Endometrium Of Endometriosis Patients Research Articles

Extracellular vesicle-packaged PKM2 from endometriotic stromal cells promotes endometrial collagen I deposition by inhibiting autophagy in endometriosis

Aberrant endometrial collagen I deposition during the implantation window impairs endometrial stromal cell (ESC) decidualization, which may contribute to lower pregnancy rate in endometriosis (EMs) patients with in vitro fertilization (IVF) treatment. However, the underlying mechanism of eutopic aberrant endometrium collagen I deposition in EMs remains unclear. In this study, we found increased endometrial collagen I and defective decidualization in the mid-secretory phase of EMs patients, while the level of eutopic ESCs' autophagy was decreased, which was an important mechanism of intracellular collagen degradation. Lower ESCs' autophagy level may cause the endometrial collagen I deposition in EMs. Furthermore, in vivo and in vitro studies showed that the extracellular vesicles derived from the ectopic ESCs of EMs patients (EMs-EVs) encapsulated higher PKM2 inhibited autophagy of the ESCs accompanied by an increase of collagen I. We also found that the constructed EMs-EVsAd-PKM2 with PKM2 overexpression inhibited ESCs' autophagy by activating the Akt/mTOR signaling pathway. And the expressions of PKM2, p-Akt and p-mTOR were also increased in the endometrium of EMs patients. Collectively, these data showed that EMs-EVs delivering PKM2 inhibited autophagy inducing aberrant endometrial collagen I deposition via the Akt/mTOR signaling pathway to impair decidualization, which provided a potential therapeutic target for improving the IVF pregnancy rate in EMs patients.

Long noncoding RNA BMPR1B-AS1 stability regulated by IGF2BP2 affects the decidualization in endometriosis patients through the SMAD1/5/9 pathway.

Endometriosis (EMs)-related infertility commonly has decreased endometrial receptivity and normal decidualization is the basis for establishing and maintaining endometrial receptivity. However, the potential molecular regulatory mechanisms of impaired endometrial decidualization in patients with EMs have not been fully clarified. We confirmed the existence of reduced endometrial receptivity in patients with EMs by scanning electron microscopy and quantitative real-time PCR. Here we identified an lncRNA, named BMPR1B-AS1, which is significantly downregulated in eutopic endometrium in EMs patients and plays an essential role in decidual formation. Furthermore, RNA pull-down, mass spectrometry, RNA immunoprecipitation, and rescue analyses revealed that BMPR1B-AS1 positively regulates decidual formation through interaction with the RNA-binding protein insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Downregulation of IGF2BP2 led to a decreased stability of BMPR1B-AS1 and inhibition of activation of the SMAD1/5/9 pathway, an inhibitory effect which diminished decidualization in human endometrial stromal cells (hESCs) decidualization. In conclusion, our identified a novel regulatory mechanism in which the IGF2BP2-BMPR1B-AS1-SMAD1/5/9 axis plays a key role in the regulation of decidualization, providing insights into the potential link between abnormal decidualization and infertility in patients with EMs, which will be of clinical significance for the management and treatment of infertility in patients with EMs.

Endometrial stromal PRMT5 plays a crucial role in decidualization by regulating NF-κB signaling in endometriosis

Decidualization is a prerequisite for successful embryo implantation, in which elongated fibroblast-like endometrial stromal cells differentiate into more rounded decidual cells. Accumulating evidence has stressed the important role of the defective eutopic endometrium in infertility in endometriosis patients. However, the role of arginine methylation in the process of physiological decidualization and pathological decidualization defects is not clear. Here, we observed that the expression level of PRMT5, the main type II PRMT, was decreased in the endometrium of endometriosis patients, predominantly in stromal cells. Compared with the undecidualized state, PRMT5 was increased in the stromal cells of normal secretory endometrium in humans and in the decidua of normal pregnant mice or mice with artificially induced decidualization. The inhibition of PRMT5 resulted in a significant decrease in uterine weight and decidualization-related regulator expression, including FOXO1, HOXA10 and WNT4, in mice and IGFBP1 and prolactin levels in human endometrial stromal cells. Transcriptome analysis showed that decreased PRMT5 activity led to NF-κB signaling activation by inducing p65 translocation to the nucleus, which was also observed in endometriosis patients. Finally, overexpression of PRMT5 rescued the defective expression of IGFBP1 and prolactin in primary endometrial stromal cells from endometriosis patients. Our results indicate that promotion of PRMT5 may provide novel therapeutic strategies for the treatment of decidualization defects in infertile women, such as those with endometriosis.

Increased Angiogenesis and Lymphangiogenesis in Adenomyosis Visualized by Multiplex Immunohistochemistry.

