Endometrial Cell Cultures Research Articles

Soluble factors formed during the healing of the endometrium suppress its "fibrosis” in vitro

During each period, the uterine mucosa of women of reproductive age heals without fibrosis. Previously, we established that the soluble factors that are released in this way have an antifibrotic effect on the culture of the human endometrial mesenchymal stromal cells. The objective of this work was to evaluate the antifibrotic properties of these factors on the in vitro endometrial fibrosis model. Serum menstrual and peripheral blood were obtained from a healthy donor in one day. Mesenchymal stromal cells of the endometrium were also isolated from menstrual blood. Simulation of endometrial fibrosis in vitro was carried out by differentiation of endometrial mesenchymal stromal cells into myofibroblasts under the action of TGF-ß1 (5 ng/ml). Evaluation of the effectiveness of the menstrual blood serum antifibrotic effect on the endometrial mesenchymal stromal cells and myofibroblasts derived from them was carried out by analyzing the expression of а-smooth muscle actin by immunofluorescence. Serum of peripheral blood with equal protein concentration was used as a control. Menstrual blood serum reduces the number of stress-fibrils positive for а-smooth muscle actin (a marker of myofibroblasts), both in the culture of endometrial mesenchymal stromal cells, and in in vitro modeling of endometrial fibrosis using TGF-ß1. These results indicate the presence of soluble factors in the serum of menstrual blood with antifibrotic properties. Perhaps their identification will explain the mechanisms of endometrial healing not accompanied by fibrosis. In addition, it can help to identify the causes of fibrosis of the uterine lining in gynecological diseases and develop effective methods for their treatment.

Inflammatory cytokine profile of co‑cultivated primary cells from the endometrium of women with and without endometriosis.

Endometriosis is a chronic gynecological disorder defined as the presence of endometrial tissue within extra-uterine sites. The primary symptoms are infertility and chronic pain. The inflammatory environment and aberrant immune responses in women with endometriosis may be directly associated with the initiation and progression of endometriotic lesions. In the present study, the secretion of inflammatory cytokines was evaluated in cultures of primary endometrial cells (ECs) isolated from the endometrium of women with and without endometriosis. The presence of endometriotic cells leads to alterations in the secretory profile of healthy ECs. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was significantly increased in endometriotic and co-cultured cells compared with healthy ECs. IL-6 expression was strongly correlated with IL-8 expression in endometriotic cells. IL-1β expression was increased on day 10 of co-culture to 48.30 pg/ml and may be associated with the long-term co-culture, rather than IL-6 and IL-8 expression. IL-6 expression was strongly correlated with cell number, whereas IL-8 expression was moderately correlated with cell number. Additionally, it was observed that co-cultured cells exhibited a different population of cells, with expression of the mesenchymal stem cell marker cell surface glycoprotein MUC18, indicating a putative role of endometrial mesenchymal stem cells in the secretion of cytokines and disease development. These results indicate a predominant role of primary endometriotic cells in the secretion of cytokines, which contributes to the disrupted peritoneal and endometrial environment observed in the women with endometriosis.

Heterogeneous nuclear ribonucleoprotein C1 may control miR-30d levels in endometrial exosomes affecting early embryo implantation.

