Could an aberrant microRNA (miRNA) expression profile be responsible for the changes in the angiogenic and fibrinolytic states observed in endometriotic lesions? This study revealed characteristic miRNA expression profiles associated with endometriosis in endometrial tissue and endometriotic lesions from the same patient and their correlation with the most important angiogenic and fibrinolytic factors. An important role for dysregulated miRNA expression in the pathogenesis of endometriosis is well documented. However, to the best of our knowledge, there are no reports of the relationship between angiogenic and fibrinolytic factors and miRNAs when endometrial tissue and different types of endometriotic lesions from the same patient are compared. Case–control study that involved 51 women with endometriosis and 32 women without the disease (controls). The miRNA expression profiles were determined using the GeneChip miRNA 2.0 Affymetrix array platform, and the results were analysed using Partek Genomic Suite software. To validate the obtained results, 12 miRNAs differentially expressed were quantified by using miRCURY LNA™ Universal RT microRNA PCR. Levels of vascular endothelial growth factor (VEGF-A), thrombospondin-1 (TSP-1), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) proteins were quantified by ELISA. Patient endometrial tissue showed significantly lower levels of miR-202-3p, miR-424-5p, miR-449b-3p and miR-556-3p, and higher levels of VEGF-A and uPA than healthy (control) endometrium. However, tissue affected by ovarian endometrioma showed significantly lower expression of miR-449b-3p than endometrium from both controls and patients, and higher levels of PAI-1 and the angiogenic inhibitor TSP-1. A significant inverse correlation between miR-424-5p and VEGF-A protein levels was observed in patient endometrium, and an inverse correlation between miR-449b-3p and TSP-1 protein levels was observed in ovarian endometrioma. Peritoneal implants had significantly higher levels of VEGF-A than ovarian endometrioma samples. Functional studies are needed to confirm the specific targets of the miRNAs differently expressed. Differences in miRNA levels could modulate the expression of VEGF-A and TSP-1, which may play an important role in the pathogenesis of endometriosis. The higher angiogenic and proteolytic activities observed in eutopic endometrium from patients might facilitate the implantation of endometrial cells at ectopic sites. This work was supported by research grants from ISCIII-FEDER (PI11/0091, Red RIC RD12/0042/0029), Consellería de Educación-Generalitat Valenciana (PROMETEO/2011/027), Beca de Investigación Fundación Dexeus para la Salud de la Mujer (2011/0469), and by Fundación Investigación Hospital La Fe (2011/211). A.B-B. has a Contrato Posdoctoral de Perfeccionamiento Sara Borrell-ISCIII (CD13/00005). J.M-A. has a predoctoral grant PFIS-ISCIII (FI12/00012). The authors have no conflicts of interest to declare.

Endometriosis is a common, multifactorial disease in which angiogenesis may be involved in the growth of endometrium outside the uterus. microRNAs (miRNAs) are 21-22 nucleotide non-coding RNAs that regulate gene expression and play fundamental roles in biological processes. The objective of this study was to analyze several miRNAs related to angiogenesis and the angiogenic factors, vascular endothelial growth factor-A (VEGF-A) and thrombospondin-1 (TSP-1), in endometriotic lesions (ovarian endometrioma, peritoneal lesion and rectovaginal nodule) and eutopic endometrium from women with endometriosis. TaqMan real-time PCR was used to assess the expression of the miRNAs (miR-15b, -16, -17-5p, -20a, -21, -125a, -221 and -222), while VEGF-A and TSP-1 mRNA were assessed by real-time PCR, with SYBR Green I and VEGF-A and TSP-1 protein levels were quantified by ELISA. Included in the study were 58 women with endometriosis and 38 control women. In paired samples, ovarian endometrioma showed significantly lower VEGF-A mRNA (P = 0.02) and protein (P = 0.002) expression than eutopic endometrium and higher expression of miR-125a (P = 0.003) and miR-222 (P <0.001). However, ovarian endometrioma had significantly higher expression of the angiogenic inhibitor TSP-1 and lower expression of miR-17-5p than eutopic endometrium (P < 0.001). Moreover, a significant inverse correlations between miR-222 and VEGF-A protein levels (-0.267, P = 0.018) and between miR-17-5p and TSP-1 protein levels (-0.260, P=0.022) were observed. Peritoneal lesions showed a significant increase in VEGF-A in comparison with ovarian endometrioma (P < 0.01). Expression levels of miRNAs related to angiogenesis were different in eutopic endometrium from that observed in ovarian endometrioma. This could influence the expression of angiogenic factors and play a role in the pathogenesis of endometriosis.

