Endometrial Stromal Cells Of Women Research Articles

P-517 Downregulated INHBB in endometrial tissue of recurrent implantation failure patients impeded decidualization through the ADCY1/cAMP signalling pathway

Abstract Study question To identify the mechanism of Inhibin Subunit Beta B (INHBB) in the regulation of human endometrial stromal cells (HESCs) decidualization in recurrent implantation failure (RIF). Summary answer INHBB was decreased in endometrial stromal cells of women with RIF, which attenuated decidualization in vitro by suppressing ADCY1-induced cAMP production and cAMP-mediated signalling. What is known already INHBB encodes a preprotein subunit of inhibin/ activin, functional cytokines belonging to the transforming growth factor-β (TGF-β) family. INHBB mRNAs were expressed in human decidualized endometrium from the first trimester of pregnancy and increased as the gestation progressed. Furthermore, INHBB expression increases in the mouse uterus in areas undergoing decidualization. These studies indicated that INHBB might play an essential role in endometrial decidualization. Study design, size, duration The Medical Ethics Committee of Nanjing Drum Tower Hospital approved this study (No. 2013-081-01). Written informed consent was obtained from each participant. Between January 2020 and November 2021, 28 infertile and 18 fertile control women aged 20-40 years with normal and regular menstrual cycles (25-35 days) and no history of steroid hormone medication in the last 3 months were included in the study. Participants/materials, setting, methods RNA-seq was conducted to identify the differentially expressed genes in the endometria from control and RIF patients. RT-qPCR, WB, and immunohistochemistry were performed to analyse the expression levels of INHBB in decidualised HESCs. RT-qPCR and immunofluorescence were used to detect changes in decidualization after knockdown INHBB. Then, RNA-seq was used to dig out the mechanism of INHBB regulating decidualisation. Main results and the role of chance Our results showed significantly reduced expression of INHBB in endometrial stromal cells of women with RIF. In addition, INHBB was increased in the endometrium of the secretory phase and significantly induced in in-vitro decidualization of HESCs. Notably, with RNA-seq and siRNA-mediated knockdown approaches, we demonstrated that the INHBB-ADCY1-mediated cAMP signalling pathway regulates the reduction of decidualization. We found a positive association between the expression of INHBB and ADCY1 in endometria with RIF (R2= 0.3785, P = 0.0005). Limitations, reasons for caution Our results indicated that INHBB regulates cAMP level by inducing the expression of ADCY1, while the regulation mechanism between INHBB and ADCY1 in endometrial decidualization requires further exploration. Wider implications of the findings The decline of INHBB in HESCs suppressed ADCY1-induced cAMP production and cAMP-mediated signalling, which attenuated decidualization in RIF patients, indicating that INHBB is an essential component in the decidualization process. Trial registration number not applicable

Downregulated INHBB in endometrial tissue of recurrent implantation failure patients impeded decidualization through the ADCY1/cAMP signalling pathway

PurposeThis study aims to identify the mechanism of Inhibin Subunit Beta B (INHBB), a member of the transforming growth factor-β (TGF-β) family involved in the regulation of human endometrial stromal cells (HESCs) decidualization in recurrent implantation failure (RIF).MethodsRNA-seq was conducted to identify the differentially expressed genes in the endometria from control and RIF patients. RT-qPCR, WB, and immunohistochemistry were performed to analyse the expression levels of INHBB in endometrium and decidualised HESCs. RT-qPCR and immunofluorescence were used to detect changes in the decidual marker genes and cytoskeleton after knockdown INHBB. Then, RNA-seq was used to dig out the mechanism of INHBB regulating decidualization. The cAMP analogue (forskolin) and si-INHBB were used to investigate the involvement of INHBB in the cAMP signalling pathway. The correlation of INHBB and ADCY expression was analysed by Pearson’s correlation analysis.ResultsOur results showed significantly reduced expression of INHBB in endometrial stromal cells of women with RIF. In addition, INHBB was increased in the endometrium of the secretory phase and significantly induced in in-vitro decidualization of HESCs. Notably, with RNA-seq and siRNA-mediated knockdown approaches, we demonstrated that the INHBB-ADCY1-mediated cAMP signalling pathway regulates the reduction of decidualization. We found a positive association between the expression of INHBB and ADCY1 in endometria with RIF (R2 = 0.3785, P = 0.0005).ConclusionsThe decline of INHBB in HESCs suppressed ADCY1-induced cAMP production and cAMP-mediated signalling, which attenuated decidualization in RIF patients, indicating that INHBB is an essential component in the decidualization process.

