier functions, especially the adherens and tight junctions. Moreover, PCX suppresses multiple factors such as LIF and signaling pathways including Wnt and calcium signaling that support receptivity but stimulates anti-implantation genes such as LEFTY2. Functional studies confirmed that PCX promotes epithelial barrier functions by increasing key epithelial junction proteins such as E-cadherin and claudin 4. PCX thus promotes an anti-adhesive and impermeable epithelium while impedes pro-implantation factors to negatively control endometrial receptivity for implantation.

The Plasminogen Activator System, Glucocorticoid, and Mineralocorticoid Receptors in the Primate Endometrium During Artificial Menstrual Cycles.

As a key mechanism in fibrinolysis and tissue remodeling, the plasminogen activator system has been suggested in the process of endometrial shedding and tissue remodeling. Previous studies have explored the role of estrogen, progesterone, and androgen receptors as well as elements of the renin-angiotensin-aldosterone system in shaping the morphology of the endometrium. This study investigates the distribution and concentrations of the mineralocorticoid receptor, glucocorticoid receptor, tissue plasminogen activator, urokinase plasminogen activator, and plasminogen activator inhibitor-1 within the endometrial stroma, glandular, and endothelial cells of the primate endometrium during artificial menstrual cycles. Our immunohistochemistry quantification shows mineralocorticoid and glucocorticoid receptors are ubiquitously distributed within the macaque endometrium with their patterns of expression following similar fluctuations to urokinase and tissue plasminogen activators particularly within the endometrial vasculature. These proteins are present in endometrial vasculature in high levels during the proliferative phase, decreasing levels during the secretory phase followed by rising levels in the menstrual phase. These similarities could suggest overlapping pathways and interactions between the plasminogen activator system and the steroid receptors within the endometrium. Given the anti-inflammatory properties of glucocorticoids and the role of plasminogen activators in endometrial breakdown, the glucocorticoid receptor may be contributing to stabilizing the endometrium by regulating plasminogen activators during the proliferative phase and menstruation. Furthermore, given the anti-mineralocorticoid properties of certain anti-androgenic progestins and their reduced unscheduled uterine bleeding patterns, the mineralocorticoid receptor may be involved in unscheduled endometrial bleeding.

Mice Uterine Stem Cells are Affected by Neonatal Endocrine Disruption & Initiate Uteropathies in Adult Life Independent of Circulatory Ovarian Hormones.

It is generally believed that ovarian hormones regulate uterine functions and their altered levels result in various uteropathies like non-receptive uterus, endometrial hyperplasia, adenomyosis, endometriosis, leiomyomas and cancer. Uterus harbors two populations of stem cells including pluripotent, very small embryonic-like stem cells (VSELs) and tissue-specific progenitors (endometrial stem cells, EnSCs). Unlike endometrial mesenchymal stem/ stromal cells, VSELs/EnSCs express ERα, ERβ and PR which makes them directly vulnerable to perinatalendocrine insults. Present study was undertaken to evaluate whether uteropathies occur due to altered hormones and/or intrinsic changes in stem/progenitor cells. Mice pups, exposed to estradiol (20µg/pup/day) on postnatal days 3-7 or vehicle, were subjected to bilateral ovariectomy on day 30 and later exposed sequentially to estradiol and progesterone resulting in receptive uterus in control mice. Despite similar hormonal exposure, endocrine disruption resulted in non-receptive uterus with noticeable endometrial and myometrial hyperplasia and up-regulation of stem cell markers (Oct-4A, Oct-4, Sox2, Nanog). Glands were poorly formed and 'defective' epithelial progenitors were found disseminated into myometrium and blood vessels revealing how adenomyosis and endometriosis possibly initiate. Progesterone resistance and estradiol dominance due to downregulation of Erα & Pr and upregulation of Erβ transcripts was observed in both intact uterus and stem cells enriched from uterus. Transcripts specific for DNA mismatch repair axis (Pcna, NP95 and Dnmt1), repair enzymes (Brca-1, Rad51 and Mlh1) were dysregulated whereas Ki67 was ten-folds increased suggestive of genomic instability. Study reveals role of stem cells in initiating uteropathies during adult life independent of circulatory ovarian hormones. Endocrine disruption affects tissue resident stem/progenitor cells (VSELs/EnSCs) in both endometrium and myometrium, result in epithelial cells hyperplasia, non-receptive endometrium, adenomyosis and defective stem cells and epithelial progenitors were detected in the perimetrium from where they can mobilize to ectopic sites to initiate endometriosis. Study shows stem cell basis for various uteropathies. VSEL: Very small embryonic like stem cell; EnSC: Endometrial stem cell; E + P: Estradiol + Progesterone; E: Endometrium; P: Perimetrium; M: Myometrium; ACD: Asymmetrical cell division; SCD: Symmetrical cell division; CE: Clonal expansion; G: Gland; S: Stromal cell; US: Undifferentiated stromal cell; LE: Luminal epithelium; GE: Glandular epithelium; EP: Epithelial progenitors; SMC: Spindle-shaped myometrial cell; OMC: Oval-shaped myometrial cell.