Is there a specific mechanism to load the microRNA (miRNA), hsa-miR-30d, into exosomes to facilitate maternal communication with preimplantation embryos? The heterogeneous nuclear ribonucleoprotein C1 (hnRNPC1) is involved in the internalization of endometrial miR-30d into exosomes to prepare for its subsequent incorporation into trophectoderm cells. Our group previously described a novel cell-to-cell communication mechanism involving the delivery of endometrial miRNAs from the maternal endometrium to the trophectoderm cells of preimplantation embryos. Specifically, human endometrial miR-30d is taken up by murine blastocysts causing the overexpression of certain genes involved in embryonic adhesion (Itb3, Itga7 and Cdh5) increasing embryo adhesion rates. Transfer of maternal miR-30d to preimplantation embryos was confirmed by co-culture of wild-type (WT) and miR-30d knockout (KO) murine embryos with primary cultures of human endometrial epithelial cells (hEECs) in which mir-30d was labeled with specific Molecular Beacon (MB) or SmartFlare probes. Potential molecules responsible for the miR-30d loading into exosomes were purified by pull-down analysis with a biotinylated form of miR-30d on protein lysates from human endometrial exosomes, identified using mass spectrometry and assessed by flow cytometry, western blotting and co-localization studies. The role of hnRNPC1 in the miR-30d loading and transportation was interrogated by quantification of this miRNA in exosomes isolated from endometrial cells in which hnRNPC1 was transiently silenced using small interference RNA. Finally, the transfer of miR-30d to WT and KO embryos was assessed upon co-culture with sihnRNPC1 transfected cells. Murine embryos from miR-30d WT and KO mice, (strain MirC26tm1Mtm/Mmjax), were obtained by oviduct flushing of superovulated females. Endometrial Exosomes were purified by ultracentrifugation of supernatants from primary cultures of hEECs or Ishikawa cells. MB and Smartflare miR-30d probes were detected by confocal and/or transmission electron microscopy (TEM). hEECs and exosomes derived from them were subjected to pull-down with a biotinylated form of miR-30d. Captured proteins were identified by mass spectrometry (MS/MS). Western blotting was performed to detect hnRNPC1 and CYR61 in whole lysates, subcellular fractions and secreted vesicles from hEECs. Co-localization studies of the selected proteins with the exosomal marker CD63 were performed. FACS analysis was carried out to determine the presence of hnRNPC1 inside exosomes. Silencing of hnRNPC1 was conducted in the Ishikawa Cell Line with the Smart Pool Accell HNRNPC siRNA at a final concentration of 50 nM. RT-qPCRs were done to determine the messenger levels of miR-30d in cells and exosomes. Co-cultures of WT and KO embryos were established with Ishikawa cells double-transfected with sihnRPNC1 and MB probes. MS/MS analysis allowed us to identify hnRNPC1 as a possible protein to influence miR-30d loading into exosomes. Co-localization studies of hnRNPC1 with CD63 and FACS analyses suggested the presence of hnRNPC1 inside exosomes. Silencing of hnRNPC1 in Ishikawa cells resulted in a sharp decrease of the levels of miR-30d in both epithelial-like cells (P = 0.0001) and exosomes (P = 0.0152), suggesting its potential role in miR-30d biogenesis and transfer. Co-culture assays of miR-30d KO embryos with sihnRNPC1 hEECs revealed a decrease in embryo-miR-30d acquisition during the adhesion and invasion stages. In turn, transient silencing of hnRNPC1 results in a significant decrease of blastocyst adhesion compared to mock transfection conditions using Block-it, in both WT [Mean ± SD; 67 ± 10.0% vs. 38 ± 8.5%(P = 0.0006)] and miR-30d KO embryos [Mean ± SD; 50 ± 11.5% vs. 26 ± 8.8% (P = 0.0029) (n = 2); 14 embryos transferred per condition tested]. MS/MS data are available via ProteomeXchange with identifier PXD008773. The Ishikawa Cell Line was used as a model of hEECs in silencing experiments due to the low survival rates of primary hEECs after transfection. The data show that hnRNPC1 may be involved in the internalization of miR-30d inside exosomes. The decreased rates of embryo adhesion in endometrial epithelial-like cells transiently silenced with sihnRNPC1evidence that hnRNPC1 could be an important player in the maternal-embryo communication established in the early stages of implantation. This work was supported by the Miguel Servet Program Type I of Instituto de Salud Carlos III [CP13/00038]; FIS project [PI14/00545] to F.V.; the 'Atracció de Talent' Program from VLC-CAMPUS [UV-INV-PREDOC14-178329 to NB]; a Torres-Quevedo grant (PTQ-13-06133) by the Spanish Ministry of Economy and Competitiveness to IM and MINECO/FEDER Grant [SAF2015-67154-R] to C.S. The authors declare there is no conflict of interest.

Comparison of CD9 & CD146 markers in endometrial stromal cells of fertile & infertile females.

Background & objectives:CD9 and CD146 are important adhesion molecules that play a role in the implantation of an embryo. This study was undertaken to correlate the expression of these markers in fertile and infertile women's endometrial stromal cells.Methods:Human endometrial stromal cell culture from endometrial biopsies of fertile (n=50) and infertile females (n=50) was performed and primary cell lines were established. Expression of CD9 and CD146 was studied for all the 100 cell lines with the help of flow cytometry. Gene expression of CD9 and CD146 was performed by real-time polymerase chain reaction.Results:There was a significant difference in endometrial stromal cells of fertile and infertile females. Flow cytometric results revealed significantly lower expression of CD9 (P=0.0126) and CD146 (P=0.0006) in the infertile endometrial stromal cells as compared to fertile endometrial stromal cells. These results were comparable with real-time data.Interpretation & conclusions:This study showed that endometrial stromal cells from infertile females had lower expression of adhesion molecules, CD9 and CD146. Our findings suggest that CD9 and CD146 may have a role in infertility. Infertile female's endometrial stromal cells have decreased expression of CD9 and CD146 which can be the cause of infertility related to implantation failure.

