Cells Of Endometrium Research Articles

Dysregulated uterine natural killer cells and vascular remodeling in women with recurrent pregnancy losses.

Angiogenesis and vascular remodeling in secretory endometrium represent one of the crucial steps in pregnancy establishment, for which uterine NK (uNK) cells have an important role. Impairment of these steps may proceed to implantation and instigate initial pathology of recurrent pregnancy losses (RPL). In this study, we aim to investigate vascular development and density of uNK cells in secretory endometrium of women with RPL. Mid-secretory phase endometrial tissues from women with RPL (n=15) and fertile controls (n=7) were investigated. CD56+ and CD16+ uNK cells, CD31+ vascular endothelial cells and smooth muscle myosin (SMM)+ . Vascular smooth muscle cells (VSMC) expressing SMM were investigated using immunohistochemistry and western blot. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was used as well. CD56+ uNK number was significantly higher in women with RPL compared to controls (P<0.0001). uNK cell density by immunohistochemistry was positively correlated with CD56 mRNA expression by qRT-PCR (r2 =0.43, P=0.0137). The number of blood vessels represented by the expression of either CD31 or SMM was higher in women with RPL as compared to controls (P<0.05 and P<0.0001, respectively), and correlated with the number of uNK cell (r2 =0.18, P<0.04, and r2 =0.65, P<0.0001, respectively). The wall thickness of spiral arteries was significantly higher in women with RPL as compared with that of controls (P=0.0027). Increased uNK cells in mid-secretory endometrium are associated with increased vascularization and defective vascular transformation of spiral arteries in women with RPL.

Role of miR‑449b‑3p in endometriosis via effects on endometrial stromal cell proliferation and angiogenesis.

Endometriosis is a common gynecological disease and the pathogenesis is not clearly understood. Previous studies have demonstrated via microarray techniques that microRNA (miR)-449b was significantly downregulated both in ectopic and eutopic endometrium in patients with endometriosis. In the present study, the aberrant expression of miR-449b was further confirmed by reverse transcription-quantitative polymerase chain reaction. It was demonstrated that miR-449b-3p was downregulated in ectopic and eutopic tissues from women with endometriosis, and the same expression pattern was observed in endometrial stromal cells (ESCs) of eutopic endometrium from women with endometriosis and normal endometrium from women without endometriosis. Functional analysis, including an MTT assay, apoptosis conducted by flow cytometry, capillary-like tube formation assay and invasion assay, indicated that the upregulated expression of miR-449b-3p inhibited the proliferation of ESCs and that the supernatants of miR-449b-overexpressing ESCs inhibited the formation of tubular structures in human umbilical vein endothelial cells, whereas it has no effect on ESC apoptosis and invasiveness. These results suggest that the aberrant expression of miR-449b-3p was involved in the development and progression of endometriosis.

Correlation between replicative senescence of endometrial gland epithelial cells in shedding and non-shedding endometria and endometriosis cyst during menstruation

To investigate correlation between abnormal replicative senescence of endometrial gland epithelial cells (EGECs) in shedding and non-shedding endometria and endometriosis cyst during menstruation. Musashi-1 expression, β-catenin expression, and EGECs ultrastructure in shedding and non-shedding endometrium when menstruation were observed through real-time PCR and transmission electron microscopy technologies. (1) Musashi-1 and β-catenin exhibited a high expression in shedding and non-shedding endometria in experimental group, showing a positive correlation between each other; and were significantly higher than that in control group. However; there was no correlation between these two in control group. (2) Transmission electron microscopy results: In experimental group, organelles in EGECs in shedding endometrium obtained were abundant on the first day of menstruation, nuclei were irregular, double nucleoli could be observed, and chromatin was rich. Furthermore, morphology of EGECs in non-shedding endometrium was irregular, organelles were abundant, basement membrane was irregular with abnormal curvature, and a large amount of collagenic fibers were found in intercellular spaces. On the fifth day of menstruation, the cilia and microvilli on secretory cells in endometrium increased and prolongated, and organelles became extremely rich. EGECs have potentials of division, proliferation, invasion and migration; and is associated with formation of endometriosis cysts.