MicroRNAs (miRNAs) are 21-22 nucleotide non-coding RNAs that regulate gene expression and play fundamental roles in biological processes. These small molecules bind to target mRNAs, leading to translational repression and/or mRNA degradation. Aberrant miRNA expression is associated with several human diseases such as cancer, cardiovascular disorders, inflammatory diseases and gynecological pathology. The present article reviews the role of miRNAs in four gynecological disorders that affect the ovary or the uterus, one benign and frequent disease (endometriosis) that is classified as a tumor-like lesion and three malignant gynecological diseases (endometrial, cervical and ovarian cancers). Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent benign gynecological diseases. Similarly to tumor metastasis, endometriotic implants require neovascularization to proliferate, invade the extracellular matrix and establish an endometriotic lesion. Despite its high prevalence and incapacitating symptoms, the exact pathogenic mechanism of endometriosis remains unsolved. A relationship between endometriosis and gynecological cancer, especially ovarian cancer, has been reported. Endometriosis is a multifactorial and polygenic disease, and emerging data provide evidence that a dysregulation of miRNA expression may be involved. miRNAs appear to be potent regulators of gene expression in endometriosis, raising the prospect of using miRNAs as biomarkers and therapeutic tools in this disease. In cancer, miRNAs have an important role as regulatory molecules, acting as oncogenes (oncomiRs) or tumor suppressors. Endometrial cancer is one of the most frequent gynecological malignancies in the developed countries. Cervical cancer, also one of the most common cancers in women, is associated with high-risk human papillomaviruses although this infection alone may not be enough to induce the malignant transformation. Ovarian cancer is the fifth leading cause of all cancer-related deaths among women. Over 80% of cases are diagnosed at an advanced stage, with a reduced five-year survival rate. Recent studies have shown that miRNAs are aberrantly expressed in different human cancer types, including endometrial, cervical and ovarian cancer, and that specific dysregulated miRNAs may act as biomarkers of patients’ outcome. Recently, miRNAs have been detected in serum and plasma, and circulating miRNA expression profiles have now been associated with a range of different tumor types. Their accessibility in peripheral blood and stability given the fact that miRNAs circulate confined within exosomes, make researchers foster hope in their role as emerging biomarkers of cancer and other disorders. The development of therapies that might block the expression or mimic the functions of miRNAs could represent new therapeutic strategies for any of the aforementioned gynecological disorders.

MicroRNAs (miRNAs) are small non-coding RNAs (18–22 nt) that function as modulators of gene expression. Since their discovery in 1993 in C. elegans, our knowledge about their biogenesis, function, and mechanism of action has increased enormously, especially in recent years, with the development of deep-sequencing technologies. New biogenesis pathways and sources of miRNAs are changing our concept about these molecules. The study of the miRNA contribution to pathological states is a field of great interest in research. Different groups have reported the implication of miRNAs in pathologies such as cancer, diabetes, cardiovascular, and gynecological diseases. It is also well-known that miRNAs are present in biofluids (plasma, serum, urine, semen, and menstrual blood) and have been proposed as ideal candidates as disease biomarkers. The goal of this review is to highlight the current knowledge in the field of miRNAs with a special emphasis to their role in endometriosis and the newest investigations addressing the use of miRNAs as biomarkers for this gynecological disease.

Which is the role of microRNAs (miRNAs) related to several angiogenesis regulators such as VEGF-A (Vascular endothelial growth factor-A) and TSP-1 (Thrombospondin-1) in endometrial cancer? A dysregulated expression of miRNAs related to angiogenesis and an increase in the VEGF-A levels were observed in endometrial cancer in comparison with control. The different expression of miRNAs could modulate the expression of angiogenic and antiangiogenic factors, which may play an important role in the pathogenesis of endometrial cancer. Dysregulated miRNA expression has been previously evaluated in endometrial adenocarcinoma. To the best of our knowledge, there are no studies on the relationship between angiogenic factors and miRNAs in endometrial cancer. Case–control study: 41 patients with histologically proven endometrioid endometrial cancer and 56 women without endometrial cancer. RNAs isolated from tissue samples were analyzed using the GeneChip miRNA 2.0 Array platform (Affymetrix). TaqMan qRT–PCR was used to assess the expression of the selected miRNAs related to angiogenesis (miR-15b, -16, -17-5p, -20a, -21, -125a, -200b, -210, -214*, -221, -222 and -424), and VEGF-A and TSP-1 mRNAs were assessed by qRT–PCR using SYBR Green. Protein levels were quantified by ELISAs. Compared with the miRNAs in the control endometrium, eight miRNAs (miR-15b, -17-5p, -20a, -125a, -214*, -221, -222 and -424) were significantly down-regulated and two miRNAs (miR-200b and -210) were significantly up-regulated in the cancerous endometrium. A significant increase in VEGF-A mRNA and protein expression and in TSP-1 protein levels (P <0.01) was observed in endometrial cancer. Moreover, significant inverse correlations between VEGF-A protein levels and miR-20a, -125a, -214*, -221, -222 and -424 were detected. In contrast, a positive correlation was observed between VEGF-A and miR-200b and -210. Furthermore, stage IB endometrial cancer was associated with a higher VEGF-A protein/mRNA ratio and lower miR-214*, -221 and -222 expression in comparison with stage IA. Future functional studies (e.g. miRNA inhibition or ectopic overexpression) in cell culture models are needed to confirm the VEGF targeting by the miRNAs found in the present study. The findings of the present study have potential implications for diagnostics and therapeutics of endometrial carcinoma. This work was supported by research grants from the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, PI080185, PI0110091) and Red RECAVA (RD06/0014/0004), by Consellería de Sanidad (AP-141/11) and Consellería de Educación (PROMETEO/2011/027), Generalitat Valenciana, by Beca Fibrinolisis 2009 and Becario 2010, 2011 from Fundación Española de Trombosis y Hemostasia and by the Fundación Investigación Hospital La Fe, Spain. None of the authors have any conflicts of interest.