TNF-α Regulated Endometrial Stroma Secretome Promotes Trophoblast Invasion

Successful implantation requires the coordinated migration and invasion of trophoblast cells from out of the blastocyst and into the endometrium. This process relies on signals produced by cells in the maternal endometrium. However, the relative contribution of stroma cells remains unclear. The study of human implantation has major technical limitations, therefore the need of in vitro models to elucidate the molecular mechanisms. Using a recently described 3D in vitro models we evaluated the interaction between trophoblasts and human endometrial stroma cells (hESC), we assessed the process of trophoblast migration and invasion in the presence of stroma derived factors. We demonstrate that hESC promotes trophoblast invasion through the generation of an inflammatory environment modulated by TNF-α. We also show the role of stromal derived IL-17 as a promoter of trophoblast migration through the induction of essential genes that confer invasive capacity to cells of the trophectoderm. In conclusion, we describe the characterization of a cellular inflammatory network that may be important for blastocyst implantation. Our findings provide a new insight into the complexity of the implantation process and reveal the importance of inflammation for embryo implantation.

Identification and Validation of the Signatures of Infiltrating Immune Cells in the Eutopic Endometrium Endometria of Women With Endometriosis.

Endometriosis is an oestrogen-dependent chronic inflammatory process with primary symptoms including dysmenorrhea, chronic pelvic pain, and infertility. The immune environment of the endometrium is essential for successful embryo implantation and ongoing pregnancy. In this study, we assessed the composition, density, and distribution of infiltrating immune cells in the endometria of women with endometriosis. Gene expression profiles of endometrial samples were downloaded from the Gene Expression Omnibus (GEO) database. We found that the TNF signalling pathway, the IL-17 signalling pathway, and the MAPK signalling pathway were significantly enriched in the eutopic endometria of women with endometriosis. The fractions and proportion of infiltrating immune cells were estimated by the CIBERSORT, MCP-counter, and ImmuCellAI methods. We found that the proportions of CD8+ T cells, activated NK cells, and follicular helper T cells were significantly higher in the endometria of women with endometriosis than in the endometria of normal controls, while the proportions of M2 macrophages and resting mast cells were significantly lower in the eutopic endometria. In GSE120103 (n = 36), we found that elevated CD8+ T cells in endometriosis increased the risk of infertility (P = 0.0019). The area under the receiver operating characteristic (ROC) curve (AUC) of CD8+ T cells to distinguish fertile and infertile endometriosis was 0.914. In clinical samples (n = 40), we found that the proportions of CD8+ T cells and CD56+ NK cells were significantly higher in the eutopic endometria of women with endometriosis than in the endometria of normal controls, while the proportion of CD163+ macrophages were lower in the eutopic endometria. The AUCs of CD8+ T cells and CD163+ macrophages were 0.727 and 0.833, respectively, which indicated that CD8 and CD163 were potential diagnostic markers for endometriosis. In conclusion, our results demonstrated that increased CD8+ T cells and CD56+ NK cells and decreased CD163+ macrophages within the eutopic endometria of women with endometriosis reveal a proinflammatory feature in the endometrial immune environment and that elevated CD8+ T cells increase the risk of infertility in women with the disease.