Oviductal glycoprotein 1 (OVGP1) is expressed by endometrial epithelium that regulates receptivity and trophoblast adhesion

To study the regulation and functions of oviductal glycoprotein 1 (OVGP1) in endometrial epithelial cells. Expression of OVGP1 in mouse endometrium during pregnancy and in the endometrial epithelial cell line (Ishikawa) was studied by immunofluorescence, Western blotting, and RT-PCR. Regulation of OVGP1 in response to ovarian steroids and human chorionic gonadotropin (hCG) was studied by real-time RT-PCR. OVGP1 expression was knockdown in Ishikawa cells by shRNA, and expression of receptivity associated genes was studied by real-time RT-PCR. Adhesion of trophoblast cell line (JAr) was studied by in vitro adhesion assays. OVGP1 was localized exclusively in the luminal epithelial cells of mouse endometrium at the time of embryo implantation. Along with estrogen and progesterone, hCG induced the expression of OVGP1 in Ishikawa cells. Knockdown of OVGP1 in Ishikawa cells reduced mRNA expression of ITGAV, ITGB3, ITGA5, HOXA10, LIF, and IL15; it increased the expression of HOXA11, MMP9, TIMP1, and TIMP3. Supernatants derived from OVGP1 knockdown Ishikawa cells reduced the adhesiveness of JAr cells in vitro. Expression of OVGP1 mRNA was found to be significantly lowered in the endometrium of women with recurrent implantation failure. OVGP1 is specifically induced in the luminal epithelium at the time of embryo implantation where it regulates receptivity-related genes and aids in trophoblast adhesion.

Pathogenomics of Endometriosis Development.

For over 100 years, endometriosis, as a chronic, estrogen-dependent, inflammatory, heritable disease affecting approximately 5–10% of women in reproductive age has been the focus of clinicians and scientists. In spite of numerous environmental, genetic, epigenetic, endocrine, and immunological studies, our knowledge of endometriosis is still fragmentary, and its precise pathophysiology and pathogenomics remain a mystery. The implementation of new technologies has provided tremendous progress in understanding the many intrinsic molecular mechanisms in the development of endometriosis, with progenitor and stem cells (SCs) of the eutopic endometrium as the starting players and endometriotic lesions as the final pathomorphological trait. Novel data on the molecular, genetic, and epigenetic mechanisms of the disease are briefly outlined. We hypothesize the existence of an endometriosis development genetic program (EMDP) that governs the origin of endometrium stem cells programmed for endometriosis (1), their transition (metaplasia) into mesenchymal SCs (2), and their invasion of the peritoneum and progression to endometriotic lesions (3). The pros and cons of the recent unifying theory of endometriosis are also discussed. Complex genomic and epigenetic interactions at different stages of the endometriosis process result in different forms of the disease, with specific features and clinical manifestations. The significance of the EMDP in elaborating a new strategy for endometriosis prediction, prevention, and treatment is discussed.

Partial regeneration of uterine horns in rats through adipose-derived stem cell sheets.

Severe uterine damage and infection lead to intrauterine adhesions, which result in hypomenorrhea, amenorrhea and infertility. Cell sheet engineering has shown great promise in clinical applications. Adipose-derived stem cells (ADSCs) are emerging as an alternative source of stem cells for cell-based therapies. In the present study, we investigated the feasibility of applying ADSCs as seed cells to form scaffold-free cell sheet. Data showed that ADSC sheets expressed higher levels of FGF, Col I, TGFβ, and VEGF than ADSCs in suspension, while increased expression of this gene set was associated with stemness, including Nanog, Oct4, and Sox2. We then investigated the therapeutic effects of 3D ADSCs sheet on regeneration in a rat model. We found that ADSCs were mainly detected in the basal layer of the regenerating endometrium in the cell sheet group at 21 days after transplantation. Additionally, some ADSCs differentiated into stromal-like cells. Moreover, ADSC sheets transplanted into partially excised uteri promoted regeneration of the endometrium cells, muscle cells and stimulated angiogenesis, and also resulted in better pregnancy outcomes. Therefore, ADSC sheet therapy shows considerable promise as a new treatment for severe uterine damage.

