The role of unfolded protein response in the pathogenesis of endometriosis: contribution of peritoneal fluid.

RESEARCH QUESTION: Endoplasmic reticulum stress (ERS) is caused by the accumulation of the misfolded or unfolded proteins in the endoplasmic reticulum and induces the unfolded protein response (UPR). Peritoneal fluid is important in the pathogenesis of endometriosis. In this study, the role of UPR associated with ERS in endometriosis, and peritoneal fluid, were investigated. DESIGN: Normal, eutopic and ectopic endometrium tissues were divided into menstrual cycle phases, and endometrial stromal cells (ESC) were treated with 10-20% concentration of control peritoneal fluid and peritoneal fluid obtained from women with endometriosis for 10, 30 and 60 min, and 24 and 48 h. The UPR signalling proteins were analysed immunohistochemically and immunocytochemically. Data were compared statistically. RESULTS: p-IRE1 was increased in ectopic glandular and stromal cells in the early proliferative phase compared with normal and eutopic endometrium. p-PERK increased in ectopic glandular and stromal cells in the late proliferative phase compared with normal endometrium. ATF6 was increased in ectopic glandular epithelium compared with normal endometrium in the proliferative phases, versus eutopic endometrium in the late secretory phase. p-IRE1 and p-PERK were increased in high concentrations of ESC treated with peritoneal fluid obtained from women with endometriosis for 10, 30 and 60 min compared with controls. In ESC treated with peritoneal fluid from women with endometriosis, p-IRE1 decreased at 24-48 h compared with 30 min. CONCLUSIONS: In endometriosis, UPR pathways are activated as highly dependent on cell type and phase. Also, p-PERK and p-IRE1 increased because of exposure to high-dose peritoneal fluid from women with endometriosis in stromal cells. Our findings provide a basis for further studies searching for a potential biomarker for the diagnosis of endometriosis.

A potential role of Sirtuin3 and its target enzyme activities in patients with ovarian endometrioma.

OBJECTIVE: Sirtuin3 (SIRT3) is a NAD+-dependent major mitochondrial deacetylase. In this study, we aimed to investigate SIRT3 levels and their target enzyme activities, including glutamate dehydrogenase (GDH), succinate dehydrogenase (SDH), and manganese superoxide dismutase (MnSOD), also to determine the antioxidant capacity and oxidative stress in tissue, mitochondria and serum samples in ovarian endometrioma patients. METHODS: We collected serum and endometrioma tissue samples from 30 patients. In the control group, we collected serum and eutopic endometrial tissue samples from 26 women without endometriosis. RESULTS: SIRT3 levels were significantly decreased in endometrioma tissue samples compared to the control group. There was no statistically significant difference in SIRT3 levels between patient and control serum samples. Furthermore, there was a decrease in GDH and SDH enzyme activities in both endometrioma tissue homogenate and mitochondria. MnSOD activity was decreased in tissue homogenate but increased in mitochondria and there was no difference in serum. While total SOD activity was decreased, CuZnSOD activity was increased in both tissue and serum samples. Besides these, total antioxidant capacity and advanced oxidation protein products (AOPP) levels were decreased in endometrioma tissue and mitochondria, but there was no difference in serum. CONCLUSIONS: Our results suggested that decreased levels of SIRT3 in endometrioma may be an important factor in the weakening of mitochondrial energy metabolism and antioxidant defense in endometriosis. We think that SIRT3 deficiency may be an important factor underlying the pathogenesis of endometriosis. More detailed studies are needed to reveal the relationship between SIRT3 and metabolism and oxidative stress in ovarian endometrioma.

miR-488 mediates negative regulation of the AKT/NF-κB pathway by targeting Rac1 in LPS-induced inflammation.