The effects of resveratrol on the expression of VEGF, TGF-\u03b2, and MMP-9 in endometrial stromal cells of women with endometriosis

Resveratrol is a phytochemical with anti-angiogenic, anti-inflammatory, and antioxidant properties. The present study has evaluated the effect of resveratrol on the expression of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and matrix metalloproteinase-9 (MMP-9) as factors related to endometriosis progression. Thirteen eutopic (EuESCs) and 8 ectopic (EESCs) endometrial stromal cells from women with endometriosis and 11 control endometrial stromal cells (CESCs) were treated with resveratrol (100 µM) for 6, 24 and 48 h. The gene and protein expression levels of VEGF, TGF-β, and MMP-9 were measured using real-time PCR and ELISA methods, respectively. Results showed that the basal gene and protein expression of VEGF and MMP-9 were higher in EESCs compared to EuESCs and CESCs (P < 0.01 to < 0.001 and P < 0.05 to < 0.01 respectively). Also, resveratrol treatment decreased the gene and protein expression of VEGF and MMP-9 in EuESCs, EESCs and CESCs (P < 0.05 to < 0.01 and P < 0.05 to < 0.01 respectively) and gene and protein expression of TGF-β in EESCs and EuESCs (P < 0.05 to < 0.01). The effect of resveratrol in reduction of VEGF gene expression was statistically more noticeable in EESCs compared to EuESCs and CESCs (P < 0.05). According to the findings, resveratrol may ameliorate endometriosis progression through reducing the expression of VEGF, TGF-β, and MMP-9 in endometrial stromal cells (ESCs).

Differential expression of microRNA in exosomes derived from endometrial stromal cells of women with endometriosis-associated infertility.

What is the expression pattern of microRNA (miRNA) in exosomes isolated from eutopic endometrial stromal cells (EuESC) of women with endometriosis-associated infertility? Small RNA sequencing was conducted in exosomes isolated from EuESC of women with endometriosis-associated infertility (n = 3) and normal endometrial stromal cells (NESC) of fertile women without endometriosis (n = 3). The differentially expressed miRNA in exosomes derived from EuESC and NESC were identified. The functions of the differentially expressed miRNA were analysed by gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Small RNA sequencing showed that the percentages of exosomal miRNA in the total small RNA isolated from EuESC and NESC were not significantly different (P = 0.7804). A total of 49 differentially expressed miRNA (fold change >1.5 and P<0.05) were identified, including 26 up-regulated and 23 down-regulated in EuESC exosomes as compared with NESC exosomes. Functional analysis revealed that 12 miRNA were predicted to target homeobox A10 (HOXA10) and/or the leukaemia inhibitory factor (LIF) 3' untranslated region (UTR). Both HOXA10 and LIF mRNA expression levels were significantly decreased in EuESC compared with NESC (P = 0.0222 and 0.0395, respectively). In addition, the predicated target genes of these differentially expressed exosomal miRNA were significantly (P<0.05) enriched in 76 pathways, including the MAPK and Wnt signalling pathways. The differential expression patterns of exosomal miRNA were identified. Many exosomal miRNA may be involved in regulating the endometrial receptivity of women with endometriosis-associated infertility.

Increased expression of YAP (yes-associated protein) is associated with the decreased cell autophagy in the eutopic endometrial stromal cells of women with endometriosis

Increased expression of YAP (yes-associated protein) is associated with the decreased cell autophagy in the eutopic endometrial stromal cells of women with endometriosis

Increased expression of YAP is associated with decreased cell autophagy in the eutopic endometrial stromal cells of endometriosis