P65: Intrauterine application of stimulated autologous PBMC leads to beneficial changes in the phenotypic distribution of uNK cells in periimplantation endometrium

American Journal of Reproductive ImmunologyVolume 80, Issue S1 p. 85-85 ABSTRACTS P65: Intrauterine application of stimulated autologous PBMC leads to beneficial changes in the phenotypic distribution of uNK cells in periimplantation endometrium First published: 26 June 2018 https://doi.org/10.1111/aji.64_12984Citations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume80, IssueS1Special Issue: American-Sino Joint Meeting of Reproductive Immunology (ASRI-CSI 2018), June 28-1 July, 2018 Shanghai, ChinaJune 2018Pages 85-85 RelatedInformation

Follicle stimulating hormone receptor protein is expressed in ovine uterus during the estrous cycle and utero-placenta during early pregnancy: An immunohistochemical study

Follicle stimulating hormone (FSH) is a well characterized gonadotropin that controls primarily development and functions of ovarian follicles in mammalian species. FSH binds to a specific G protein-coupled receptor (FSHR) belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although the primary location of FSHR is in the gonads (mainly in ovarian follicles), FSHR protein and/or mRNA have also been detected in extragonadal female reproductive tissues including embryo, placenta, endometrium, cervix, ovarian cancer tissues, and/or endometriotic lesions in several species. To determine the pattern of FSHR expression in the uterus and placenta, uterine tissues were collected at the early, mid- and/or late luteal phases of the estrous cycle from non-treated or FSH-treated ewes, and utero-placental tissues were collected during early pregnancy followed by immunohistochemistry and image generation. FSHR was immunolocalized to several uterine and utero-placental compartments including luminal epithelium, endometrial glands and surrounding stroma, myometrium, and endothelium and vascular smooth muscle cells in endometrium, myometrium and mesometrium. Intensity of staining and distribution of FSHR in selected compartments differed and seems to depend on the stage of the estrous cycle or pregnancy, and FSH-treatment. These novel data demonstrate differential expression of FSHR protein indicating that FSH plays a specific role in regulation of uterine and utero-placenta functions in sheep.

A prophylaxis of relapses and rehabilitation in women of reproductive age underwent leiomyoma surgery on a background of metabolic syndrome