Endometritis is an inflammatory change in the structure of the endometrium due to various causes and is a common cause of infertility. Studies have confirmed that microRNAs (miRNAs) play a key regulatory role in various inflammatory diseases. However, the miRNA-mediated mechanism of endometrial inflammation induced by lipopolysaccharides (LPS) remains unclear. In this study, real-time quantitative polymerase chain reaction, Western blot analysis, immunofluorescence and Rac family small GTPase 1 (Rac1) interference were used to reveal the overexpression of miR-488 in the LPS-induced bovine uterus, and the effect of protein kinase B κ-light chain enhancement of the nuclear factor-activated B cells (AKT/NF-κB) pathway in intimal epithelial cells. The results showed that the expression of inflammatory cytokines such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α in the experimental group was significantly lower than that in the control group when miR-488 was overexpressed. Similar results were observed in the expression levels of p-AKT, p-IKK, and p-p65 proteins. In addition, the dual-luciferase reporter system confirmed that miRNA-488 may directly target the 3'-untranslated region of Rac1. In turn, the expression of Rac1 was inhibited. Moreover, the nuclear translocation of NF-κB was inhibited, and meanwhile, the accumulation of reactive oxygen species (ROS) in the cells was reduced. Thus, we provide basic data for the negative regulation of miR-488 in LPS-induced inflammation by inhibiting ROS production and the AKT/NF-kB pathway in intimal epithelial cells.

Aldo-keto reductase 1C3-Assessment as a new target for the treatment of endometriosis.

Endometriosis is a common gynecological disorder, which is treated surgically and/ or pharmacologically with an unmet clinical need for new therapeutics. A completed phase I trial and a recent phase II trial that investigated the steroidal aldo-keto reductase 1C3 (AKR1C3) inhibitor BAY1128688 in endometriosis patients prompted this critical assessment on the role of AKR1C3 in endometriosis. This review includes an introduction to endometriosis with emphasis on the roles of prostaglandins and progesterone in its pathophysiology. This is followed by an overview of the major enzymatic activities and physiological functions of AKR1C3 and of the data published to date on the expression of AKR1C3 in endometriosis at the mRNA and protein levels. The review concludes with the rationale for using AKR1C3 inhibitors, a discussion of the effects of AKR1C3 inhibition on the pathophysiology of endometriosis and a brief overview of other drugs under clinical investigation for this indication.

PI3K/AKT pathway is altered in the endometriosis patient's endometrium and presents differences according to severity stage.

Based on the inflammatory nature and hormone-dependency of endometriosis, PI3K/AKT signaling appears to influence its progression. Could the endometriosis stages be linked to differential changes in PI3K/AKT pathway regulation? The objective is to evaluate the expression of PI3K, PTEN, AKT and p-AKT in endometrial human biopsies, according to the presence or absence of the disease, and to assess the underlying differences regarding the endometriosis stages. Biopsy specimens of the ectopic and eutopic endometrium were obtained from twenty women with untreated peritoneal endometriosis as well as endometrium biopsies from nine controls. Our study revealed an increased expression of PI3K in eutopic and ectopic endometrium from patients with endometriosis, and a reduced expression of PTEN and increased levels of AKT phosphorylation, compared to control endometrium. Both eutopic and ectopic endometrium from patients with minimal-mild endometriosis expressed a significant reduced PTEN level compared to the respective endometrium from patients with moderate-severe endometriosis. The ratio p-AKT/total AKT showed higher levels of AKT phosphorylation in endometriotic tissue from patients with minimal-mild endometriosis. This study has firmly confirmed the alteration in PI3K/AKT pathway regulation and demonstrated clear differences between the stages of endometriosis, emphasizing the importance of this pathway in the first stage of the disease.

Expression of Matrix Metalloproteinases and Their Inhibitors in Endometrium: High Levels in Endometriotic Lesions.

Endometriosis is a condition defined as presence of endometrium outside of the uterine cavity. These endometrial cells are able to attach and invade the peritoneum or ovary, thus forming respectively the deep infiltrating endometriosis (DIE) and the ovarian endometrioma (OMA), the ectopic lesions feature of this pathology. Endometriotic cells display high invasiveness and share some features of malignancy with cancer cells. Indeed, the tissue remodeling underlining lesion formation is achieved by matrix metalloproteinases (MMPs) and their inhibitors. Therefore, these molecules are believed to play a key role in development and pathogenesis of endometriosis. This study investigated the molecular profile of metalloproteinases and their inhibitors in healthy (n = 15) and eutopic endometrium (n = 19) in OMA (n = 10) and DIE (n = 9); moreover, we firstly validated the most reliable housekeeping genes allowing accurate gene expression analysis in these tissues. Gene expression, Western blot, and immunofluorescence analysis of MMP2, MMP3, and MMP10 and their tissue inhibitors TIMP1 and TIMP2 demonstrated that these enzymes are finely tuned in these tissues. In OMA lesions, all the investigated MMPs and their inhibitors were significantly increased, while DIE expressed high levels of MMP3. Finally, in vitro TNFα treatment induced a significant upregulation of MMP3, MMP10, and TIMP2 in both healthy and eutopic endometrial stromal cells. This study, shedding light on MMP and TIMP expression in endometriosis, confirms that these molecules are altered both in eutopic endometrium and endometriotic lesions. Although further studies are needed, these data may help in understanding the molecular mechanisms involved in the extracellular matrix remodeling, a crucial process for the endometrial physiology.

