Human Ovarian Endometriosis Research Articles

Total Flavonoids of Polygala fallax Hemsl Induce Apoptosis of Human Ectopic Endometrial Stromal Cells through PI3K/AKT/Bcl-2 Signaling Pathway

Objective: The objective of this study was to explore the inhibitory effect of total flavonoids of Polygala fallax Hemsl (PFHF) on human ectopic endometrial stromal cells (HEcESCs) and its mechanism. Design: The apoptosis, cell cycle, migration, and invasion ability of HEcESCs (Fresh human ovarian endometriosis tissue was used for primary culture) after PFHF treatment were detected, and the mechanism of action was explored. Materials: The Polygala fallax Hemsl (PFH), RPMI 1640 culture medium, Dulbecco’s modified Eagle’s medium (DMEM)/F-12, fetal bovine serum, penicillin/streptomycin, cell counting kit-8 (CCK-8) kit, trypsin, phenylmethylsulfonyl fluoride, radioimmunoprecipitation assay tissue/cell lysate, bicinchoninic acid protein concentration detection kits, protein loading buffer, the apoptosis and cell cycle extraction kits, the matrix glue, TRIzol Universal Reagent, the reverse transcription kit, AB HS Green qPCR Mix, the ECL chromogenic solution, enzyme labeling instrument, flow cytometry, automatic real-time fluorescence quantitative PCR instrument, Goat anti-rabbit, rabbit anti-β-actin, vimentin, phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), B-cell lymphoma-2 (Bcl-2), Bcl-extra long (Bcl-xl), Bcl-2 associated death promoter (Bad) antibody, Alexa Fluor 594-labeled secondary antibody, the inverted microscope, the constant temperature carbon dioxide cell incubator. Setting: Five parts included introduction, materials and methods, results, discussion, and conclusion. Methods: The potential targets and pathways of PFHF in the treatment of endometriosis were predicted by network pharmacology. The effect of PFHF on the proliferation, apoptosis and cell cycle, migration, and invasion of HEcESCs was detected by CCK-8 method, flow cytometry, and Transwell chamber experiment. Label-free quantitative proteomics based on mass spectrometry was used to analyze the protein mass spectrum of differential expression of HEcESCs before and after PFHF, and the biological information was analyzed. The effects of PFHF on the mRNA and protein expression of pathway-related genes predicted in HEcESCs were detected by reverse transcription-quantitative polymerase chain reaction and Western blotting. Results: The network pharmacology predicts that PFHF treats endometriosis through PI3K/AKT signaling pathway. Compared with control group (DMEM/F-12 medium alone), the high dose PFHF can significantly reduce the viability, migration, and invasion of HEcESCs, increase the apoptosis rate of HEcESCs, and make the HEcESCs accumulated in G0/G1 phase in a time- and dose-dependent manner (p < 0.05). The analysis of label-free quantitative proteomics indicated that PFHF flavonoids may induce apoptosis of EESCs through PI3K/AKT signaling pathway. The results of RT-qPCR and Western blotting showed that the expressions of PI3K, AKT, Bcl-2, and Bcl-xl were significantly downregulated, while the bad expression was upregulated in HEcESCs treated with PFHF (p < 0.05). Limitations: This research investigated the effects of PFHF on the stromal endometriotic cells only. So it is unknown how PFHF can affect the entire endometriotic lesion. And the research is carried out in vitro, which gives no impression about the bioavailability of the flavonoids. Conclusion: PFHF reduces the expression of PI3K, AKT, Bcl-2, and Bcl-xl through the PI3K/AKT/Bcl-2 signaling pathway to inhibit HEcESCs proliferation, migration, and invasion and promote their apoptosis.

Detention and mapping of iron and toxic environmental elements in human ovarian endometriosis: A suggested combined role

BackgroundEndometriosis is a disease affecting 10–15 % of women worldwide, consisting in the ectopic growth of endometrial cells outside the uterine cavity.Whist the pathogenetic mechanisms of endometriosis remain elusive and contemplating even environmental causes, iron deposits are common in endometrial lesions, indicating an altered iron metabolism at this level. This study was undertaken to reveal a possible relationship between iron dysmetabolism and accumulation of environmental metals. MethodsBy combining histological and histochemical analysis (H&E and Perl's staining) with μ- and nano- synchrotron-based (SR-based) X-ray Fluorescence (XRF) microscopy, we investigated the distribution of iron and other elements in the ovarian endometriomas of 12 endometriosis patients and in 7 healthy endometrium samples. ResultsXRF microscopy expanded the findings obtained by Perl's staining, revealing with an exceptional sensitivity intracellular features of iron accumulation in the epithelial endometrium, stroma and macrophages of the endometriotic lesions. XRF evidenced that iron was specifically accumulated in multiple micro aggregates, reaching concentrations up to 10–20 % p/p. Moreover, by XRF analysis we revealed for the first time the retention of a number of exogenous and potentially toxic metals such as Pb, Br, Ti, Al Cr, Si and Rb partially or totally co-localizing with iron. ConclusionμXRF reveals accumulation and colocalization of iron and environmental metals in human ovarian endometriosis, suggesting a role in the pathogenesis of endometriosis.