Basic research has shown that signal pathways play significant roles in the development and progression of endometriosis (EMS). We first reported that the Hippo-YAP (Yes-associated protein) pathway promotes cell proliferation and anti-apoptosis in endometrial stromal cells (ESCs) of EMS. Cell autophagy has been found to be involved in the endometrial regulation and the pathophysiology of EMS. We speculated that there may be an elaborate dialogue between Hippo-YAP and autophagy pathway in EMS. To explore this, we performed molecular biology experiments to investigate the expressions of YAP pathway and cell autophagy markers (mTOR, LC-3) in ESCs of women with or without EMS and detected the protein levels of autophagy markers after verteporfin and rapamycin treatments and the transfection with YAP-knockdown vector in the eutopic ESCs, respectively. We found that the mRNAs of YAP and mTOR were increased in the eutopic ESCs compared with controls, but no statistically difference, while the protein levels were significantly increased in the eutopic ESCs. Conversely, the ratio of the autophagy marker protein LC3-II/LC3-I was significantly decreased in the eutopic ESCs compared with controls. Moreover, verteporfin treatment interfered with the YAP function, but it had no effect on mTOR expression and cell autophagy level. Rapamycin treatment and YAP knockdown in the eutopic ESCs both inhibited the expression of YAP and increased the ratio of LC3-II/LC3-I significantly. These results demonstrate that the decreased cell autophagy level is associated with the increased expression of YAP and YAP may participate in the mTOR-autophagy pathway in the eutopic ESCs of endometriosis.

SAT-198 Increased Expression of YAP Is Associated with the Decreased Cell Autophagy in the Eutopic Endometrial Stromal Cells of Women with Endometriosis

Endometriosis (EMS) is a debilitating gynecologic disease characterized by the presence of endometrial glands and stroma outside the uterus. Symptoms caused by this disease have serious implications for women's lives and health. Although it has been widely studied recent years, the exact pathogenesis of endometriosis is not fully clear. Basic researches have shown that the signal pathways play significant roles in the development and progression of EMS (1). We first reported the Hippo-YAP (Yes-associated protein) pathway promotes cell proliferation and anti-apoptosis in endometrial stromal cells (ESCs) of EMS (2). It is well known that autophagy with normal metabolic condition is important to sustain cell growth and function. It has been reported that cell autophagy involves in the pathogenesis of EMS and endometrial regulation through multi-level and multipath interference in EMS (3). Wilkinson et al. found that phosphorylation of LC3 by STK3/STK4, the mammalian homologs of Hippo which can antagonize YAP activity, is an essential step in the autophagy process (4). It hints that there may be an elaborate dialogue between Hippo-YAP and autophagy. To explore the role of YAP in the regulation of cell autophagy in the eutopic ESCs, we performed in vitro studies to investigate the expressions of YAP pathway and cell autophagy markers (mTOR, LC-3) in ESCs of women with or without EMS and detect the protein levels of autophagy markers in the eutopic ESCs after the transfection with YAP-knockdown vector and Verteporfin treatment, the inhibitor of YAP-TEAD complex, in ESCs, respectively. The mRNA levels of YAP and mTOR were all increased in the eutopic ECSs of women with EMS compared with controls (1.56 ± 0.37 vs. 1.04 ± 0.17; 0.49 ± 0.12 vs. 0.31 ± 0.08; P>0.05). Similarly, the protein levels of YAP (0.85 ± 0.13 vs. 0.35 ± 0.04; P<0.05) and mTOR (0.81 ± 0.03 vs. 0.37 ± 0.01; P<0.01) were significantly increased in the eutopic ECSs of women with EMS compared with controls. To the contrary, the ratio of the autophagy marker protein LC3-II/LC3-I (1.36 ± 0.06 vs. 2.17 ± 0.19; P<0.05) was significantly decreased in the eutopic ECSs of women with EMS compared with controls. Furthermore, YAP knockdown and Verteporfin treatment in the eutopic ESCs increased the level of cell autophagy with an increased ratio of the autophagy marker protein LC3-II/LC3-I (1.15 ± 0.05 vs. 0.87± 0.04; P<0.05). Therefore, our study demonstrates that the decreased cell autophagy level is associated with the increased expression of YAP in the eutopic ESCs and it may be negatively regulated by YAP. Reference: (1) Choi et al., Mol Hum Reprod. 2014, 20(4): 309-17. (2) Song et al., J Clin Endocrinol Metab. 2016, 101: 1552-61. (3) Mei et al., Hum Reprod. 2015, 30: 1677-89. (4) Wilkinson et al., Mol Cell. 2015; 57:55-68.

Endometrial stromal cell proteome mapping in repeated implantation failure and recurrent pregnancy loss cases and fertile women.