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.

Antioxidant ameliorating effects against H2O2-induced cytotoxicity in primary endometrial cells

Oxidative stress and a disrupted antioxidant system are involved in a variety of pregnancy complications. In the present study, the role of vitamin E (Vit E) and folate as radical scavengers on the GSH homeostasis in stress oxidative induced in rat endometrial cells was investigated. Primary endometrial stromal cell cultures treated with 50 and 200 µM of H2O2 and evaluated the cytoprotective effects of Vit E (5 µM) and folate (0.01 µM) in H2O2-treated cells for 24 h. Following the exposure of endometrial cells to H2O2 alone and in the presence of Vit E and/or folate, cell survival, glutathione peroxidase (GPx) and glutathione reductase activities and the level of reduced glutathione (GSH) were measured. Cell adhesions comprise of cell attachment and spreading on collagen were determined. Flow cytometric analysis using annexin V was used to measure apoptosis. H2O2 treatment showed a marked decrease in cell viability, GPx and GR activities and the level of GSH. Although Vit E or folate had some protective effect, combination therapy with Vit E and folate attenuated all the changes due to H2O2 toxicity. An increasing number of alive cells was showed in the cells exposed to H2O2 (50 µM) accompanied by co-treatment with Vit E and folic acid. The present findings indicate that co-administration of Vit E and folate before and during pregnancy may maintain a viable pregnancy and contribute to its clinical efficacy for the treatment of some idiopathic infertility.

Profile of MicroRNA Expression in Endometrial Cell during In Vitro Culture According to Progesterone Concentration.

Artificial uterus using endometrium implant can be a novel treatment strategy for infertile women with refractory endometrial dysfunction. At early pregnancy, the function of uterine endometrial cells for the communication between the conceptus of pre-implantation period and maternal reproductive system is essential. MicroRNA (miR) expression profile of endometrial cells according to progesterone, a crucial pregnancy-maintaining hormone, provides important data for in vitro endometrial cell culture strategy that is useful for engineering artificial uteri using endometrial implants. The present study aimed to evaluate the miR expression profile of in vitro cultured endometrial cells under hormonal milieu mimicking early pregnancy period in terms of progesterone concentration. We cultured murine uterine endometrial cells, human uterine endometrial carcinoma cells, and immortalized human uterine endometrial cells using different progesterone concentrations, and analyzed the expression of miRs critical for early pregnancy. The expression of miR-20a, -21, -196a, -199a, and -200a was differently regulated according to progesterone concentration in different endometrial cell lines. The analysis of candidate target genes showed that the expression of phosphatase and tensin homolog, mucin 1 (MUC1), progesterone receptor, transforming growth factor β receptor II, matrix metallopeptidase-9 was up-regulated by progesterone treatment in mouse and human endometrial cell lines. These results indicate that physiological concentration range (10-7 and 10-9 M) of progesterone affect the survival and target gene expression via modulating miR expression. Taken together, progesterone can be a crucial factor in regulating miR expression on in vitro cultured endometrial cells.

Proangiogenic Activity of Endometrial Epithelial and Stromal Cells in Response to Estradiol in Gelatin Hydrogels.

Biomaterial vascularization remains a major focus in the field of tissue engineering. Biomaterial culture of endometrial cells is described as a platform to inform the design of proangiogenic biomaterials. The endometrium undergoes rapid growth and shedding of dense vascular networks during each menstrual cycle mediated via estradiol and progesterone in vivo. Cocultures of endometrial epithelial and stromal cells encapsulated within a methacrylamide-functionalized gelatin hydrogel are employed. It is reported that proangiogenic gene expression profiles and vascular endothelial growth factor production are hormone dependent in endometrial epithelial cells, but that hormone signals have no effect on human telomerase reverse transcriptase (hTERT)-immortalized endometrial stromal cells. This study subsequently examines whether the magnitude of epithelial cell response is sufficient to induce changes in human umbilical vein endothelial cell network formation. Incorporation of endometrial stromal cells improves vessel formation, but co-culture with endometrial epithelial cells leads to a decrease in vascular formation, suggesting the need for stratified cocultures of endometrial epithelial and stromal cells with endothelial cells. Given the transience of hormonal signals within 3D biomaterials, the inclusion of sex hormone binding globulin (SHBG) to alter the bioavailability of estradiol within the hydrogel is reported, demonstrating a strategy to reduce diffusive losses via SHBG-mediated estradiol sequestration.