Use of selective PGE2 receptor antagonists on human endometriotic stromal cells and peritoneal macrophages.

Non-hormonal therapeutic strategies for endometriosis are needed. The aim of this study was to characterize the effects of prostaglandin (PG)E2 receptor inhibitors to explore their potential as novel therapeutic strategies for endometriosis. The expression of PGE2 receptors (EP2 and EP4) in donated tissues from human ovarian endometriosis, adenomyosis and peritoneal endometriosis was examined using immunohistochemistry. Human endometriotic stromal cells (ESC) isolated from ovarian endometriotic tissue and peritoneal macrophages were treated with EP2 and EP4 antagonists. cAMP accumulation and the effect of EP antagonists were measured using cAMP assays. DNA synthesis in ESC was detected using bromodeoxyuridine incorporation analysis. Interleukin (IL)-6 and IL-8 protein levels in ESC supernatants were measured using ELISAs. mRNA expression level for aromatase by ESC, and selected cytokines by peritoneal macrophages was measured using RT–PCR. EP2 and EP4 receptors were expressed in cells derived from control and diseased tissue, ovarian endometriotic, adenomyotic and peritoneal lesions. A selective EP2 antagonist reduced DNA synthesis, cAMP accumulation and IL-1β-induced proinflammatory cytokine secretion and aromatase expression. A selective EP4 antagonist negated IL-1β-induced IL-6 secretion and aromatase expression. In peritoneal macrophages, EP expression was elevated in endometriosis samples but the EP4 antagonist reduced cAMP levels and expression of vascular endothelial growth factor, chemokine ligand 2 and chemokine ligand 3 mRNA. EP2 and EP4 are functioning in endometriosis lesions and peritoneal macrophages, and their selective antagonists can reduce EP-mediated actions, therefore, the EP antagonists are potential therapeutic agents for controlling endometriosis.

Identification of germ cell-specific VASA and IFITM3 proteins in human ovarian endometriosis.

BackgroundEndometriosis is a gynaecological disorder that affects 6–10 % of female population. It is characterized by the presence of endometrial tissue outside the uterus, most often in the pelvic peritoneum or ovaries. Recent studies have indicated that mesenchymal endometrial stem cells might get involved in endometriosis progression. Although germ line stem cells have been proved to exist in the ovary, their involvement in ovarian endometriosis has not been investigated. In this preliminary report we aimed to identify germinal stem cell markers in ovarian endometriosis.FindingsTen paraffin-embedded ovarian endometriosis samples were screened for germ cell-specific proteins DDX4 (VASA) and IFITM3, and its relation with stem cell marker OCT4, proliferation marker PCNA and estrogen receptor alpha (ESR1), by immunohistochemistry, immunofluorescence and PCR. DDX4 and IFITM3 proteins were expressed in isolated cells and clusters of cells in the cortical region of ovarian endometriotic cysts. DDX4 and IFITM3 co-localized in cells from endometriotic stroma, and DDX4/IFITM3-expressing cells were positive for ESR1, OCT4 and PCNA. No cells expressing neither DDX4 nor IFITM3 were detected in normal endometrial tissue.ConclusionThe identification of germ cell-specific proteins DDX4 and IFITM3 provides the first evidence of ovarian-sourced cells in ovarian endometriotic lesions and opens up new directions towards understanding the still confusing pathogenesis of endometriosis.