Inhibition of Cyclooxygenase-2 Suppresses the Recruitment of Endothelial Progenitor Cells in the Microvasculature of Endometriotic Lesions.

The incorporation of endothelial progenitor cells (EPCs) into newly developing blood vessels contributes to the vascularization of endometriotic lesions. We analyzed whether cyclooxygenase (COX)-2 signaling regulates this vasculogenic process. Endometriotic lesions were surgically induced in irradiated FVB/N mice, which were reconstituted with bone marrow from FVB/N-TgN [Tie2/green fluorescent protein (GFP)] 287 Sato mice. The animals received ß-estradiol 17-valerate once a week and were treated daily with the selective COX-2 inhibitor parecoxib (25 mg/kg) or vehicle (control) for 7 and 28 days. Analyses involved the determination of lesion growth, cyst formation, homing of GFP+/Tie2+ EPCs, numbers of circulating EPCs, vascularization, cell proliferation, apoptosis, and immune cell infiltration by means of high-resolution ultrasonography, caliper measurements, flow cytometry, histologic analysis, and immunohistochemical analysis. In parecoxib-treated mice, blood circulating EPCs were higher, but numbers of recruited EPCs in endometriotic lesions were significantly lower when compared with controls. This finding was associated with an impaired early vascularization and stromal tissue growth as well as reduced glandular secretory activity of the lesions. Parecoxib-treated lesions further contained less proliferating and more apoptotic cells and exhibited lower numbers of infiltrating macrophages and neutrophilic granulocytes. These findings demonstrate that the inhibition of COX-2 suppresses vasculogenesis in endometriotic lesions, which may contribute to an impaired lesion vascularization and growth.

Cai's Neiyi Prescription promotes apoptosis and inhibits inflammation in endometrial stromal cells with endometriosis through inhibiting USP10.

To observe the effect of Cai's Neiyi Prescription (CNYP) on the apoptosis and inflammation in endometrial stromal cells with endometriosis (EM) both in vivo and in vitro, EM model rats and endometrial stromal cells were treated with CNYP and the level of USP10, p-ERK1/2, ERK1/2, and apoptosis-related protein as well as the levels of proinflammatory factors were measured by Western blotting and ELISA, respectively. Rats with surgically induced EM showed increased USP10 expression and ERK/2 activation. Intragastric administration of CNYP granule significantly inhibited EM-induced ERK1/2 activation and expression of USP10 and Bcl-2, but increased the expression of Bax and Caspase-7 in EM-induced rats. CNYP granule administration also inhibited EM-induced inflammation in rats. Moreover, the ectopic endometrial stromal cells isolated from EM patients demonstrated decreased ERK1/2 activation and expression of USP10 and Bcl-2 and increased expression of Bax and Caspase-7 after cultured in DMEM containing CNYP-medicated rat serum, which were reversed by USP10 overexpression and were enhanced by USP10 siRNA. USP10 overexpression also inhibited while USP10 siRNA enhanced the CNYP-induced inhibition of inflammation in ectopic endometrial stromal cells. Taken together, our results suggest that CNYP granule promotes apoptosis and inhibits inflammation in endometrial stromal cells with EM through inhibiting USP10.

Impaired Expression of Ectonucleotidases in Ectopic and Eutopic Endometrial Tissue Is in Favor of ATP Accumulation in the Tissue Microenvironment in Endometriosis.