Аim – to work out the algorithm of prophylaxis of leiomyoma (LM) relapses and rehabilitation of women of reproductive age with metabolic syndrome (MS) in postoperative period. Materials and methods. In research group 130 women with LM and MS (research group) or without MS (comparison group) were involved. The control group consisted of 50 somatically healthy women with normal menstrual and reproductive functions. МS was set on according the following "combined" criteria – criteria of WHO 1999 (impaired glucose tolerance) and criteria of IDF (2009) taking into account the age-related levels of lipids and arterial blood pressure. In venous blood plasma we determined total testosterone, DHEA-S, estradiol, progesterone, FSH, LH, prolactin, using solid-phase ELISA on the "Cobas e 411" and reagents of DRG Diagnostics, Germany. The content of total cholesterol, triglycerides and high-density lipoproteins (HDLP) was determined by a colorimetry method using the "DiaSys" sets (Germany). Before operative interventions the histological and immunohistochemical research was conducted after the aspiration biopsy or separate diagnostic scraping of uterus. Endoscopic surgery was provided in research and comparison group using "Karl Storz" equipment (Germany) on the generally accepted methodology. Results and discussion. The volume of operative intervention and operative access was determined depending on the previous results of endometrial biopsy, sizes and localization of nodules. During the study of the hormonal homoeostasis we revealed increase of estradiol (research group – 429.4±31.2 and comparison group – 320.4±25.3 vs 228.3±24.1 nmol/l in control; р 0.05), but the decline of this index was determined in women of research group and comparison group on the 21-th day of cycle. In addition, pays attention on itself increase of androgens in women with LM and MS as compared to other groups of patients. Revealed increase of LDLP (1.2 times), triglycerides (1.7 times), total cholesterol (1.4 times) and decreased HDLP (1.4 times) reflect negatively on the reproductive function. The immunohistochemical research showed that in the case of MS there was a positive reaction on androgen receptors in leiomyoma cells, particularly in the vessel wall myocytes. We offered our patients the moderate hypocaloric diet and the individually worked out program of fitness. Metformin was added to traditional therapy. The treatment complex lasted from 3 to 6 months. As a result, we observed the decrease of body weight by 10.5±1.8 kg on average, that corresponded 11.4%. Waist circumference decreased in all patients and in average of group consisted 10.2±3.2 сm; hip circumference decreased by 6.6±4.2 сm suggesting the visceral fat lost. We established statistically significant decrease of LDLP cholesterol that undoubtedly is the additional positive result of provided therapy. Efficiency of the offered diagnostic and curative complex for women with LM and MS was estimated by absence of relapses of LM and by incidence of pregnancies. So at the first year of supervision there were no relapses of LM in ІА subgroup (aromatase inhibitor added to traditional treatment), indeed in ІІA subgroup (traditional treatment only) relapses occurred in 6 patients (20%). The pregnancy occurred in 6 (20.0±2.1%) women of ІІА subgroup, and frequency calculated for one cycle of stimulation consisted 16.7%. The stimulation of ovulation using the aromatase inhibitor 2.5 mg/d was more effective: the pregnancy occurred in 11 (36.6±2.4%) women of ІА subgroup, accordingly frequency calculated for one cycle of stimulation – 30.0 %, that 1.8 times more effective due to offered method of treatment. Conclusions. Comorbidity of benign diseases of uterus and MS in women of reproductive age takes place on a background of significant estradiol increase. Immunohistochemical feature of this comorbidity is expression of androgenic receptors in the separate stromal cells of endometrium and cellular myomas. Improvement in diagnostic and treatment algorithm (using aromatase inhibitor for stimulation of ovulation) resulted in prophylaxis of LM relapses and increase of pregnancies as compared to traditional treatment. We offer the algorithm of management of women of reproductive age with benign diseases of uterus and MS: I stage: complex clinical and laboratory assessment; ІІ stage: correction of revealed hormonal and metabolic violations at MS; ІІI stage: add the aromatase inhibitor to the standard treatment for restoration of reproductive function.

Immunolocalization of angiogenic growth factors in the ovine uterus during the oestrus cycle and in response to Steroids.

The vascular changes associated with endometrial maturation in preparation for embryo implantation depend on numerous growth factors, known to regulate key angiogenic events. Primarily, the vascular endothelial growth factor (VEGF) family promotes vascular growth, whilst the angiopoietins maintain blood vessel integrity. The aim was to analyse protein levels of VEGFA ligand and receptors, Angiopoietin-1 and 2 (ANG1/2) and endothelial cell receptor tyrosine kinase (TIE-2) in the ovine endometrium in the follicular and luteal phases of the oestrus cycle and in response to ovarian steroids. VEGFA and its receptors were localized in both vascular cells and non-vascular epithelium (glandular and luminal epithelium) and stroma cells. VEGFA and VEGFR2 proteins were elevated in vascular cells in follicular phase endometrium, compared to luteal phase, most significantly in response to oestradiol. VEGFR1 was expressed by epithelial cells and endothelial cells and was stimulated in response to oestradiol. In contrast, Ang-1 and Ang-2 proteins were elevated in luteal phase endometrium compared to follicular phase, and in response to progesterone, evident in vascular smooth muscle cells and glands which surround TIE-2-expressing blood vessels. Our findings indicate that VEGFA is stimulated by oestradiol, most predominantly in follicular phase endometrium, and Ang-1 and 2 are stimulated by progesterone and were increased during the luteal phase of the oestrus cycle, during the time of vascular maturation.

Endometrial natural killer (NK) cells reveal a tissue-specific receptor repertoire.