Involvement of estrogen receptor-related receptors in human ovarian endometriosis

To determine whether decreased estrogen receptor alpha (ER-α) expression in endometriotic lesions could be balanced by an increased expression of estrogen receptor-related receptors (ERRs). To evaluate whether ERR-α expression is influenced by hormonal change in fertile and menopausal women. Prospective controlled study. University Hospital, Department of Gynecology. Twenty-five women: 20 women of reproductive age with (n = 10) and without (control; n = 10) endometriosis and 5 menopausal women. Real-time polymerase chain reaction (qPCR). Immunohistochemistry. The ER and ERR expression levels were studied by reverse transcriptase-qPCR, ELISA, and immunohistochemistry using endometriotic and normal endometrial tissues. The ERR-α protein distribution was performed by immunohistochemistry in fertile and menopausal women. Increased levels of ER-β were associated with ER-α, ERR-α, and ERR-γ reductions in ectopic tissue but not in eutopic and normal endometria. Similar levels of ERR-β were found in women with and without endometriosis. The ERR-α expression was similar in proliferative and secretory endometrial samples, whereas a down-regulation of this receptor was found in atrophic tissue. Our data confirm the up-regulation of ER-β as the principal receptor involved in the progression of human endometriosis. In addition, we found that ERR-α seems to be unresponsive to hormonal changes during the menstrual cycle.

Expression patterns of the steroid receptor coactivator family in human ovarian endometriosis

Endometriosis is an estrogen-dependent disease that causes pelvic pain and infertility. In this study, to examine the estrogen-dependent mechanisms of human endometriosis, we focused on the expression patterns of the steroid receptor coactivator (SRC) family of cofactors for nuclear steroid receptors and estrogen receptor α (ERα). The expression patterns of SRC-1, transitional intermediary factor 2 (TIF2), SRC-3, and ERα, were analyzed by immunohistochemistry of normal endometrium and ovarian endometriotic tissue. In addition, reverse transcription polymerase chain reaction (RT-PCR) for the SRCs was performed for ovarian endometriosis. SRCs were expressed in all examined tissues. The expression levels of SRC-1 and the number of SRC-1-positive cells in ovarian endometriosis were greater than those of TIF2 and SRC-3. In addition, immunohistochemistry showed that ERα was colocalized with SRC-1 in almost all glandular and many stromal cells in ovarian endometriotic tissue. The present study demonstrates the expression pattern of SRCs in ovarian endometriosis. It appears that SRC-1 is predominant among the other SRC family members and colocalizes with ERα. Although further study is needed, SRC-1 may affect the transcriptional activity of ERα in human ovarian endometriosis.

DFF45 expression in ovarian endometriomas

Objective Endometriosis, defined as a spread of endometrium outside the uterus cavity, affects up to 30% women of reproductive age, with the ovaries being its most common localization. In the ectopic lesions, endometrial cells show abnormal proliferation and impaired apoptosis. The DNA destruction during apoptosis is a direct result of activation of the DFF40/DFF45 complex. DFF40 (DNA fragmentation factor of 40 kDa) is responsible for direct DNA fragmentation while DFF45 (DNA fragmentation factor of 45 kDa) acts not only as a DFF40 inhibitor, but also as its chaperone. Therefore, the presence of DFF45 is required for proper DFF40 synthesis. The aim of this study was to determine the DFF45 level in human ovarian endometriosis. Study design The endometriosis samples were collected from 43 affected women, while the 81 normal endometrial specimens were obtained from the control group. Western blot and immunohistochemistry tests were used to determine the DFF45 level in examined tissues. Results The expression of DFF45 in normal human endometrium and ovarian endometriosis was confirmed using both the Western blot and the immunohistochemistry tests. In normal eutopic proliferatory endometrium, a lower DFF45 expression was observed compared with secretory endometrium, while no cyclic changes in DFF45 expression were observed in the ovarian endometriomas. In the normal eutopic endometrium, stronger DFF45 staining was noted in the endometrial glands in comparison to the stroma, irrespective of menstrual cycle phase. However, in the ovarian endometriosis no difference between the glandular layer and stroma in DFF45 immunoreactivity was appreciated. The lowest level of DFF45 was observed in ovarian endometriosis when compared with both normal eutopic proliferatory and secretory endometria using the Western blot and immunohistochemistry analysis. Conclusions A decreased level of DFF45 observed in ovarian endometriosis may be a part of an apoptosis-resistant mechanism enhancing the disease progression.

Involvement of regulatory T cells and Th17 cells in ovarian endometriosis and ovarian epithelial tumors