Endometriosis is a prevalent disease defined by the presence of endometrial tissue outside the uterus. Adenosine triphosphate (ATP), as a proinflammatory molecule, promotes and helps maintain the inflammatory state of endometriosis. Moreover, ATP has a direct influence on the two main symptoms of endometriosis: infertility and pain. Purinergic signaling, the group of biological responses to extracellular nucleotides such as ATP and nucleosides such as adenosine, is involved in the biology of reproduction and is impaired in pathologies with an inflammatory component such as endometriosis. We have previously demonstrated that ectonucleotidases, the enzymes regulating extracellular ATP levels, are active in non-pathological endometria, with hormone-dependent changes in expression throughout the cycle. In the present study we have focused on the expression of ectonucleotidases by means of immunohistochemistry and in situ activity in eutopic and ectopic endometrial tissue of women with endometriosis, and we compared the results with endometria of women without the disease. We have demonstrated that the axis CD39-CD73 is altered in endometriosis, with loss of CD39 and CD73 expression in deep infiltrating endometriosis, the most severe, and most recurring, endometriosis subtype. Our results indicate that this altered expression of ectonucleotidases in endometriosis boosts ATP accumulation in the tissue microenvironment. An important finding is the identification of the nucleotide pyrophophatase/phosphodiesterase 3 (NPP3) as a new histopathological marker of the disease since we have demonstrated its expression in the stroma only in endometriosis, in both eutopic and ectopic tissue. Therefore, targeting the proteins directly involved in ATP breakdown could be an appropriate approach to consider in the treatment of endometriosis.

Granulosa-Lutein Cell Sirtuin Gene Expression Profiles Differ between Normal Donors and Infertile Women.

Sirtuins are a family of deacetylases that modify structural proteins, metabolic enzymes, and histones to change cellular protein localization and function. In mammals, there are seven sirtuins involved in processes like oxidative stress or metabolic homeostasis associated with aging, degeneration or cancer. We studied gene expression of sirtuins by qRT-PCR in human mural granulosa-lutein cells (hGL) from IVF patients in different infertility diagnostic groups and in oocyte donors (OD; control group). Study 1: sirtuins genes' expression levels and correlations with age and IVF parameters in women with no ovarian factor. We found significantly higher expression levels of SIRT1, SIRT2 and SIRT5 in patients ≥40 years old than in OD and in women between 27 and 39 years old with tubal or male factor, and no ovarian factor (NOF). Only SIRT2, SIRT5 and SIRT7 expression correlated with age. Study 2: sirtuin genes' expression in women poor responders (PR), endometriosis (EM) and polycystic ovarian syndrome. Compared to NOF controls, we found higher SIRT2 gene expression in all diagnostic groups while SIRT3, SIRT5, SIRT6 and SIRT7 expression were higher only in PR. Related to clinical parameters SIRT1, SIRT6 and SIRT7 correlate positively with FSH and LH doses administered in EM patients. The number of mature oocytes retrieved in PR is positively correlated with the expression levels of SIRT3, SIRT4 and SIRT5. These data suggest that cellular physiopathology in PR's follicle may be associated with cumulative DNA damage, indicating that further studies are necessary.

The levels of matrix metalloproteinase-9 and neutrophil gelatinase-associated lipocalin in different stages of endometriosis.

This study was designed to explore matrix metalloproteinase-9 (MMP-9), neutrophil gelatinase-associated lipocalin (NGAL) levels and MMP-9/NGAL ratio in women with and without endometriosis diagnosed surgically and/or histopathologically. The correlation between biomarkers and the severity of the disease is analysed. The revised American Fertility Society classification system was used to determine the severity of endometriosis. Serum MMP-9 and Ca125, urine NGAL levels were measured in all participants. Serum MMP-9 levels were significantly higher in the study group (n = 60) compared to controls (n = 31) (15.0 pg/mL (6.0-143.0) vs. 12.0 (4.0-18.0), respectively; p=.002). MMP-9 levels were significantly higher in severe endometriosis compared to mild endometriosis subgroups (p<.001). No significant difference was found between NGAL levels in study and control groups (p>.05). The diagnostic value of MMP-9 and NGAL is not superior than CA-125 for endometriosis. Nevertheless, MMP-9 might be a potential predictive marker for advanced stage of the disease. Impact Statement What is already known on this subject? The gold standard diagnostic test for diagnosis of endometriosis is laparoscopy combined with histopathological confirmation of eutopic endometrial glands and/or stroma. Both invasiveness and possible accompanying complications limit the preference regarding the surgical approach. Among non-invasive markers none has been accepted as gold standard neither for diagnosis nor for determining the severity of the disease. MMPs are extracellular endopeptidases, which have a significant role in degradation and remodelling of extracellular matrix for cellular migration and invasion. Among these, MMP-9 has been shown to be higher in eutopic/ectopic endometrial tissue in women with endometriosis and has been suggested to have a role in pathogenesis of endometriosis by promoting invasion of the endometriotic lesions. NGAL is an acute phase protein, which is involved in a variety of physiological and pathophysiological processes. The molecule has also been revealed to correlate with endometriosis pathophysiology through the epithelial-mesenchymal transition process which is the basis for the onset of endometriosis. But also, NGAL which composes a complex with MMP-9 (MMP-9 and NGAL complex), has been shown to protect MMP-9 from autodegradation in vitro which might be a contributing factor for endometriosis pathophysiology. What the results of this study add? MMP-9 cut-off level for prediction of severe endometriosis is a novel finding obtained from this study with acceptable sensitivity and specificity. On the other hand, NGAL seems to have no significant value either for diagnosis of for determining severity of the disease. After all, MMP-9 might be an easy use acceptable biomarker for endometriosis but further studies on larger populations are needed. What the implications are of these findings for clinical practice and/or further research? MMP might be a potential non-invasive predictive marker for advanced stage disease.