Are there proteomic differences between endometrial stromal cells of repeated implantation failure (RIF), recurrent pregnancy loss (RPL) and normal fertile women, and is there differential protein expression upon decidualization? This exploratory study investigated the proteome of in-vitro cultured endometrial stromal cells of women with RIF (n = 4), women with RPL (n = 3) and normal fertile women (n = 4), comparing day 0 with 5 days of decidualization. Total proteins extracted from cell lysates were analysed by high-definition mass spectrometry. Data analysis was performed using significance analysis of microarray in R (P<0.05; false discovery rate [FDR] 10%). In the RIF group, ANXA6, PSMC5 and FSCN1 were up-regulated (1.9-fold, 2.5-fold and 1.9-fold, respectively), whereas PBXIP1 was down-regulated (7.7-fold) upon decidualization. In the RPL group, RPS25 and ACADVL were down-regulated (1.9-fold and 2.4-fold, respectively; FDR 10%) between the non-decidualized and the decidualized samples. In the normal fertile group VIM and RPL23A were down-regulated (1.9-fold and 2.4-fold, respectively). Comparing ratios of expression of decidualized over non-decidualized samples in the different groups revealed six differentially expressed proteins: DUX4L2, CNPY4, PDE7A, CTSK, PCBP2 and PSMD4. Comparison of RPL versus normal fertile in the decidualized condition revealed serotransferrin to be differentially expressed. The changes in expression levels for serotransferrin, ANX6, ACDVL and VIM were confirmed by western blot. Results show a varying response of endometrial stromal cells in distinct clinical groups (RIF, RPL and normal fertile) upon in-vitro decidualization. Serotransferrin could serve as a marker for the aberrant decidualization process in RPL.

Decreased Notch pathway signaling in the endometrium of women with endometriosis impairs decidualization.

Endometriosis is a common gynecological disease affecting one in 10 women of reproductive age and is a major cause of pelvic pain and impaired fertility. Endometrial stromal cells of women with endometriosis exhibit a reduced response to in vitro decidualization. NOTCH1 is critical for decidualization of both mouse and human uterine stromal cells. This study aimed to determine whether decidualization failure in women with endometriosis is a consequence of impaired Notch signaling. We investigated expression levels of Notch signaling components in the endometrium of women and baboons with or without endometriosis. We identified NOTCH1-regulated genes during decidualization of human uterine fibroblast (HuF) cells by microarray and quantified their expression levels in in vitro-decidualized endometrial stromal cells isolated from women with or without endometriosis. Notch signaling receptors NOTCH1 and NOTCH4, ligands JAGGED2 and DLL4, as well as direct target genes HES5 and HEY1 were decreased in the eutopic endometrium of women and baboons with endometriosis. Notch signaling was decreased in stromal cells isolated from women with endometriosis, which was associated with impaired in vitro decidualization. Genes that were down-regulated by NOTCH1 silencing in decidualized HuF cells were also decreased in decidualized endometrial stromal cells of women with endometriosis. FOXO1 acts as a downstream target of Notch signaling and endometriosis is associated with decreased expression of NOTCH1-regulated, FOXO1-responsive genes during decidualization. Decreased Notch signaling is associated with endometriosis and contributes to impaired decidualization through the down-regulation of FOXO1.

CAPN 7 promotes the migration and invasion of human endometrial stromal cell by regulating matrix metalloproteinase 2 activity

BackgroundMatrix metalloproteinase 2 (MMP-2) has been reported to be an important regulator of cell migration and invasion through degradation of the extracellular matrix (ECM) in many diseases, such as cancer and endometriosis. Here, we found calcium-activated neutral protease 7 (CAPN 7) expression was markedly upregulated in the eutopic endometrium and endometrial stromal cells of women diagnosed with endometriosis. Our studies were carried out to detect the effects of CAPN 7 on human endometrial stromal cell (hESC) migration and invasion.MethodsWestern blotting and quantitative real-time PCR were used to detect the expression of CAPN 7 in endometriosis patients and normal fertile women. Scratch-wound-healing and invasion chamber assay were used to investigate the role of CAPN 7 in hESC migration and invasion. Western blotting, quantitative real-time PCR and zymography were carried out to detect the effect of CAPN 7 on the expressions and activity of MMP-2.ResultsCAPN 7 was markedly up-regulated in endometriosis, thereby promoting the migration and invasion of hESC. CAPN 7 overexpression led to increased expression of MMP-2 and tissue inhibitor of metalloproteinases 2 (TIMP-2); CAPN 7 knockdown reversed these changes. CAPN 7 increased MMP-2 activity by increasing the ratio of MMP-2 to TIMP-2. We also found that OA-Hy (an MMP-2 inhibitor) decreased the effects of CAPN 7 overexpression on hESC migration and invasion by approximately 50% and 55%, respectively. Additionally, a coimmunoprecipitation assay demonstrated that CAPN 7 interacted with activator protein 2α (AP-2α): an important transcription factor of MMP-2.ConclusionsCAPN 7 promotes hESC migration and invasion by increasing the activity of MMP-2 via an increased ratio of MMP-2 to TIMP-2.