Is the natural killer (NK) cell receptor repertoire of endometrial NK (eNK) cells tissue-specific? The NK cell receptor (NKR) expression profile in pre-pregnancy endometrium appears to have a unique tissue-specific phenotype, different from that found in NK cells in peripheral blood, suggesting that these cells are finely tuned towards the reception of an allogeneic fetus. NK cells are important for successful pregnancy. After implantation, NK cells encounter extravillous trophoblast cells and regulate trophoblast invasion. NK cell activity is amongst others regulated by C-type lectin heterodimer (CD94/NKG2) and killer cell immunoglobulin-like (KIR) receptors. KIR expression on decidual NK cells is affected by the presence of maternal HLA-C and biased towards KIR2D expression. However, little is known about NKR expression on eNK cells prior to pregnancy. In this study, matched peripheral and menstrual blood (a source of endometrial cells) was obtained from 25 healthy females with regular menstrual cycles. Menstrual blood was collected during the first 36 h of menstruation using a menstrual cup, a non-invasive technique to obtain endometrial cells. KIR and NKG2 receptor expression on eNK cells was characterized by 10-color flow cytometry, and compared to matched pbNK cells of the same female. KIR and HLA-C genotypes were determined by PCR-SSOP techniques. Anti-CMV IgG antibodies in plasma were measured by chemiluminescence immunoassay. KIR expression patterns of eNK cells collected from the same female do not differ over consecutive menstrual cycles. The percentage of NK cells expressing KIR2DL2/L3/S2, KIR2DL3, KIR2DL1, LILRB1 and/or NKG2A was significantly higher in eNK cells compared to pbNK cells, while no significant difference was observed for NKG2C, KIR2DL1/S1, and KIR3DL1. The NKR repertoire of eNK cells was clearly different from pbNK cells, with eNK cells co-expressing more than three NKR simultaneously. In addition, outlier analysis revealed 8 and 15 NKR subpopulation expansions in eNK and pbNK cells, respectively. In contrast to the pbNK cell population, the expansions present in the eNK cell population were independent of CMV status and HLA-C genotype. Moreover, the typical NKG2C imprint induced by CMV infection on pbNK cells was not observed on eNK cells from the same female, suggesting a rapid local turnover of eNK cells and/or a distinct licensing process. Based on our previous work and the parameters studied here, menstrual blood-derived eNK cells closely resemble biopsy-derived eNK cells. However, sampling is not done at the exact same time during the menstrual cycle, and therefore we cannot exclude some, as yet undetected, differences. Our data reveals that NK cells in the pre-implantation endometrium appear to have a dedicated tissue-specific phenotype, different from NK cells in peripheral blood. This may indicate that eNK cells are finely tuned to receive an allogeneic fetus. Studying the endometrial NKR repertoire of women with pregnancy related problems could provide clues to understand the pathogenesis of pregnancy complications. No external funding was obtained for the present study. None of the authors has any conflict of interest to declare. NA.

Therapeutic effect of human umbilical cord-derived mesenchymal stem cells on injured rat endometrium during its chronic phase

BackgroundRepair deficiency after endometrial injury is an important reason for intra-uterine adhesions, amenorrhea, and infertility in females. Bone marrow-derived mesenchymal stem cell (BMSC) transplantation is effective in repairing the damaged endometrium. However, the possibility of using umbilical cord-derived MSCs (UC-MSCs) to treat endometrial injury is rarely reported.MethodsEthanol (95%) was injected into rat uterus to establish a model of endometrial injury. UC-MSCs were injected through the tail vein, either as a single, twice, or thrice administration. Functional restoration of the uterus was assessed by testing embryo implantation rates. Endometrial morphological alteration was observed by hematoxylin and eosin staining. Endometrial fibrosis, markers of epithelial and stromal cells of endometrium, cell proliferation and angiogenesis, and inflammatory factors were detected using immunohistochemistry, Western blotting, and quantitative reverse-transcription polymerase chain reaction.ResultsEndometrial morphology and embryo implantation rates were significantly improved on day 8 of transplantation among single-, twice-, or thrice-administered rats. Moreover, UC-MSCs could alleviate fibrosis in general, and reduced the expression of fibrosis markers, α-smooth muscle actin (α-SMA) and transforming growth factor (TGF)-β. The cell proliferation marker Ki-67 had a positive expression in the injured endometrium after UC-MSC transplantation. The endometrial stromal marker vimentin and epithelial marker cytokeratin-19 (CK-19) expressions were visibly increased. The expression of vascular markers CD31, vascular endothelial growth factor (VEGF)A, and matrix metalloprotein (MMP)9 was generally upregulated. Proinflammatory factors interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-2 were significantly downregulated in the rats administered UC-MSCs twice and thrice.ConclusionsUC-MSC transplantation contributed to the repair of endometrial injury and restoration of fertility, likely through the suppression of excessive fibrosis and inflammation, and enhancement of endometrial cell proliferation and vascular remodeling.