Evaluation of Total, Active, and Specific Myeloperoxidase Levels in Women with and without Endometriosis.

Myeloperoxidase (MPO) is a proinflammatory enzyme and a marker for neutrophil activation and oxidative stress. Since oxidative stress and inflammation are linked to the pathogenesis of endometriosis, we hypothesized that the total, active, and specific (active/total) MPO levels were significantly different in plasma of women with and without endometriosis. Samples were selected from our biobank from women with endometriosis (n = 212) and controls without endometriosis (n = 121) across the menstrual cycle. Total MPO plasma levels were measured by immunoassay and MPO activity by enzymatic assay. Total and active MPO levels did not differ significantly among endometriosis cases and controls, whereas the specific MPO activity was significantly lower in women with endometriosis than that in controls (p = 0.0159). After the subdivision of control patients into women with a normal pelvis and women with other benign gynecological disorders, a significant difference was observed only between women with endometriosis and women with other benign gynecological disorders (p = 0.0266). In conclusion, systemic MPO levels may not be suited as a single biomarker for endometriosis. Our data support the involvement of MPO in other gynecological disorders but do not provide any evidence for an association with endometriosis.

Aromatase as a target for treating endometriosis.

Endometriosis is a common gynecological disease that causes various clinical symptoms, such as chronic pelvic pain, dysmenorrhea and infertility, seriously affecting women's health and their quality of life. The symptoms and endometriotic lesions are relieved, in many cases, after menopause, when estrogen levels are lowered. Therefore, endometriosis is considered to be estrogen-dependent. Aromatase, the enzyme responsible for the last step of estrogen biosynthesis converting testosterone and androgen to estrogen, was previously reported to be more abundant in endometriotic tissues than in the normal endometrium, leading to an increased local estrogen concentration. Therefore, aromatase is considered a key therapeutic target for regulating local estrogen biosynthesis in endometriosis. A more complete understanding of the mechanisms that modulate aromatase and its activity is required to develop novel estrogen-targeted therapies for endometriosis. In this review article, we outline the current understanding of the pathological processes involved in estrogen production in endometriosis and propose novel strategies to treat this disorder.

Immunohistochemical Features of O6-Methylguanine-DNA Methyltransferase Expression during Ovarian Endometriosis.

A comparative immunohistochemical study for the expression of O6-methylguanine-DNA methyltransferase (MGMT) was performed in tissues of the eutopic endometrium and ovarian endometriosis. The highest level of MGMT expression in eutopic endometrial tissue was observed in epitheliocyte nuclei during the proliferative phase. In regions of endometriosis the expression of MGMT in epitheliocyte nuclei was shown to increase during stages I and II, but decreased in stages III and IV. The progression of endometriosis was accompanied by a gradual increase of study parameters in the nuclei and cytoplasm of stromal cells. These changes reflect the impairment of DNA reparation, which probably serves as a stage in the development and progression of endometriosis.

Is cytochrome P450 3A4 regulated by menstrual cycle hormones in control endometrium and endometriosis?