Endometrial Stromal Cells of Women with Recurrent Miscarriage Fail to Discriminate between High- and Low-Quality Human Embryos

BackgroundThe aetiology of recurrent miscarriage (RM) remains largely unexplained. Women with RM have a shorter time to pregnancy interval than normally fertile women, which may be due to more frequent implantation of non-viable embryos. We hypothesized that human endometrial stromal cells (H-EnSCs) of women with RM discriminate less effectively between high-and low-quality human embryos and migrate more readily towards trophoblast spheroids than H-EnSCs of normally fertile women.Methodology/Principal FindingsMonolayers of decidualized H-EnSCs were generated from endometrial biopsies of 6 women with RM and 6 fertile controls. Cell-free migration zones were created and the effect of the presence of a high-quality (day 5 blastocyst, n = 13), a low-quality (day 5 blastocyst with three pronuclei or underdeveloped embryo, n = 12) or AC-1M88 trophoblast cell line spheroid on H-ESC migratory activity was analyzed after 18 hours. In the absence of a spheroid or embryo, migration of H-EnSCs from fertile or RM women was similar. In the presence of a low-quality embryo in the zone, the migration of H-EnSCs of control women was inhibited compared to the basal migration in the absence of an embryo (P<0.05) and compared to the migration in the presence of high-quality embryo (p<0.01). Interestingly, the migratory response H-EnSCs of women with RM did not differ between high- and low-quality embryos. Furthermore, in the presence of a spheroid their migration was enhanced compared to the H-EnSCs of controls (p<0.001).ConclusionsH-EnSCs of fertile women discriminate between high- and low-quality embryos whereas H-EnSCs of women with RM fail to do so. H-EnSCs of RM women have a higher migratory response to trophoblast spheroids. Future studies will focus on the mechanisms by which low-quality embryos inhibit the migration of H-EnSCs and how this is deregulated in women with RM.

A polymorphism in a let-7 microRNA binding site of KRAS in women with endometriosis.

Endometriosis is found in 5–15% of women of reproductive age and is more frequent in relatives of women with the disease. Activation of KRAS results in de novo endometriosis in mice, however, activating KRAS mutations have not been identified in women. We screened 150 women with endometriosis for a polymorphism in a let-7 microRNA (miRNA) binding site in the 3'-UTR of KRAS and detected a KRAS variant allele in 31% of women with endometriosis as opposed to 5% of a large diverse control population. KRAS mRNA and protein expression were increased in cultured endometrial stromal cells of women with the KRAS variant. Increased KRAS protein was due to altered miRNA binding as demonstrated in reporter assays. Endometrial stromal cells from women with the KRAS variant showed increased proliferation and invasion. In a murine model, endometrial xenografts containing the KRAS variant demonstrated increased proliferation and decreased progesterone receptor levels. These findings suggest that an inherited polymorphism of a let-7 miRNA binding site in KRAS leads to abnormal endometrial growth and endometriosis. The LCS6 polymorphism is the first described genetic marker of endometriosis risk.