There is evidence for increased angiogenesis in the (ectopic) endometrium of adenomyosis patients under the influence of vascular endothelial growth factor (VEGF). VEGF stimulates both angiogenesis and lymph-angiogenesis. However, information on lymph vessels in the (ectopic) endometrium of adenomyosis patients is lacking. In this retrospective matched case-control study, multiplex immunohistochemistry was performed on thirty-eight paraffin embedded specimens from premenopausal women who had undergone a hysterectomy at the Amsterdam UMC between 2001 and 2018 to investigate the evidence for (lymph) angiogenesis in the (ectopic) endometrium or myometrium of patients with adenomyosis versus controls with unrelated pathologies. Baseline characteristics of both groups were comparable. In the proliferative phase, the blood and lymph vessel densities were, respectively, higher in the ectopic and eutopic endometrium of patients with adenomyosis than in the endometrium of controls. The relative number of blood vessels without α-smooth muscle actinin (α SMA) was higher in the eutopic and ectopic endometrium of adenomyosis patients versus controls. The level of VEGF staining intensity was highest in the myometrium but did not differ between patients with adenomyosis or controls. The results indicate increased angiogenesis and lymphangiogenesis in the (ectopic) endometrium affected by adenomyosis. The clinical relevance of our findings should be confirmed in prospective clinical studies.

CSF-1-induced DC-SIGN+ macrophages are present in the ovarian endometriosis

BackgroundResearchers have found that macrophages are the predominant cells in the peritoneal fluid (PF) of endometriosis patients. CSF-1 has been found to accumulate in the lesions and PF of endometriosis patients, and CSF-1 induces THP-1-derived macrophages to polarize toward a CD169+ DC-SIGN+ phenotype. Does the cytokine CSF-1 induce monocytes to differentiate into macrophages with a DC-SIGN+ phenotype in endometriosis?MethodsThe level of CSF-1 in the endometrium of control subjects, and the eutopic, and ectopic endometrium of endometriosis patients was evaluated by real-time polymerase chain reaction (qRT–PCR) and was determined by enzyme-linked immunosorbent assay (ELISA) in the PF of control and endometriosis patients. CSF-1 expression was examined with a MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel. DC-SIGN+ macrophages were detected by immunohistochemical staining of tissues and flow cytometric analysis of the PF of control subjects (N = 25) and endometriosis (N = 35) patients. The phenotypes and biological activities of CSF-1 -induced macrophages were compared in an in vitro coculture system with peripheral blood lymphocytes from control subjects.ResultsIn this study, we found that the proportion of DC-SIGN+ CD169+ macrophages was higher in the abdominal immune microenvironment of endometriosis patients. CSF-1 was primarily secreted from ectopic lesions and peritoneum in mice with endometriosis. In addition, CSF-1 induced the polarization of macrophages toward a DC-SIGN+ CD169+ phenotype; this effect was abolished by the addition of an anti-CSF-1R antibody. CSF-1 induced the generation of DC-SIGN+ macrophages, leading to a depressed status of peripheral blood lymphocytes, including a high percentage of Treg cells and a low percentage of CD8+ T cells. Similarly, blockade with the anti-CSF-1R antibody abrogated this biological effect.ConclusionsThis is the first study on the role of DC-SIGN+ macrophages in the immune microenvironment of endometriosis. Further study of the mechanism and biological activities of CSF-1-induced DC-SIGN+ macrophages will enhance our understanding of the physiology of endometriosis.Graphical

Role of WNT4, HOXA10 and TWIST1 genes in the pathogenesis of external genital endometriosis and uterine leiomyoma