Oestrogen, progesterone and stem cells: the discordant trio in endometriosis?

Oestrogen-progesterone signalling is highly versatile and critical for the maintenance of healthy endometrium in humans. The genomic and nongenomic signalling cascades initiated by these hormones in differentiated cells of endometrium have been the primary focus of research since 1920s. However, last decade of research has shown a significant role of stem cells in the maintenance of a healthy endometrium and the modulatory effects of hormones on these cells. Endometriosis, the growth of endometrium outside the uterus, is very common in infertile patients and the elusiveness in understanding of disease pathology causes hindrance in selection of treatment approaches to enhance fertility. In endometriosis, the stem cells are dysfunctional as it can confer progesterone resistance to their progenies resulting in disharmony of hormonal orchestration of endometrial homeostasis. The bidirectional communication between stem cell signalling pathways and oestrogen-progesterone signalling is found to be disrupted in endometriosis though it is not clear which precedes the other. In this paper, we review the intricate connection between hormones, stem cells and the cross-talks in their signalling cascades in normal endometrium and discuss how this is deregulated in endometriosis. Re-examination of the oestrogen-progesterone dependency of endometrium with a focus on stem cells is imperative to delineate infertility associated with endometriosis and thereby aid in designing better treatment modalities.

Identification of perivascular and stromal mesenchymal stem/progenitor cells in porcine endometrium.

Mammalian uterus contains a population of mesenchymal stem/progenitor cells that likely contribute to endometrial regeneration during each reproductive cycle. In human and mouse, they reside in perivascular, epithelial and stromal compartments of the endometrial functionalis and basalis. Here, we aimed to identify tissue resident cells expressing mesenchymal stem cell markers CD29, CD44, CD90, CD105, CD140b and CD146 in the porcine endometrium. We used single immunofluorescence and Western blotting. Each of these markers was detected in small cells surrounding endometrial blood vessels. CD105 and CD146 were also expressed in single stromal cells. A few stromal and perivascular cells showed the presence of pluripotency marker Oct4 in the cytoplasm, but not in the nucleus, which may imply they are not truly pluripotent. Endometrial cell cultures were examined for the expression of CD29, CD44, CD90, CD105 and CD140b proteins and tested in wound-healing assay and culture model of chemotaxis. In conclusion, our results demonstrate perivascular location of prospective mesenchymal stem/progenitor cells in the porcine endometrium and may suggest that stromal CD105+ and CD146+ cells represent more mature precursors originating from their perivascular ancestors.

Expression of NKp46 on NK cells in endometrium in women with endometriosis

Expression of NKp46 on NK cells in endometrium in women with endometriosis

Bone Marrow Stem Cells Do Not Contribute to Endometrial Cell Lineages in Chimeric Mouse Models.