The estrogen-metabolizing activities of cytochrome P450 (CYP) enzymes have been implicated in endometriosis. However, their regulation in various sources of endometrial tissue under different hormonal conditions has not been clarified. Our objective was to study the hormone regulation of a specific CYP enzyme, namely CYP3A4, in control (n = 15) and endometriosis patients (n = 42). To this end, we evaluated mRNA expression (using real-time PCR) of CYP3A4 in tissue samples classified according to the phase of menstrual cycle at which they were obtained as confirmed by the related circulating hormone levels. Protein expression was also evaluated by Western Blot. In order to further investigate the hormonal regulation of CYP3A4, stromal cells from ovarian endometriotic lesions were cultured with the prevailing hormones of the distinct phases of the menstrual cycle. We observed that all control and endometriosis tissues express CYP3A4. Nevertheless, changes in CYP3A4 gene expression related to cycle phase were only seen in the control eutopic endometrium and not in samples from endometriosis patients, with an increase in the luteal phase. Stromal cells isolated from ovarian endometriotic lesions expressed CYP3A4 and their exposure to luteal phase-mimicking hormones (estradiol + progesterone) reduced CYP3A4 mRNA in parallel with a diminished expression of the corresponding receptors, estrogen receptor alpha and progesterone receptor. Our findings suggest that steroid hormones are able to regulate CYP3A4 mRNA expression, although the circulating levels of these hormones can only regulate control endometrium and not endometriosis tissues, probably because of dysregulated local steroid concentration in these latter samples.

In Vitro and In Vivo Effects of Tumor Suppressor Gene PTEN on Endometriosis: An Experimental Study.

BACKGROUND Endometriosis can cause dysmenorrhea and infertility. Its pathogenesis has not yet been clarified and its treatment continues to pose enormous challenges. The protein tyrosine phosphatase (PTEN) gene is a tumor suppressor gene. The aim of this study was to investigate the role and significance of PTEN protein in the occurrence, development, and treatment of endometriosis through changes in apoptosis rate, cell cycle, and angiogenesis. MATERIAL AND METHODS PTEN was overexpressed and silenced in lentiviral vectors and inserted into primary endometrial cells. The changes in cell cycle and apoptosis in the different PTEN expression groups were evaluated using flow cytometry. Vessel growth mimicry was observed using 3-dimensional culture. A human-mouse chimeric endometriosis model was constructed using SCID mice. Hematoxylin and eosin staining and immunohistochemistry were used to detect pathological changes in ectopic endometrial tissues and the expression of VEGF protein in a human-mouse chimeric endometriosis mouse model. RESULTS PTEN overexpression significantly increased apoptosis and inhibited the cell cycle compared with the silenced and control groups. Furthermore, cells expressing low PTEN levels were better able to undergo vasculogenic mimicry, and exhibited significantly increased angiogenesis compared to cells overexpressing PTEN. We found that ectopic foci were more easily formed in the endometrial tissue of SCID mice with low PTEN expression, and the VEGF expression in this group was relatively high. CONCLUSIONS PTEN inhibits the occurrence and development of endometriosis by regulating angiogenesis and the apoptosis and cell cycle of endometrial cells; therefore, we propose that the PTEN gene can be used to treat endometriosis.

MiR-191 modulates malignant transformation of endometriosis through regulating TIMP3.

BACKGROUND: Although aberrant expression of several miRNAs was found during the pathological development of endometriosis to endometriosis-associated ovarian cancer (EAOC), their roles are not fully understood. miR-191 is a miRNA significantly upregulated in endometriosis and EAOC patients. However, its downstream network is still not clear. This study explored its role in malignant transformation of endometriosis to EAOC. MATERIAL AND METHODS: Tissues from 12 healthy controls, 12 patients with endometriomas, and 12 patients with EAOC were used to verify miR-191 expression by using qRT-PCR. Endometriosis cell line CRL-7566 and ovarian endometrioid carcinoma cell line CRL-11731 were used to explore the downstream regulative function of miR-191. RESULTS: By using tissue and serum samples from healthy, endometriosis, and EAOC participants, we confirmed that miR-191 expression was significantly higher in endometriosis and EAOC participants. Interestingly, we also observed that TIMP3 expression was negatively correlated with miR-191 expression. Overexpressing miR-191 in CRL-7566 significantly increased cell proliferation and invasion, while miR-191 knockdown in CRL-11731 cells significantly decreased cell proliferation and invasion. These modulating effects of miR-191 are achieved through its regulation of TIMP3. CONCLUSIONS: miR-191 can directly regulate TIMP3 expression, thereby affecting cell proliferation rate and invasion ability. The miR-191-TIMP3 axis might be critical in the malignant transformation of endometriosis to EAOC.