BACKGROUND: Uterine leiomyoma and endometriosis are the most common gynecological diseases in women of reproductive age. A number of data indicate that there are common elements in the pathogenesis of these hyperproliferative conditions. This article is devoted to comparative analysis of the role of the WNT4, HOXA10 and TWIST1 genes in the development of uterine leiomyoma and external genital endometriosis.
 AIM: The aim of this study was to evaluate the frequency of polymorphic variants rs7521902 (WNT4) and rs4721745 (TWIST1) in patients with uterine leiomyoma, external genital endometriosis and in the comparison group; to determine the frequency of rare allelic variants of the HOXA10 gene in patients with external genital endometriosis; and to study the expression of these genes in the endometrium in patients with uterine leiomyoma, EGE and in the comparison group.
 MATERIALS AND METHODS: The polymorphic variants of the WNT4 and TWIST1 genes were studied by real-time PCR in patients with external genital endometriosis, uterine leiomyoma and in the comparison group. In patients with EGE and women in the comparison group, the second exon of the HOXA10 gene was sequenced. Real-time PCR with reverse transcription analysis of the expression of the WNT4, TWIST1 and HOXA10 genes in endometrial samples from the patients of the study groups was performed.
 RESULTS: The frequencies of polymorphic variants rs7521902 (WNT4) and rs4721745 (TWIST1) in patients with uterine leiomyoma, external genital endometriosis and in the comparison group did not differ significantly. Minor alleles of the HOXA10 gene were not identified in patients with external genital endometriosis. Expression of the WNT4 gene in the endometrium of patients with external genital endometriosis was independent of menstrual cycle phase and was reduced by 1.9 times compared to the endometrium of women with uterine leiomyoma. Expression of the HOXA10 gene in the endometrium of endometriosis patients on days 20-23 of the menstrual cycle was significantly reduced compared to the women in the comparison group. Expression of the TWIST1 gene was not altered in the endometrium of patients with uterine leiomyoma and external genital endometriosis.
 CONCLUSIONS: We did not identify associations of the studied polymorphic variants of the WNT4 and TWIST genes and minor variants of the HOXA10 gene with uterine leiomyoma and external genital endometriosis. The expression of the WNT4 and HOXA10 genes is reduced in the endometrium in patients with external genital endometriosis, but not in women with uterine leiomyoma. Changes in expression patterns of the studied genes in the endometrium differ significantly in these two diseases.

Decreased mixed lineage leukemia 1 is involved in endometriosis-related infertility.

The aberrant histone methylation patterns contribute to the pathogenesis of endometriosis (EM). Mixed lineage leukemia 1 (MLL1), a histone methyltransferase, is crucial for gene expression by catalyzing the trimethylation of histone 3 lysine 4 (H3K4me3) in gene promoter. This study aimed to explore whether MLL1 is involved in EM-related infertility. The expressions of MLL1 and H3K4me3 were analyzed in the eutopic endometria from EM women with infertility (n = 22) and the normal endometria from EM-free women (n = 22). Mouse EM model was established. The MLL1 and H3K4me3 expression patterns in mice endometria of early pregnancy were also investigated. Immortalized human endometrial stromal cells (iESCs) were cultured and underwent in vitro decidualization. The chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed to find the target gene of MLL1 during decidual process. Results showed that both MLL1 and H3K4me3 decreased in the eutopic endometrium from EM patients compared to that in the normal endometrium. During early pregnancy and the decidual process, MLL1 and H3K4me3 were significantly upregulated in stromal cells. ChIP-seq and ChIP-qPCR found that the cytochrome c oxidase subunit 4I 2 (COX4I2) was directly targeted by MLL1. The dominance of COX4I2-containing enzyme induced the expression of hypoxia-inducible factor-2α (HIF-2α), whose expression in the peri-implantation endometrium is essential for embryo implantation. Further results showed that MLL1 was directly regulated by progesterone (P4) - P4 receptors (PRs). Our study proved that MLL1 was involved in EM-related infertility, which may provide a novel approach to treat the nonreceptive endometrium in EM patients.

Uterine bathing with sonography gel prior to IVF/ICSI-treatment in patients with endometriosis, a multicentre randomised controlled trial.