Studies from five independent laboratories conclude that bone marrow stem cells transdifferentiate into endometrial stroma, epithelium, and endothelium. We investigated the nature of bone marrow-derived cells in the mouse endometrium by reconstituting irradiated wild type recipients with bone marrow containing transgenic mTert-green fluorescent protein (GFP) or chicken β-actin (Ch β-actin)-GFP reporters. mTert-GFP is a telomerase marker identifying hematopoietic stem cells and subpopulations of epithelial, endothelial, and immune cells in the endometrium. Ch β-actin-GFP is a ubiquitous reporter previously used to identify bone marrow-derived cells in the endometrium. Confocal fluorescence microscopy for GFP and markers of endometrial and immune cells were used to characterize bone marrow-derived cells in the endometrium of transplant recipients. No evidence of GFP+ bone marrow-derived stroma, epithelium, or endothelium was observed in the endometrium of mTert-GFP or Ch β-actin-GFP recipients. All GFP+ cells detected in the endometrium were immune cells expressing the pan leukocyte marker CD45, including CD3+ T cells and F4/80+ macrophages. Further examination of the Ch β-actin-GFP transplant model revealed that bone marrow-derived F4/80+ macrophages immunostained weakly for CD45. These macrophages were abundant in the stroma, infiltrated the epithelial and vascular compartments, and could easily be mistaken for bone marrow-derived endometrial cells. We conclude that it is unlikely that bone marrow cells are able to transdifferentiate into endometrial stroma, epithelium, and endothelium. This result has important therapeutic implications, as the expectation that bone marrow stem cells contribute directly to endometrial regeneration is shaping strategies designed to regenerate endometrium in Asherman's syndrome and to control aberrant endometrial growth in endometriosis. Stem Cells 2018;36:91-102.

Aberrant expression of epithelial leucine-rich repeat containing G protein–coupled receptor 5–positive cells in the eutopic endometrium in endometriosis and implications in deep-infiltrating endometriosis

To characterize leucine-rich repeat containing G protein-coupled receptor 5-positive (LGR5+) cells from the endometrium of women with endometriosis. Prospective experimental study. University hospital/fertility clinic. Twenty-seven women with endometriosis who underwent surgery and 12 healthy egg donors, together comprising 39 endometrial samples. Obtaining of uterine aspirates by using a Cornier Pipelle. Immunofluorescence in formalin-fixed paraffin-embedded tissue from mice and healthy and pathologic human endometrium using antibodies against LGR5, E-cadherin, and cytokeratin, and epithelial and stromal LGR5+ cells isolated from healthy and pathologic human eutopic endometrium by fluorescence-activated cell sorting and transcriptomic characterization by RNA high sequencing. Immunofluorescence showed that LGR5+ cells colocalized with epithelial markers in the stroma of the endometrium only in endometriotic patients. The results from RNA high sequencing of LGR5+ cells from epithelium and stroma did not show any statistically significant differences between them. The LGR5+ versus LGR5- cells in pathologic endometrium showed 394 differentially expressed genes. The LGR5+ cells in deep-infiltrating endometriosis expressed inflammatory markers not present in the other types of the disease. Our results revealed the presence of aberrantly located LGR5+ cells coexpressing epithelial markers in the stromal compartment of women with endometriosis. These cells have a statistically significantly different expression profile in deep-infiltrating endometriosis in comparison with other types of endometriosis, independent of the menstrual cycle phase. Further studies are needed to elucidate their role and influence in reproductive outcomes.

Expression of natural cytotoxicity receptors and cytokine production on endometrial natural killer cells in women with recurrent pregnancy loss or implantation failure, and the expression of natural cytotoxicity receptors on peripheral blood natural killer cells in pregnant women with a history of recurrent pregnancy loss.

Natural cytotoxicity receptors (NCR) are unique markers that regulate natural killer (NK) cell cytotoxicity and cytokine production. In this study, we investigated the expression of NCR (NKp46, NKp44, and NKp30) and cytokine production in NK cells derived from the uterine endometrium of women with recurrent pregnancy loss (RPL). We also investigated the expression of NCR in peripheral blood NK cells in pregnant women with and without a history of RPL. The expression of NCR (NKp46, NKp44, and NKp30) in NK cells (CD56dim and CD56bright ) in the uterine endometrium was analyzed using 3-color flow cytometry. Cytokine (tumor necrosis factor-α and interferon-γ) production was also analyzed. NK cells from the mid-secretory endometrium of 28 women with RPL, 34 women with implantation failure, and 74 controls were collected and mechanically dispersed using a tissue grinder. The expression of NCR in peripheral blood NK cells from pregnant women with (n = 17) and without (n = 91) a history of RPL was analyzed. The percentages of NKp46+ NK cells were significantly lower in both women with RPL and pregnant women with a history of RPL. The percentages of tumor necrosis factor-α- and/or interferon-γ-producing uterine endometrial NK cells were significantly lower in women with RPL compared with controls. The changes in NCR expression and cytokine production, especially decreased NKp46 expression in endometrial NK cells, suggests the presence of abnormal NK cell regulation in women with reproductive failures.