Inhibitory effect of curcumin in human endometriosis endometrial cells via downregulation of vascular endothelial growth factor.

Endometriosis, which affects up to 10% of women of reproductive age, is defined as endometrial-like gland and stroma tissue growths outside the uterine cavity. Despite increasing research efforts, there are no current effective treatment methods for this disease, therefore investigations for therapeutic strategies are of primary concern. In preliminary work, the authors demonstrated that curcumin inhibits endometriosis invivo. The present invitro study aimed to investigate the association between endometriotic stromal cells and curcumin and to clarify the underlying mechanism of action. A total of 14patients with endometriosis were enrolled in the present study. The purity of endometrial stromal cell cultures was proven by standard immunofluorescent staining of vimentin. The cell proliferation and curcumin effects on endometrial stromal cells were assessed by the MTT assay and Hematoxylin and Eosin staining. For cell cycle analysis, phase distribution was detected by flow cytometry. Vascular endothelial growth factor (VEGF) protein expression was examined using immunohistochemistry staining. Apoptosis was assessed using Annexin V‑fluorescein isothiocyanate staining. The results indicated that the treatment of curcumin decreased human ectopic and eutopic stromal cell growth. Following treatment with curcumin, human endometriotic stromal cells demonstrated an increased percentage of G1‑phase cells and decreased percentages of S‑phase cells, particularly in the group treated with 50µmol/l curcumin. Treatment with curcumin additionally decreased expression of VEGF. The data provide evidence that curcumin reduces cell survival in human endometriotic stromal cells, and this may be mediated via downregulation of the VEGF signaling pathway.

Effect of Taoren Quyu Decoction on human endometrial cells and its anti-endometriosis activity in rats.

To study the effect of Taoren Quyu Decoction (TQD) on endometrial cells in patients with endometriosis (EMs) and EMs in rats. A total of 60 female Wistar rats were randomly divided into 4 groups, namely, normal group, model group, positive group and TQD group, each group having 15 rats. Except the normal group, EMs model was established in the other three groups by transplanting the rat autologous endometrium. After 4 weeks of intragastric administration, blood, eutopic and ectopic endometrial tissues of rats in each group were collected to detect the serum levels of estrogen (E2), cancer antigen 125 (CA125), endometrial antibody (EMAb), and expressions of microvessel density (MVD), vascular endothelial growth factor (VEGF) and angiopoietin (Ang-2). The volume of endometriosis cyst was determined simultaneously. For the invitro culture of human endometrial cells, 4 groups, namely, normal group, model group, positive group and TQD group were used. The positive group and TQD group were treated with danazol and TQD respectively. Then 24h after the treatment, the expressions of survivin and tumor suppressor gene (p53) of each group were detected. The volumes of the endometriosis cysts in the positive group and the TQD group were significantly reduced compared with the model group (P<0.05). The serum levels of E2, CA125 and EMAb, and the expressions of MVD, VEGF and Ang-2 in the model group were significantly increased compared with the normal group (P<0.05); while they were all significantly reduced in the positive group and TQD group (P<0.05). Compared with the normal group, the expression of survivin in the model group was significantly up-regulated (P<0.05), and expression of p53 was significantly reduced (P<0.05); compared with the model group, the expressions of survivin in the positive and TQD groups were significantly decreased (P<0.05), and expression of p53 was significantly up-regulated (P<0.05). The difference between positive group and TQD group was not statistically significant (P>0.05). TQD has a significant anti-EMs effect, and its mechanism of action may be related to anti-angiogenesis and promoting apoptosis of ectopic endometrial cell.