STUDY QUESTIONWhat is the effect of uterine bathing with sonography gel prior to IVF/ICSI-treatment on live birth rates after fresh embryo transfer in patients with endometriosis?SUMMARY ANSWERAfter formal interim analysis and premature ending of the trial, no significant difference between uterine bathing using a pharmacologically neutral sonography gel compared to a sham procedure on live birth rate after fresh embryo transfer in endometriosis patients (26.7% vs. 15.4%, relative risk (RR) 1.73, 95% confidence interval (CI) 0.81–3.72; P-value 0.147) could be found, although the trial was underpowered to draw definite conclusions.WHAT IS KNOWN ALREADYImpaired implantation receptivity contributes to reduced clinical pregnancy rates after IVF/ICSI-treatment in endometriosis patients. Previous studies have suggested a favourable effect of tubal flushing with Lipiodol® on natural conceptions. This benefit might also be explained by enhancing implantation through endometrial immunomodulation. Although recent studies showed no beneficial effect of endometrial scratching, the effect of mechanical stress by intrauterine infusion on the endometrium in endometriosis patients undergoing IVF/ICSI-treatment has not been investigated yet.STUDY DESIGN, SIZE, DURATIONWe performed a multicentre, patient-blinded, randomised controlled trial in which women were randomly allocated to either a Gel Infusion Sonography (GIS, intervention group) or a sham procedure (control group) prior to IVF/ICSI-treatment. Since recruitment was slow and completion of the study was considered unfeasible, the study was halted after inclusion of 112 of the planned 184 women.PARTICIPANTS/MATERIALS, SETTING, METHODSWe included infertile women with surgically confirmed endometriosis ASRM stage I–IV undergoing IVF/ICSI-treatment. After informed consent, women were randomised to GIS with intrauterine instillation of ExEm-gel® or sonography with gel into the vagina (sham). This was performed in the cycle preceding the embryo transfer, on the day GnRH analogue treatment was started. The primary endpoint was live birth rate after fresh embryo transfer. Analysis was performed by both intention-to-treat and per-protocol.MAIN RESULTS AND THE ROLE OF CHANCEBetween July 2014 to September 2018, we randomly allocated 112 women to GIS (n = 60) or sham procedure (n = 52). The live birth rate after fresh embryo transfer was 16/60 (26.7%) after GIS versus 8/52 (15.4%) after the sham (RR 1.73, 95% CI 0.81–3.72; P-value 0.147). Ongoing pregnancy rate was 16/60 (26.7%) after GIS versus 9/52 (17.3%) in the controls (RR 1.54, 95% CI 0.74–3.18). Miscarriage occurred in 1/60 (1.7%) after GIS versus 5/52 (9.6%) in the controls (RR 0.17, 95% CI 0.02–1.44) women. Uterine bathing resulted in a higher pain score compared with a sham procedure (visual analogue scale score 2.7 [1.3–3.5] vs. 1.0 [0.0–2.0], P < 0.001). There were two adverse events after GIS compared with none after sham procedures.LIMITATIONS, REASONS FOR CAUTIONThe study was terminated prematurely due to slow recruitment and trial fatigue. Therefore, the trial is underpowered to draw definite conclusions regarding the effect of uterine bathing with sonography gel on live birth rate after fresh embryo transfer in endometriosis patients undergoing IVF/ICSI-treatment.WIDER IMPLICATIONS OF THE FINDINGSWe could not demonstrate a favourable effect of uterine bathing procedures with sonography gel prior to IVF/ICSI-treatment in patients with endometriosis.STUDY FUNDING/COMPETING INTEREST(S)Investigator initiated study. IQ Medical Ventures provided the ExEm FOAM® kits free of charge, they were not involved in the study design, data management, statistical analyses and/or manuscript preparation, etc. C.B.L. reports receiving grants from Ferring, Merck and Guerbet, outside the submitted work. C.B.L. is Editor-in-Chief of Human Reproduction. V.M. reports grants and other from Guerbet, outside the submitted work. B.W.M. reports grants from NHMRC (GNT1176437), personal fees from ObsEva, Merck and Merck KGaA, Guerbet and iGenomix, outside the submitted work. N.P.J. reports research funding from Abb-Vie and Myovant Sciences and consultancy for Vifor Pharma, Guerbet, Myovant Sciences and Roche Diagnostics, outside the submitted work. K.D. reports personal fees from Guerbet, outside the submitted work. The other authors do not report any conflicts of interest. No financial support was provided.TRIAL REGISTRATION NUMBERNL4025 (NTR4198)TRIAL REGISTRATION DATE7 October 2013DATE OF FIRST PATIENT’S ENROLMENT22 July 2014

Analysis of Reciprocally Dysregulated miRNAs in Eutopic Endometrium Is a Promising Approach for Low Invasive Diagnostics of Adenomyosis.

Endometriosis is a chronic disease characterized by the growth of endometrial tissue outside of the uterine cavity. Endometriosis affects up to 10% of women of reproductive age and has great social impact. The diagnostics of endometriosis are based on clinical appearance, ultrasound, and magnetic resonance imaging (MRI); however, a diagnosis is frequently hampered by the absence of objective criteria. Adenomyosis (AM) is a particular type of endometriosis wherein the spread of the ectopic endometrial gland is limited by the uterine myometrium. Alteration of the microRNA expression profile in the eutopic endometrium can be associated with AM, and may be assayed for diagnostic purposes. In the presented study, we aimed to explore the diagnostic potency of this approach. Eutopic endometrium specimens were collected from patients (n = 33) and healthy women (n = 30). The microRNA expression was profiled to select individual microRNAs with AM-associated expression alterations. A new method of two-tailed RT-qPCR microRNA analysis was applied to assay potential markers. The expression ratios of reciprocally dysregulated microRNAs were calculated, and the diagnostic potency of these parameters was evaluated by receiver operation curve (ROC) analysis. Mir-10b, miR-200c and miR-191 were significantly dysregulated in the eutopic endometrium of AM patients. The expression ratio of reciprocally dysregulated microRNAs allowed us to diagnose AM with a range of sensitivity from 65% to 74%, and of specificity from 72% to 86%. The analysis of microRNAs from the eutopic endometrium might present a promising low-invasive method of AM diagnostics.