Clarithromycin regresses endometriotic implants in rat endometriosis model.

The aim of this study was to investigate the effect of clarithromycin in rat endometriosis and its association with matrix metalloproteinase-9 (MMP-9) expression. After surgical induction of endometriosis, 27 rats were randomised into three groups. Size of endometriotic implants were evalutated and rats in group I (n = 9) were given 100 mg/kg/day of oral clarithromycin, rats in group II (n = 9) were given single 1 mg/kg s.c. injection of leuprolide acetate and rats in group III (n = 9) were not given any medication for 21 days. At the end of 21 days of medication, remaining 23 rats were sacrificed to evaluate morphological and histological features of implants. There was a significant difference between the groups in implant volumes (p = 0.004) before and after medication. Regression of implants were significantly higher in groups I and II than that in control group (p = 0.009 and p = 0.011, respectively). After medication, in group I the implant volume decreased from 62 (12-166) mm(3) to 26 (3-87) mm(3) (p = 0.012) and in group II the volume decreased from 224 (76-1135) mm(3) to 62 (26-101) mm(3) (p = 0.028). There was a significant difference between groups in histopathological score (p = 0.024). The epithelial immunohistochemical score of MMP-9 was significantly lower in group II than that in control group (p = 0.014). In conclusion, clarithromycin regresses endometriotic implants in rats, but not via MMP-9.

Effect of warming Yang and removing blood stasis method on matrix metalloproteinases / tissue inhibitor metalloproteinases levels secreted by cultured endometrial cells from patients with endometriosis.

OBJECTIVE: To investigate the effect of Chinese medicines using the warming Yang and removing blood stasis method on levels of matrix metalloproteinases (MMPs)/tissue inhibitor metalloproteinases (TIMPs) secreted by cultured endometrial cells from patients with endometriosis. METHODS: Ectopic and eutopic endometrial cells obtaind from 15 endometriosis patients were cultured in vitro, and divided randomly into five groups: high dose; moderate dose; low dose; nemestran; blank control. The three dose groups were treated with a decoction prepared according to the principle of warming Yang and removing blood stasis; nemestran and 0.9% NaCl were administered to the nemestran group and balnk control group, respectively. Eutopic endometrial cells obtaind from 10 hysteromyoma patients were cultured in vitro, as the normal control group, 0.9% NaCl were administered to the normal control group. Cell culture supernatants were collected and levels of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), tissue inhibitor metalloproteinase-1 (TIMP-1) and tissue inhibitor metalloproteinase-2 (TIMP-2) detected by enzyme-linked immuno sorbent assay (ELISA). RESULTS: Compared with the normal control group, levels of MMP-1, MMP-2, and MMP-9 in eutopic and ectopic endometrium cell supernatants in the blank control group were increased, whereas levels of TIMP-1 and TIMP-2 were decreased (P < 0.05). Compared with the blank control group, levels of MMP-1 and MMP-2 in ectopic and eutopic endometrium cell supernatants cultured in low-dose, middle-dose, and high-dose groups were decreased, whereas levels of TIMP-1 and TIMP-2 were increased significantly (P < 0.05). CONCLUSION: The warming Yang and removing blood stasis method affects expression of MMPs and TIMPs.

Expression, activation, and role of AKT isoforms in the uterus.

The three isoforms of AKT: AKT1, AKT2, and AKT3, are crucial regulators of both normal and pathological cellular processes. Each of these isoforms exhibits a high level of homology and functional redundancy with each other. However, while being highly similar and structurally homologous, a rising amount of evidence is showing that each isoform possesses specific targets as well as preferential subcellular localization. The role of AKT has been studied extensively in reproductive processes, but isoform-specific roles are yet to be fully understood. This review will focus on the role of AKT in the uterus and its function in processes related to cell death and proliferation such as embryo implantation, decidualization, endometriosis, and endometrial cancer in an isoform-centric manner. In this review, we will cover the activation of AKT in various settings, localization of isoforms in subcellular compartments, and the effect of isoform expression on cellular processes. To fully understand the dynamic molecular processes taking place in the uterus, it is crucial that we better understand the physiological role of AKT isoforms as well as their function in the emergence of diseases.