Hepatoma-derived growth factor: Protein quantification in uterine fluid, gene expression in endometrial-cell culture and effects on in vitro embryo development, pregnancy and birth

Hepatoma-derived growth factor (HDGF) is present in the endometrium of cows and other mammals. Recombinant HDGF (rHDGF) improves bovine blastocyst development in vitro. However, specific culture conditions and essential aspects of HDGF uterine physiology are yet unknown. In this work we quantified total HDGF protein in uterine fluid (UF) by multiple reaction monitoring (MRM), and analyzed effects of rHDGF on specific embryonic stages with Day-6 bovine embryos cultured in vitro with and without BSA, and on pregnancy viability and calf phenotypes after embryo transfer to recipients. In addition, mRNA abundance of HDGF in endometrial cells co-cultured with one male or one female embryo was quantified. In the presence of BSA, rHDGF had no effect on blastocyst development; however, in BSA-free culture rHDGF mainly promoted development of early blastocysts in contrast with morulae. As the presence of HDGF contained in commercial BSA replacements was suspected, western blot confirmed HDGF identification in BSA both with and without fatty acids. Total HDGF quantified by MRM tended to increase in UF without vs. UF with embryos (P = 0.083). Pregnancy and birth rates, birth weight and calf measurements did not differ between embryos cultured with rHDGF and controls without rHDGF. However, HDGF abundance in cultured epithelial, endometrial cells tended to increase (P < 0.08) in culture with one male embryo. rHDGF acts selectively on specific embryonic stages, but care should be taken with specific macromolecular supplements in culture. The endometrial expression of HDGF can be regulated by the embryonic sex. The use of rHDGF is compatible with pregnancy and birth of normal calves.

Sexual Dimorphism of miRNAs Secreted by Bovine In vitro-produced Embryos.

Sexual dimorphism of bovine blastocysts has previously been observed through differences in development, cell death, metabolism, telomere length, DNA methylation, and transcriptomics. However, dimorphism in the secretion of miRNAs to culture media has not yet been evaluated. The objectives of this study were to determine if sex-specific blastocyst miRNA secretion occurs and to further investigate the role these miRNAs may have in the interaction between a blastocyst and the maternal environment. In vitro embryo culture was performed and media from male and female blastocysts was collected into sex-specific pools. Profiling of 68 miRNAs revealed a total of eight miRNAs that were differentially expressed between female and male-conditioned media. Validation by qPCR confirmed higher expression of miR-22 (P < 0.05), miR-122 (P < 0.05), and miR-320a (P < 0.05) in female media for three additional biological replicates. To examine the potential roles of secreted miRNAs to the media in communication with the maternal environment, miR-22, miR-122, and miR-320a were each supplemented to four replicates of primary bovine endometrial epithelial cell culture. Uptake of miR-122 (P < 0.05) and miR-320a (P < 0.05) was detected, and a trend of uptake was detected for miR-22 (P > 0.05). Further, expression of the progesterone receptor transcript, a predicted target of all three miRNAs, was found to be upregulated in the cells following supplementation of miR-122 (P < 0.05) and miR-320a (P < 0.05), and a trend upregulation of the transcript was observed following miR-22 (P > 0.05) supplementation. This work demonstrates that male and female conceptuses are able to differentially secrete miRNAs at the blastocyst stage and that these miRNAs have the ability to induce a transcriptomic response when applied to maternal cells. This knowledge builds on the known dimorphic differences in conceptuses at the blastocyst stage and demonstrates a role for blastocyst-secreted miRNAs in cell–cell communication.

Chronic endometritis modifies decidualization in human endometrial stromal cells

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

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for &lt;em&gt;In Vitro&lt;/em&gt; Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Expression of Inflammatory and Neurogenic Mediators in Adenomyosis.