Prolonged anovulation causes aberrant endometrial gene promoter methylation changes in women with polycystic ovary syndrome

OBJECTIVE: Abnormal expression of steroid hormone receptors in the endometrium of women with polycystic ovary syndrome (PCOS) has been suggested to adversely affect endometrial function. Although aberrant epigenetic regulation of endometrial gene expression has been demonstrated in endometriosis and endometrial adenocarcinoma, epigenetic regulation of endometrial gene expression in PCOS has not been evaluated. We hypothesize that abnormal endometrial gene expression in PCOS may be associated with epigenetic modification of gene promoter sites.DESIGN: Laboratory comparison study.MATERIALS AND METHODS: Endometrial specimens were collected in PCOS (n=3) and control (n=3) subjects. The subjects with PCOS had a proliferative phase that ranged from 5-120 days. Endometrial stromal cells were isolated and grown in culture with subsequent isolation of RNA and DNA. Expression levels of AR, ERβ, and HOXA10 were determined by real time RT-PCR. Results for individual PCOS patients were compared to averaged control results. Differences in methylation patterns at CpG island sites of the promoters for AR, ERβ, and HOXA10 were determined by EpiTyper. Unpaired student t test and one way ANOVA were used for statistical analysis.RESULTS: Compared to controls, PCOS was associated with increased expression of AR and ERβ and decreased expression of HOXA10. Longer proliferative phase duration in PCOS patients was positively associated with the observed changes in gene expression. AR and ERβ promoters were found to be demethylated at specific CpG sites in PCOS patients. No difference was noted in HOXA10 promoter methylation between control and PCOS groups.CONCLUSIONS: Aberrant steroid hormone receptor expression in endometrial stromal cells of women with PCOS is associated with changes in gene promoter methylation. This expression appears to be more pronounced in those subjects who have demonstrated proliferative changes over a longer period of time. OBJECTIVE: Abnormal expression of steroid hormone receptors in the endometrium of women with polycystic ovary syndrome (PCOS) has been suggested to adversely affect endometrial function. Although aberrant epigenetic regulation of endometrial gene expression has been demonstrated in endometriosis and endometrial adenocarcinoma, epigenetic regulation of endometrial gene expression in PCOS has not been evaluated. We hypothesize that abnormal endometrial gene expression in PCOS may be associated with epigenetic modification of gene promoter sites. DESIGN: Laboratory comparison study. MATERIALS AND METHODS: Endometrial specimens were collected in PCOS (n=3) and control (n=3) subjects. The subjects with PCOS had a proliferative phase that ranged from 5-120 days. Endometrial stromal cells were isolated and grown in culture with subsequent isolation of RNA and DNA. Expression levels of AR, ERβ, and HOXA10 were determined by real time RT-PCR. Results for individual PCOS patients were compared to averaged control results. Differences in methylation patterns at CpG island sites of the promoters for AR, ERβ, and HOXA10 were determined by EpiTyper. Unpaired student t test and one way ANOVA were used for statistical analysis. RESULTS: Compared to controls, PCOS was associated with increased expression of AR and ERβ and decreased expression of HOXA10. Longer proliferative phase duration in PCOS patients was positively associated with the observed changes in gene expression. AR and ERβ promoters were found to be demethylated at specific CpG sites in PCOS patients. No difference was noted in HOXA10 promoter methylation between control and PCOS groups. CONCLUSIONS: Aberrant steroid hormone receptor expression in endometrial stromal cells of women with PCOS is associated with changes in gene promoter methylation. This expression appears to be more pronounced in those subjects who have demonstrated proliferative changes over a longer period of time.

Macrophage migration inhibitory factor (MIF)-induced signaling pathways in human endometrial stromal cells of women with and without endometriosis