PCR Genetic Expression of Interleukin 37 in the Eutopic and Ectopic Endometrium of Women with Endometriosis

Background: In endometriosis, there is a chronic immune and inflammatory reaction. Objective: To study the PCR genetic expression of interleukin 37 in the eutopic and ectopic endometrium of women with endometriosis in comparison with controls. Patient and Methods: Forty-six patients were included in the study. Twenty-three women diagnosed as endometriosis and twenty-three women diagnosed as free of endometriosis were subjected to laparoscopy for any other gynecological cause. Interleukin 37 gene expression was studied in ectopic and eutopic endometrium using polymerase chain reaction. Results: Ectopic endometrium expressed high levels of mRNA compared to eutopic endometrium in endometriosis patients. Eutopic endometrium of endometriosis patients expressed high levels of mRNA compared to eutopic endometrium of controls. Conclusion: The finding of high mRNA produced in endometrial tissue suggests that interleukin 37 may be important in the chronic inflammation occurring during endometriosis.

Expression and significance of autophagy genes LC3, Beclin1 and MMP-2 in endometriosis.

Expression of autophagy-related proteins, microtubule-associated protein light chain 3 (LC3) and Beclin1, and matrix metalloproteinase-2 (MMP-2) was investigated in serum and peritoneal fluid of patients with endometriosis (EM). The messenger ribonucleic acid (mRNA) expression levels of MMP-2, LC3 and Beclin1 in endometrial tissues of EM patients and correlation of these genes with EM and their significance were evaluated. The serum, peritoneal fluid and endometrial tissues of 84 patients treated in The First Affiliated Hospital of Qiqihar Medical University (Qiqihar, China) from March 2016 to March 2017 were collected. The serum, peritoneal fluid and endometrial tissues of 42 EM patients were used as the experimental group, while those of 42 non-EM patients were used as the control group. The levels of LC3, Beclin1 and MMP-2 in serum and peritoneal fluid of EM patients and non-EM patients were quantitatively detected via enzyme-linked immunosorbent assay (ELISA), followed by comparative analysis based on data in both groups. In addition, mRNA expression of LC3, Beclin1 and MMP-2 in the endometrium in both groups were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and differences in expression of these genes between the groups were analyzed and evaluated. Correlation of LC3, Beclin1 and MMP-2 with EM was explored. Results of ELISA showed that levels of LC3 and Beclin1 in the EM group were significantly lower than those in the control group, while levels of MMP-2 in serum and peritoneal fluid of the EM group were significantly higher than those in the control group (P<0.05). Results of RT-qPCR revealed that mRNA expression of LC3 and Beclin1 in the endometrium of patients in the EM group were obviously decreased compared with those in the control group, while the expression of MMP-2 was high, and differences in expression were statistically significant (P<0.05). The expression of MMP-2 is high, and expression of LC3 and Beclin1 is low in serum, peritoneal fluid and endometrium of EM patients, and investigating the expression of MMP-2, LC3 and Beclin1 in EM is helpful to further clarify the pathogenesis of EM, and guide the clinical diagnosis and treatment.

Soluble CD200 in secretory phase endometriosis endometrial venules may explain endometriosis pathophysiology and provide a novel treatment target

Retrograde menstruation is common and in about 10% of women, endometrial tissues implant at ectopic sites and grow as endometriosis (EM) deposits. To date, there has been no marker to identify which patients have an endometrium that will generate deposits. Both endometrial regulatory T cells (Tregs) and increased stromal cell indoleamine 2,3-dioxygenase (IDO) have been implicated, and may suppress rejection by peritoneal NK cells, neutrophils, and cytotoxic macrophages. CD200 is a tolerance signaling molecule which promotes Tregs, IDO-producing macrophages, and can directly inhibit cytolytic natural killer (NK) cells and neutrophils. To determine if CD200 might be overexpressed in the endometrium of women with endometriosis, a pilot study using quantitative immunohistochemistry was done using uterine sections and EM deposits from hysterectomy patients. Both CD200 and CD200R proteins were detectable in endometriosis (EM) deposits and in endometrial epithelium and stroma. CD200 increased slightly in secretory phase whole endometrium of EM patients, but strikingly increased soluble CD200 (sCD200) absent sCD200R within venules typified both endometriosis deposits and secretory phase endometrial stromal venules and lymphatics in EM endometria compared to secretory phase NE endometria (P = 0.000006). In our opinion, accumulation of sCD200 in secretory phase endometrial blood vessels may explain development of ectopic deposits and quantifying sCD200 in menstrual blood may cases and identify predisposition to EM. Animal model studies are required to determine if antagonizing CD200 could be therapeutic.