Adenomyosis is a uterine disorder characterized by dysmenorrhea, dyspareunia, abnormal uterine bleeding, and infertility. Pathogenesis indicates that endometrial cells invade and proliferate within myometrium, and inflammatory mediators participate to the intense painful symptoms. The aim of the present study was to investigate the messenger RNA (mRNA) and protein expression of inflammatory (interleukin 1β [IL-1β], corticotropin-releasing hormone [CRH], urocortin [Ucn]) and neurogenic (nerve growth factors [NGFs], synaptophysin [SYN], microtubule-associated protein 2 [MAP2]) factors in adenomyotic nodules. This prospective study enrolled 16 women, 8 women with nodular adenomyosis and 8 control women undergoing to hysterectomy. Specimens from adenomyotic nodules and eutopic endometrium were collected after surgery. Endometrial tissue was also obtained from the control group and also used for preparing primary culture of human endometrial stromal cells (HESCs). Messenger RNA expression of inflammatory mediators (IL-1β, CRH, and Ucn) and neurogenic factors (NGF, SYN, and MAP2) was analyzed by real-time polymerase chain reaction. The in vitro effects of CRH/Ucn on NGF or SYN mRNA expression were also investigated. Adenomyotic nodules highly expressed IL-1β, CRH, and Ucn mRNAs, as well as NGF, SYN, and MAP2 mRNAs ( P < .001 vs eutopic endometrium and control). Endometrium of women with adenomyosis showed high expression of IL-1β and CRH ( P < .001 vs control). Protein expression of CRH, NGF, and SYN in adenomyotic nodules was confirmed by immunohistochemical and immunofluorescence analyses. Urocortin increased NGF mRNA expression in cultured HESCs. The present study showed that adenomyotic nodules are novel site of expression of inflammatory and neurogenic factors, probably involved in the pathogenesis of adenomyosis.

Activation of α7-nicotinic acetylcholine receptors causes secretion of matrix metalloproteinases-9 in the human endometrium

α7-Nicotinic acetylcholine receptors (α7nAChR) have been discovered and studied in the human endometrium for the first time. The expression of these receptors at the level of mRNA has been shown in the endometrial tissue by polymerase chain reaction and at the protein level by Western blot. It has been found that the addition of carbachol, an agonist of acetylcholine receptors, or PHA 543613, a selective activator of α7nAChR, to the primary culture of endometrial cells leads to regulated pulse secretion of matrix metalloproteinase- 9.

The functional expression of transient receptor potential channels in the mouse endometrium.

Does mouse endometrial epithelial cells and stromal cells have a similar transient receptor potential (TRP)-channel expression profile and to that found in the human endometrium? Mouse endometrial epithelial and stromal cells have a distinct TRP channel expression profile analogous to what has been found in human endometrium, and hence suggests the mouse a good model to investigate the role of TRP channels in reproduction. An optimal intercellular communication between epithelial and stromal endometrial cells is crucial for successful reproduction. Members of the TRP family were recently described in the human endometrial stroma; however their functional expression in murine endometrium remains unspecified. Furthermore, epithelial and stromal cells have distinct functions in the reproductive process, implying the possibility for a different expression profile. However, knowledge about the functional expression pattern of TRP channels in either epithelial or stromal cells is not available. In this study, the expression pattern of TRP channels in the murine (C57BL/6 J strain) endometrium was investigated and compared to the human expression pattern. Therefore, expression was examined in uterine tissue isolated during the natural estrous cycle (n = 16) or during an induced menstrual cycle using the menstruating mouse model (n = 28). Next, the functional expression of TRP channels was assessed separately in endometrial epithelial and stromal cell populations. Quantitative RT-PCR was used to evaluate the relative mRNA expression of TRP channels in murine uterine tissue and cells. To further assess the functional expression in epithelial or stromal cells, primary endometrial cell cultures and Fura2-based calcium-microfluorimetry experiments were performed. The expression pattern of TRP channels during the natural estrous cycle or the induced menstrual cycle is analog to what has been shown in human samples. Furthermore, a very distinct expression pattern was observed in epithelial cells compared to stromal cells. Expression of TRPV4, TRPV6 and TRPM6 was significantly higher in epithelial cells whereas TRPV2, TRPC1/4 and TRPC6 were almost exclusively expressed in stromal cells. N/A. Although relevant mRNA levels are detected for TRPV6 and TRPM6, and TRPM4, lack of selective, available pharmacology restricted functional analysis of these ion channels. Successful reproduction, and more specifically embryo implantation, is a dynamic developmental process that integrates many signaling molecules into a precisely orchestrated program. Here, we describe the expression pattern of TRP channels in mouse endometrium that is similar to human tissue and their restricted functionality in either stromal cells or epithelial cells, suggesting a role in the epithelial-stromal crosstalk. These results will be very helpful to identify key players involved in the signaling cascades required for successful embryo implantation. In addition, these results illustrate that mouse endometrium is a valid representative for human endometrium to investigate TRP channels in the field of reproduction. The Research Foundation-Flanders (G.0856.13 N to J.V.); the Research Council of the Katholieke Universiteit Leuven (OT/13/113 to J.V. and PF-TRPLe to T.V.); the Planckaert-De Waele fund (to J.V.); Fonds Wetenschappelijk Onderzoek Belgium (to K.D.C. and A.H.). None of the authors have a conflict of interest.