Endometriosis is a chronic inflammatory disorder defined as abnormal growth of endometrial tissue at ectopic sites, most often on the ovary. Women with endometriosis are at higher risk of developing ovarian cancer. We recently generated a mouse model for ovarian endometriosis, using the previously established LSL‐KrasG12D mice, which we crossed with MUC1 transgenic mice. Following intra‐bursal administration of Cre‐encoding adenovirus, the MUC1Kras mice develop de novo, endometriosis‐like lesions. Interestingly, these mice show an increased percentage of FoxP3+ regulatory T cells in their spleens and para‐aortic lymph nodes but fewer Th17.We also investigated the pattern of FoxP3, IL‐17, TGF‐b, IL‐1 and IL‐6 mRNA expression in human ovarian endometriosis and ovarian cancer using qRT‐PCR. Our results from a total of 23 patients (normal donors, physiological ovarian cysts, endometriosis, ovarian tumors with paired ascites) show that FoxP3 was detected in the ascites of all tested patients and in the majority of tumor specimens but also in 6 out of 9 of patients with endometriosis, confirming our results in mice. IL‐17 was also up‐regulated in 55% of endometriosis cases and it was highly up‐regulated in ovarian cysts. Further studies on the immune imbalance in premalignancy (endometriosis) and ovarian cancer in mice and humans are in progress. Research support from the Continuing Medical Research Fund (AMV).

Expression analysis of the genes involved in estradiol and progesterone action in human ovarian endometriosis

Endometriosis is defined as the presence of endometrial glands and stroma within extrauterine sites, and it is well known that endometriosis is an estrogen-dependent disease. The defective formation and metabolism of steroid hormones is responsible for the promotion and development of endometriosis. In the present study we examined the mRNA levels of six enzymes that are involved in the metabolism of estrogen and progesterone – aromatase, 17β-hydroxysteroid dehydrogenase (17β-HSD) types 1, 2 and 7, sulfatase and sulfotransferase – and of the steroid receptors – estrogen receptors α and β (ERα, ERβ) and progesterone receptors A and B (PRAB) – implicated in human ovarian endometriosis. We analyzed 16 samples of ovarian endometriosis and 9 of normal endometrium. The real-time polymerase chain reaction analyses revealed that six of the nine genes investigated are differentially regulated. Aromatase, 17β-HSD types 1 and 7, sulfatase and ERβ were statistically significantly upregulated, while ERα was significantly downregulated, in the endometriosis group compared with the control group. There were no significant differences in 17β-HSD type 2, sulfotransferase and PRAB gene expression. Our results indicate that, in addition to the previously reported upregulation of aromatase, upregulation of 17β-HSD types 1 and 7 and sulfatase can also increase the local estradiol concentration. This could thus be responsible for the estrogen-dependent growth of endometriotic tissue. Surprisingly ERα was downregulated.

Dominant Expression of Progesterone Receptor Form B mRNA in Ovarian Endometriosis

This study was designed to examine the biological implications of progesterone receptor form A (PR-A) and B (PR-B) mRNA expressions in human ovarian endometriosis (ectopic endometrium). A high ratio of PR-B to PR-AB (PR-A+PR-B) mRNA expression was found in 8 of 14 cases of endometriosis, compared with the ratio in eutopic endometrium. The mean ratio in ectopic endometria was significantly (p < 0.01) higher than in eutopic endometria. The ratio in eutopic and ectopic endometria showed no significant change during the menstrual cycle. The mean ratio in ectopic endometria in the proliferative and secretory phases of the endometrium was significantly (p < 0.01) higher than in eutopic endometria. In conclusion, PR-B mRNA was relatively highly expressed in some endometriomas, which might lead to aberrations in the control of progestational effects involving responsiveness to sex steroidal growth regulation.

Expression of sex hormone-binding globulin exon VII splicing variant messenger ribonucleic acid in human ovarian endometriosis

Objective: To investigate the expression of sex hormone-binding globulin (SHBG) exon VII splicing variant messenger RNA (mRNA) in human ovarian endometriosis. Design: The expression of SHBG variant mRNA in normal uterine endometrium and endometriotic tissue was determined. Setting: Department of Obstetrics and Gynecology, Gifu University Hospital. Patient(s): Fourteen women with endometriosis and 18 women without endometriosis. Intervention(s): Normal uterine endometria and ovarian endometriotic tissues from patients who had undergone gynecological surgery were studied. Main Outcome Measure(s): Levels of SHBG wild-type and variant mRNAs were determined using the quantitative reverse transcription-polymerase chain reaction. Result(s): Analysis of the missing base pairs proved that they corresponded to the entire exon VII. There was no significant difference between the levels of SHBG wild-type mRNA in normal endometria and in endometriotic endometria, although the levels of SHBG variant mRNA in endometriotic endometria were significantly higher than that in normal endometria. The ratio of SHBG variant to wild-type mRNA levels was significantly higher in endometriotic endometria than in normal endometria. Conclusion(s): This study demonstrates the coexpression of SHBG exon VII splicing variant mRNA with its wild-type and the dominant expression of the variant in ovarian endometriosis. These results might be involved in the cellular estrogenic interaction, plausibly assisting in the development and growth of ovarian endometriosis.