OBJECTIVE: To investigate signal transduction pathways in endometrial stromal cells of women with and without endometriosis in response to MIF, a potent proinflammatory and growth-promoting factor.DESIGN: Primary cultures of endometrial stromal cells isolated from endometrial biopsies of women with and without endometriosis in proliferative and secretory cycle phase exposed to MIF and assessment of extracellular signal-related kinase (Erk)1/2 mitogen-activated protein kinase (MAPK), p38 MAPK and Akt phosphorylation.MATERIALS AND METHODS: Cells were cultured in DMEM-F12 medium with 10% FBS, 1% antibiotics, 5 μg/mL insulin and 5 μg/mL transferrin. Cells grown to confluence were preincubated for 1 h with Erk1/2 MAPK, p38 MAPK and Akt inhibitors (PD98059, SB203580 and LY294002 respectively) and stimulated with different concentrations of MIF (0-50 ng/mL) for different periods of time (0-1 h). Then, the extraction of proteins was performed and phosphorylation of Erk1/2 MAPK, p38 MAPK and Akt was analyzed by Western blot. An unpaired t test and one-way ANOVA followed by the Dunnett's test were performed for this study.RESULTS: Our study showed that MIF stimulated p38 MAPK and Akt phosphorylation in endometrial cells of women with and without endometriosis in the proliferative and the secretory phases of the menstrual cycle. In normal women, the basal level of Erk1/2 MAPK phosphorylation was lower in cells from the secretory phase of the menstrual cycle compared to the proliferative phase, but remained steadily elevated throughout the menstrual cycle in women with endometriosis. Cell exposure to MIF did not result in any detectable increase in Erk1/2 MAPK phosphorylation, whereas in normal women MIF activated Erk1/2 MAPK phosphorylation in the secretory phase. PD98059, SB203580 and LY294002 suppressed the ability of MIF to increase Erk1/2 MAPK, p38 MAPK and Akt phosphorylation, respectively.CONCLUSIONS: p38 and Akt signaling pathways are involved in MIF-mediated activation of endometrial stromal cells of women with and without endometriosis. Activation of Erk1/2 in endometrial cells throughout the menstrual cycle of women with endometriosis may, considering the role of these kinases in cell growth, angiogenesis and tissue remodeling, play an important role in the capability of endometrial cells to implant and develop in ectopic locations. OBJECTIVE: To investigate signal transduction pathways in endometrial stromal cells of women with and without endometriosis in response to MIF, a potent proinflammatory and growth-promoting factor. DESIGN: Primary cultures of endometrial stromal cells isolated from endometrial biopsies of women with and without endometriosis in proliferative and secretory cycle phase exposed to MIF and assessment of extracellular signal-related kinase (Erk)1/2 mitogen-activated protein kinase (MAPK), p38 MAPK and Akt phosphorylation. MATERIALS AND METHODS: Cells were cultured in DMEM-F12 medium with 10% FBS, 1% antibiotics, 5 μg/mL insulin and 5 μg/mL transferrin. Cells grown to confluence were preincubated for 1 h with Erk1/2 MAPK, p38 MAPK and Akt inhibitors (PD98059, SB203580 and LY294002 respectively) and stimulated with different concentrations of MIF (0-50 ng/mL) for different periods of time (0-1 h). Then, the extraction of proteins was performed and phosphorylation of Erk1/2 MAPK, p38 MAPK and Akt was analyzed by Western blot. An unpaired t test and one-way ANOVA followed by the Dunnett's test were performed for this study. RESULTS: Our study showed that MIF stimulated p38 MAPK and Akt phosphorylation in endometrial cells of women with and without endometriosis in the proliferative and the secretory phases of the menstrual cycle. In normal women, the basal level of Erk1/2 MAPK phosphorylation was lower in cells from the secretory phase of the menstrual cycle compared to the proliferative phase, but remained steadily elevated throughout the menstrual cycle in women with endometriosis. Cell exposure to MIF did not result in any detectable increase in Erk1/2 MAPK phosphorylation, whereas in normal women MIF activated Erk1/2 MAPK phosphorylation in the secretory phase. PD98059, SB203580 and LY294002 suppressed the ability of MIF to increase Erk1/2 MAPK, p38 MAPK and Akt phosphorylation, respectively. CONCLUSIONS: p38 and Akt signaling pathways are involved in MIF-mediated activation of endometrial stromal cells of women with and without endometriosis. Activation of Erk1/2 in endometrial cells throughout the menstrual cycle of women with endometriosis may, considering the role of these kinases in cell growth, angiogenesis and tissue remodeling, play an important role in the capability of endometrial cells to implant and develop in ectopic locations.