Expression and localization of ARTEMIN in the bovine uterus and embryos

Artemin a member of the glial cell line-derived neurotrophic factor (GDNF) family is present in mice and human preimplantation embryos, and reproductive tract, during early pregnancy promoting embryo development in vitro. The presence of artemin in cattle embryos and reproductive tract, however, is unknown. In the present work we identified for first time artemin in bovine uterine fluid (UF) (Western blot), endometrium (RT-PCR, Western blot and immunohistochemistry) and embryos (RT-PCR and immunohistochemistry) during early preimplantation development. In addition, GFRalpha3, a component of the artemin receptor was localized in blastocysts produced in vitro. Individually developing embryos released ARTEMIN in culture medium and triggered ARTEMIN mRNA down-regulation in epithelial cells from endometrial cell cultures. Our results suggest that ARTEMIN derived from early embryos and maternal reproductive tract may exert important roles during early development in cattle.

124 CHANGES IN GENE EXPRESSION FOLLOWING EXPOSURE OF BOVINE ENDOMETRIAL EPITHELIAL CELLS (bEEC) TO ESCHERICHIA COLI LPS; THEIR POSSIBLE EFFECT ON IMPLANTATION

Lipopolysaccharide (LPS) is a component of the outer membrane of gram-negative bacteria and is involved in postpartum uterine infection in cattle. Lipopolysaccharide causes inflammation of the endometrium and the activation of immune and pro-inflammatory pathways in uterine cells has been well documented. This study was performed to investigate the effects of LPS on epithelial cells from whole-genome information, and this abstract focuses on genes and pathways involved in the regulation of implantation. Following in vitro culture of bovine endometrial epithelial cells (bEEC), passage 4 epithelial cell samples from 3 cows were exposed to 0, 2, and 8 µg mL−1 LPS (Sigma L2630, Escherichia coli O111:B4, Sigma Chemical Co., St. Louis, MO, USA) for 24 h. At time 0 and at 24 h for each LPS dosage, RNA was extracted by using the All prep DNA/RNA Universal kit (Qiagen, Valencia, CA, USA). Samples were analysed by RNA sequencing performed in the SciLife Laboratory in Uppsala. Differentially expressed genes (DEG) were identified by using Ensemble genes as a reference. No DEG were found between 2 and 8 µg mL−1 LPS-treated samples and at 24 h 2035 DEG were identified (Benjamini-Hochberg adjusted P-value &lt; 0.05) between controls and samples treated with 2 µg mL−1 LPS. Gene ontology analysis did show that DEG were associated to immune response (up), response to stress and external stimuli (up), catalytic activity (up), cell cycle, anatomical structures especially cell membrane, and adhesion (down) pathways. In the latest, numerous specific genes in relation with implantation were highly deregulated. This includes down-regulation of 8 members of the cadherin superfamily. On the contrary, 4 members of the mucin family were strongly up-regulated by LPS (MUC1, MUC13, MUC16, F1MUC1). Molecules such as plakophilins and desmogleins involved in desmosomes, in tight junctions, and in the control of cell adhesion were also deregulated. Specific changes occurred in immune response related with implantation [strong up-regulation of the immunoglobulin superfamily members such ICAM1 (or CD54) and down-regulation of ALCAM]. A set of 10 molecules belonging to the family of integrins and their binding partners were also deregulated [for instance, down-regulation of osteopontin (SPP1)]. In addition, LPS deregulated a large set of genes binding the above molecules (such as galectins LGALS1, S3, S9) and more than 20 transcripts coding for cytokines and their receptors. A large series of interferon-induced genes (IFITS) and genes coding for interferon-induced trans membrane proteins (IFITM) were highly up-regulated by LPS. This may be of functional importance due to the fact that all those genes are normally up-regulated by interferon tau from embryonic origin. The above results show that the function of endometrial epithelial cells is profoundly affected by LPS and that most of the key signals involved in implantation are deregulated. It is likely that these LPS-induced changes strongly perturb lately endometrial responsiveness to embryos at the time of implantation. Research was done with the financial support of FP7 project “Prolific” and RMUTSV (Thailand).