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

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

Aldo-keto reductase activity after diethylhexyl phthalate exposure in eutopic and ectopic endometrial cells

ObjectiveEndometriosis is a multifactorial gynaecological disease in reproductive-age women. Endometriotic tissue is characterized by high prostaglandin levels and progesterone resistance. Human aldo-keto reductases (AKRs) convert progesterone to a less potent metabolite and cause progesterone resistance. Therefore, in this study, we evaluated whether diethylhexyl phthalate (DEHP) alters AKR expression in human ectopic and eutopic endometrium. Study designWe used microarrays and western blotting to study the effects of DEHP, and checked the presence of AKR in endometriosis patients by enzyme-linked immunosorbent assay (ELISA). ResultsCultured human endometrial cells from normal endometrium of women without endometriosis (NE), eutopic endometrium from endometriosis patients (EE), and ectopic endometrium from endometriosis patients (EC) differed in genetic expression changes after DEHP treatment. DEHP upregulated AKR1C1, AKR1C2, AKR1C3, and AKR1B10 expression in EE, while EC showed continuously increased AKR1C3 expression before and after DEHP exposure. In western blot analysis, before and after DEHP exposure, the AKR1B10 protein band was detected in NE, EE, and EC, whereas the AKR1C3 band was detected only in EC. AKR1B10 and AKR1C3 expression levels in the blood of the enrolled patients were evaluated using ELISA. AKR1B10 expression did not differ between groups (without endometriosis [N=13], 0.10 vs. with endometriosis [N=20], 0.11; P=0.27). AKR1C3 expression was significantly higher in the blood of endometriosis patients than in that of patients without endometriosis (without endometriosis, 9.1 vs. with endometriosis, 10.1; P=0.02). Analysis according to menstrual period showed significantly increased AKR1C3 levels in patients with endometriosis only during the secretory phase and not the proliferative phase (P<0.05). ConclusionDEHP induces AKR activity in the endometrium of endometriosis patients, and AKR1C3 might influence the development of endometriosis.

Down-regulation of tumor suppressor PDCD4 expression in endometrium of adenomyosis patients

ObjectiveAdenomyosis is a common benign gynecological disease which has some malignant behaviors. Programmed cell death 4 (PDCD4) is a newly identified tumor suppressor gene which lowly expresses in various cancers. However, the expression status of PDCD4 in endometrium of adenomyosis patients has not been investigated. The aim of this study is to assess the expression levels of PDCD4 in endometrium of normal controls and adenomyosis patients. MethodsThe expression of PDCD4 in endometrium of normal controls and eutopic or ectopic endometrium of patients with adenomyosis was evaluated with quantitative real-time PCR, western blot and immunohistochemistry. In addition, the levels of serum estradiol and progesterone in normal controls and adenomyosis patients were detected using electrochemiluminescence immunoassay. ResultsThe results showed that PDCD4 mainly expressed in the cytoplasma of glandular epithelium of control endometrium and varied during the cycle changes of endometrium, which may be regulated by changing concentrations of progesterone in the menstrual cycle. Compared with the proliferative phase of control endometrium, PDCD4 expression was down-regulated in proliferative phase of eutopic endometrium or ectopic endometrium, and there was no cyclic variation of PDCD4 expression in eutopic endometrium of adenomyosis patients due to progesterone resistance. ConclusionThese results suggest that PDCD4 may be involved in the pathogenesis of adenomyosis, which will provide a novel strategy for the early diagnosis and new therapeutic target of adenomyosis.

Altered expression of progesterone receptor isoforms A and B in human eutopic endometrium in endometriosis patients

Recent data implicate an altered expression of progesterone receptor isoform A (PR-A) and B (PR-B) in the endometrium of endometriosis patients. This prospective exploratory study aimed to precisely determine the PR-A and PR-B expression using immunohistochemical techniques in eutopic endometrium of women with endometriosis compared with disease-free women throughout the menstrual cycle.All symptomatic patients underwent laparoscopy for the diagnosis of endometriosis and histological confirmation of the disease (EO) whereas controls were proven disease-free (CO). In CO samples (n=10) an increased expression of PR-A and PR-B during the proliferative to early secretory phase and a decreased expression of both receptor isoforms during the mid to late secretory phase was ascertained in accordance with previous studies.In patients with endometriosis (n=16) no cycle dependent pattern of PR-A and PR-B expression was identified in contrast to patients without endometriosis. Moreover, in EO samples a huge variety of inter- and intra-individual differences in PR-A and PR-B expression were detected.These data provide further evidence that dysregulation of the PR-A and PR-B expression might contribute to the pathophysiology of endometriosis.