NLRP7 contributes to in vitro decidualization of endometrial stromal cells

BackgroundNucleotide-binding oligomerization domain (NACHT), leucine rich repeat (LRR) and pyrin domain (PYD) 7 containing protein, NLRP7, is a member of the NLR family which serves as innate immune sensors. Mutations and genetic variants of NLRP7 have been found in women with infertility associated conditions, such as recurrent hydatidiform mole, recurrent miscarriage, and preeclampsia. Decidualization of endometrial stromal cells is a hallmark of tissue remodeling to support embryo implantation and proper placental development. Given defective decidualization has been implicated in miscarriage as well as preeclampsia, we aimed to explore the link between the NLRP7 gene and decidualization.MethodsEndometrial samples obtained from pregnant women in the first trimester and non-pregnant women were used to study NLRP7 expression pattern. The human telomerase reverse transcriptase (hTERT)-immortalized human endometrial stromal cells (T-HESCs) were used to study the effect of NLRP7 on decidualization. Decidualization of T-HESCs was induced with 1 μM medroxyprogesterone acetate (MPA) and 0.5 mM 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). siRNA was used to knock down NLRP7 while lentiviral vectors were used to overexpress NLRP7 in cells. NLRP7 expression was detected by immunofluorescence, qRT-PCR, and Western blotting. Decidualization markers, Insulin-like growth factor-binding protein 1 (IGFBP-1) and prolactin (PRL), were detected by qRT-PCR and ELISA. Nuclear translocation of NLRP7 was detected by the subcellular fractionation and confocal microscopy. The effect of NLRP7 on progesterone receptor (PR) activity was evaluated by a reporter system.ResultsNLRP7 was up-regulated in the decidual stromal cells of human first-trimester endometrium. After in vitro decidualization, T-HESCs presented with the swollen phenotype and increased expressions of IGFBP-1 and PRL. Knockdown or over-expression of NLRP7 reduced or enhanced the decidualization, respectively, according to the expression level of IGFBP-1. NLRP7 was found to translocate in the nucleus of decidualized T-HESCs and able to promote PR activity.ConclusionsNLRP7 was upregulated and translocated to the nucleus of the endometrial stromal cells in an in vitro decidualization model. Overexpressed NLRP7 promoted the IGFBP-1 expression and PR reporter activation. IGFBP-1 expression decreased with the knockdown of NLRP7. Therefore, we suggest that NLRP7 contributes to in vitro decidualization of endometrial stromal cells.

Immunohistochemical Parameters of Musashi-1 in Nodular and Diffuse Adenomyosis.

Comparative immunohistochemical analysis of the expression of somatic stem cells marker Musashi-1 in the endometrium was performed during various phases of menstrual cycle and in patients with nodular and diffuse adenomyosis. The expression of Musashi-1 reflecting proliferative potential of epithelial and stromal cells was quantitatively analyzed by the optical density in the nuclei and cytoplasm of epithelial and stromal cells of the eutopic endometrium and adenomyosis foci. In the eutopic endometrium, the immunohistochemical reaction was more intensive during proliferation phase in comparison with secretion phase. Enhanced expression of Musashi-1 was observed in adenomyosis foci in comparison with endometrial cells. The most intensive staining was found in nodular adenomyosis, especially in epithelial cells during the secretion phase. These data attest to the role of somatic stem cells in the development and progression of various forms of adenomyosis.