Altered apoptosis and proliferation in endometrial stromal cells of women with adenomyosis

The eutopic endometrium in a woman suffering from adenomyosis is known to be biologically different from that of healthy women. The aim of this study was to examine the apoptosis and proliferation of eutopic endometrium from women with adenomyosis. We enrolled 23 women with adenomyosis (study group) and 21 without (control group). Eutopic endometrium was obtained and separated into single endometrial stromal cells (ESCs). ESCs were treated in vitro with hydrogen peroxide (H(2)O(2)) to examine their apoptosis using a fluorescence-activated cell sorter. Cells were also treated with estradiol (E(2)), medroxyprogesterone acetate, interleukin (IL)-6, lipopolysaccharide and interferon-gamma (IFN-gamma) to test their proliferation using a non-radioactive cell proliferation assay. The percentage of annexin V ( + )/7-amino-actinomycin D ( + ) ESCs was much lower in women with adenomyosis after 24 h culture with and without H(2)O(2) treatment when compared with the control group. ESCs of adenomyosis proliferated more rapidly than those of the control group, whether they were cultured alone or were treated with E(2), MPA, IL-6 or IFN-gamma. The immunocytochemical Ki-67 labelling index was much more prominent in adenomyotic ESCs than that of the control group (7.7% versus 1.1%, P < 0.001). Altered apoptosis and proliferation of eutopic endometrium possibly elucidate some aspects of the pathophysiology of adenomyosis. A high Ki-67 labelling index in immunocytochemistry might be a potential indicator in predicting the occurrence of adenomyosis.

A study of luteal phase expression of inhibin, activin, and follistatin subunits in the endometrium of women with recurrent miscarriage

To compare the luteal phase endometrial expression of inhibin, activin, and follistatin subunits in women with recurrent miscarriage compared to a control group. Other parameters of luteal function assessed included Doppler blood flow, serum biochemical markers, and histopathology. This was a prospective case control study. The study was conducted in a tertiary care hospital of Sheffield, United Kingdom. Thirty-three women with recurrent miscarriage and 10 women with no previous history of miscarriage (control group) were recruited. Histologic assessment of the luteal phase endometrium was done using Noyes criteria followed by immunohistochemical analysis for expression of inhibin, activin, and follistatin subunits. Doppler analysis for perifollicular and endometrial blood flow was also done. Simultaneously serum concentrations of E(2), LH, FSH, P, and inhibin B at the time of LH surge and at LH +7 days were measured. Difference in endometrial expression of inhibin, activin, and follistatin subunit in the two groups. Endometrial expression of follistatin and beta A in the endometrial stromal cells of women with recurrent miscarriage was significantly lower than in control women. There were no differences in results of the Doppler studies or the hormonal profiles between cases and controls. The lower expression of follistatin and beta A subunit in women with recurrent miscarriage may imply an altered activity of activin A at the time of decidualization, which may lead to poor pregnancy outcome in the form of miscarriage.

Decreased suppression of interleukin-6 after treatment with medroxyprogesterone acetate and danazol in endometrial stromal cells of women with adenomyosis

To examine changes in local cytokine production in the eutopic endometrium of adenomyosis after treatment with medroxyprogesterone acetate (MPA) and danazol. Prospective cohort study. Tertiary university hospital. Twenty-four women with adenomyosis (study group) and 22 women without adenomyosis (control group). Eutopic endometrium was separated into single endometrial stromal cells (ESCs), and was cultured in vitro alone, with MPA (10(-7) M), or with danazol (10(-7) M) for 8 days. Messenger RNA expression of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha in ESCs, and their concentrations in the supernatants of ESCs. A significant decrease of IL-6 concentration in the supernatant, measured by ELISA, was found with time of ESCs cultured with MPA and danazol in the control group, but not in adenomyosis. The IL-6 mRNA in ESCs determined by real-time quantitative polymerase chain, as well as its concentration in the supernatant, was much higher in adenomyosis than that in the control group after treatment with MPA and danazol for 8 days. Medroxyprogesterone acetate and danazol appeared to have a decreased effect on the suppression of IL-6 liberated by ESCs in adenomyosis. This aberrant secretion of IL-6 might play a role in the formation of ectopic endometrial implants in adenomyosis.

Effect of monocyte chemoattractant protein-1 and estradiol on the secretion of vascular endothelial growth factor in endometrial stromal cells in vitro

To explore the initial activity of endometrial stromal cells (ESCs) and the participation of monocyte chemoattractant protein-1 (MCP-1) in the histogenesis of endometriosis, vascular endothelial growth factor (VEGF) secretion of ESCs and the effect of MCP-1 on VEGF secretion of ESCs were observed in vitro. The VEGF level was detected in ESC culture media and was increased significantly when E2 or MCP-1 was added to the media, especially in the presence of E2 plus MCP-1. The VEGF secretion was higher in the ESCs of women with endometriosis than in those women without endometriosis.