Endometrial vezatin and its association with endometriosis risk.

Do endometriosis risk-associated single nucleotide polymorphisms (SNPs) found at the 12q22 locus have effects on vezatin ( ITALIC! VEZT) expression? The original genome-wide association study (GWAS) SNP (rs10859871), and other newly identified association signals, demonstrate strong evidence for ITALIC! cis-expression quantitative trait loci (eQTL) effects on ITALIC! VEZT expression. GWAS have identified several disease-risk loci (SNPs) associated with endometriosis. The SNP rs10859871 is located within the ITALIC! VEZT gene. ITALIC! VEZT expression is altered in the endometrium of endometriosis patients and is an excellent candidate for having a causal role in endometriosis. Most of the SNPs identified from GWAS are not located within the coding region of the genome. However, they are likely to have an effect on the regulation of gene expression. Genetic variants that affect levels of gene expression are called expression quantitative trait loci (eQTL). Samples for genotyping and ITALIC! VEZT variant screening were drawn from women recruited for genetic studies in Australia/New Zealand and women undergoing surgery in a tertiary care centre. Coding variants for ITALIC! VEZT were screened in blood from 100 unrelated individuals (endometriosis-dense families) from the QIMR Berghofer Medical Research Institute dataset. SNPs at the 12q22 locus were imputed and reanalysed for their association with endometriosis. Reanalysis of endometriosis risk-association was performed on a final combined Australian dataset of 2594 cases and 4496 controls. Gene expression was performed on 136 endometrial samples. eQTL analysis in whole blood was performed on 862 individuals from the Brisbane Systems Genetics Study. Endometrial tissue-specific eQTL analysis was performed on 122 samples (eutopic endometrium) collected following laparoscopic surgery. VEZT protein expression studies employed ITALIC! n = 56 (western blotting) and ITALIC! n = 42 (immunohistochemistry) endometrial samples. The women recruited for this study provided blood and/or endometrial tissue samples in a hospital setting. Genomic DNA was screened for common and coding variants. SNPs of interest in the 12q22 region were genotyped using Agena MassARRAY technology or Taqman SNP genotyping assay. Gene expression profiles from RNA extracted from blood and endometrial tissue samples were generated using Illumina whole-genome expression chips (Human HT-12 v4.0). Whole protein extracted from endometrium was used for VEZT western blots, and paraffin sections of endometrium were employed for VEZT immunohistochemistry semi-quantitative analysis. A total of 11 coding variants of ITALIC! VEZT (including one novel variant) were identified from an endometriosis-dense cohort. Polymorphic coding and imputed SNPs were combined with previous GWAS data to reanalyse the endometriosis risk association of the 12q22 region. The disease association signal at 12q22 was due to coding variants in ITALIC! VEZT or ITALIC! FGD6 (FYVE, RhoGEF and PH domain-containing 6) and SNPs with the strongest signals were either intronic or intergenic. We found strong evidence for ITALIC! VEZT cis-eQTLs with the sentinel SNP (rs10859871) in blood and endometrium, where the endometriosis risk allele (C) was associated with an increase in ITALIC! VEZT expression. We could not demonstrate this genotype-specific effect on VEZT protein expression in endometrium. However, we did observe a menstrual cycle stage specific increase in VEZT protein expression in endometrial glands, specific to the secretory phase ( ITALIC! P = 2.0 × 10(-4)). In comparison to the blood sample datasets, the study numbers of endometrial tissues were substantially reduced. Protein studies failed to complement RNA results, also likely a reflection of the low study numbers in these experiments. ITALIC! In silico prediction tools used in this investigation are typically based on cell lines different to our tissues of interest, thus any functional annotations drawn from these approaches should be considered carefully. Therefore, functional studies on VEZT and related pathway components are still warranted to unequivocally implicate a causal role for VEZT in endometriosis pathophysiology. GWAS have proven to be very valuable tools for deciphering complex diseases. Endometriosis is a text-book example of a complex disease, involving genetic, lifestyle and environmental influences. Our focused investigation of the 12q22 region validates an association with increased endometriosis risk. Endometriosis risk SNPs (including rs10859871) located within this locus demonstrated evidence for ITALIC! cis-eQTLs on ITALIC! VEZT expression. By examining women who possess an enhanced genetic risk of developing endometriosis, we have identified an effect on ITALIC! VEZT expression and therefore a potential gene/gene pathway in endometriosis disease establishment and development. Funding for this work was provided by NHMRC Project Grants GNT1012245, GNT1026033, GNT1049472 and GNT1046880. G.W.M. is supported by the NHMRC Fellowship scheme (GNT1078399). S.J.H.-C. is supported by the J.N. Peters Bequest Fellowship. The authors declare no competing interests. N/A.

MiR-183 Regulates ITGB1P Expression and Promotes Invasion of Endometrial Stromal Cells.

We applied in the previous study miRNA microarray screening analysis to identify several differentially expressed miRNAs, including miR-183 in normal, eutopic, and ectopic endometrium. Knockdown of miR-183 expression induced the invasiveness and inhibition of apoptosis in endometrial stromal cells. The current study aims to identify the miR-183 targets with relevance to cell functions in endometrial stromal cells, to verify the interaction of miR-183 with its target genes, and to confirm the role of miR-183 in the process of endometriosis. Using microarray analysis, we identified 27 differentially expressed genes (19 were upregulated and 8 downregulated), from which we selected 4 downregulated genes (ITGB1, AMIGO2, VAV3, and PSEN2) based on GO databases for functional analysis and significant pathway analysis. Western blotting analyses showed that integrin β1 (ITGB1), but not AMIGO2, was affected by miR-183 overexpression, whereas no protein expression of VAV3 and PSEN2 was detected. Luciferase reporter assay verified that ITGB1 is a target gene of miR-183. Moreover, we found that ITGB1 is overexpressed in the endometrium of endometriosis patients. Furthermore, overexpression of ITGB1 rescued the repressive effects of miR-183 on the invasiveness of endometrial stromal cells. These findings, together with the fact that ITGB1 is a critical factor for cell adhesion and invasiveness, suggest that miR-183 may be involved in the development of endometriosis by regulating ITGB1 in endometrial stromal cells.

The Effect of Oral Contraceptives Combined With Pycnogenol (Pinus Pinaster) On Aromatase and VEGF Expression in the Eutopic Endometrium of Endometriosis Patients

Oral contraceptives are effective in curbing down endometrial inflammation and decreasing endometriosisrelated dysmenorrhea. This antiinflammatory action is mediated by the progestin component of the pill and it involves the blockade of the NF-kappa.B inflammatory cascade. One of the genes activated by NF-kappa.B is the one that codes for angiogenic factors such as VEGF. In this respect, oral contraceptives would be expected to block VEGF expression in the endometrium. However, the use of oral contraceptives alone failed to significantly decrease the percentage of endometria testing positive for VEGF. Introducing pycnogenol to the treatment regimen, on the other hand, significantly reduced the percentage of VEGF-positive cases in oral contraceptive users. Since pycnogenol blocks NF-kappa.B gene transcription after its binding to DNA, the combination of oral contraceptives with pycnogenol would be expected to have a synergetic effect on the eutopic endometrium of endometriosis patients. The expression of other inflammatory-induced genes in the endometrium, such as aromatase, is also significantly diminished with the use of oral contraceptives alone. However, this inhibitory effect was significantly enhanced with the concomitant use of pycnogenol. These results suggest that the concomitant use of pycnogenol with oral contraceptives is more effective in reducing VEGF and aromatase expression in the eutopic endometrium of endometriosis patients than the use of oral contraceptives alone.

Expression of human β-defensin-2 in the eutopic and ectopic endometrial tissues in patients with endometriosis

To investigate the expression of human β-defensin-2 (hBD-2) in the endometrium of patients with endometriosis (EMS) and explore the potential role of hBD-2 in the pathogenesis of EMS. Prospective controlled study. 50 women including EMS patients undergoing laparoscopic ovarian cystectomy and non-EMS patients undergoing hysterectomy for uterine fibroids. Large university teaching hospital. Patients were divided into EMS and non-EMS groups. The gene expressions of hBD-2, interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the endometrial tissues of each group were detected with real-time quantitative polymerase chain reaction (PCR), and hBD-2 protein expression with immunohistochemical method. The gene expression levels of hBD-2, TNF-α, and IL-1β as well as the positive expression rate of hBD-2 protein in the ectopic endometrium of EMS patients were significantly higher than those in the eutopic endometrium of EMS and non-EMS patients (all P < 0.05). Correlation analysis showed that the gene expression levels of hBD-2 in the ectopic and eutopic endometrium of EMS patients were positively correlated with the gene expression levels of IL-1β and TNF-α (P < 0.01). High levels of hBD-2 gene and protein expressions in the ectopic endometrium of EMS patients may be an important contributor in the pathogenesis of EMS. TNF-α and IL-1β may promote the upregulation of hBD-2 expression.