Could peritoneal fluid (PF) from patients with endometriosis alter the microRNA (miRNA) expression profile in endometrial and endometriotic cells from patients? PF from patients with endometriosis modifies the miRNA expression profile in endometrial cells from patients. Angiogenesis is a pivotal system in the development of endometriosis, and dysregulated miRNA expression in this disease has been reported. However, to our knowledge, the effect of PF from patients on the miRNA expression profile of patient endometrial cells has not been reported. Moreover, an effect of three miRNAs (miR-16-5p, miR-29c-3p and miR-424-5p) on the regulation of vascular endothelial growth factor (VEGF)-A mRNA translation in endometrial cells from patients with endometriosis has not been demonstrated. Primary cultures of stromal cells from endometrium from 8 control women (control cells) and 11 patients with endometriosis (eutopic cells) and ovarian endometriomas (ectopic cells) were treated with PF from control women (CPF) and patients (EPF) or not treated (0PF) in order to evaluate the effect of PF on miRNA expression in these cells. MiRNA expression arrays (Affymetrix platform) were prepared from cells (control, eutopic, ectopic) treated with CPF, EPF or 0PF. Results from arrays were validated by quantitative reverse transcription–polymerase chain reaction in cultures from 8 control endometrium, 11 eutopic endometrium and 11 ovarian endometriomas. Functional experiments were performed in primary cell cultures using mimics for miRNAs miR-16-5p, miR-29c-3p and miR-424-5p to assess their effect as VEGF-A expression regulators. To confirm a repressive action of miR-29c-3p through forming miRNA:VEGFA duplexes, we performed luciferase expression assays. EPF modified the miRNA expression profile in eutopic cells. A total of 267 miRNAs were modified in response to EPF compared with 0PF in eutopic cells. Nine miRNAs (miR-16-5p, miR-21-5p, miR-29c-3p, miR-106b-5p, miR-130a-5p, miR-149-5p, miR-185-5p, miR-195-5p, miR-424-5p) that were differently expressed in response to EPF, and which were potential targets involved in angiogenesis, proteolysis or endometriosis, were validated in further experiments (control = 8, eutopic = 11, ectopic = 11). Except for miR-149-5p, all validated miRNAs showed significantly lower levels (miR-16-5p, miR-106b-5p, miR-130a-5p; miR-195-5p and miR-424-5p, P < 0.05; miR-21-5p, miR-29c-3p and miR-185-5p, P < 0.01) after EPF treatment in primary cell cultures from eutopic endometrium from patients in comparison with 0PF. Transfection of stromal cells with mimics of miRNAs miR-16-5p, miR-29c-3p and miR-424-5p showed a significant down-regulation of VEGF-A protein expression. However, VEGFA mRNA expression after mimic transfection was not significantly modified, indicating the miRNAs inhibited VEGF-A mRNA translation rather than degrading VEGFA mRNA. Luciferase experiments also corroborated VEGF-A as a target gene of miR-29c-3p. The study was performed in an in vitro model of endometriosis using stromal cells. This model is just a representation to try to elucidate the molecular mechanisms involved in the development of endometriosis. Further studies to identify the pathways involved in this miRNA expression modification in response to PF from patients are needed. This is the first study describing a modified miRNA expression profile in eutopic cells from patients in response to PF from patients. These promising results improve the body of knowledge on endometriosis pathogenesis and could open up new therapeutic strategies for the treatment of endometriosis through the use of miRNAs. This work was supported by research grants by ISCIII and FEDER (PI11/00091, PI11/00566, PI14/01309, PI14/00253 and FI12/00012), RIC (RD12/0042/0029 and RD12/0042/0050), IIS La Fe 2011-211, Prometeo 2011/027 and Contrato Sara Borrell CD13/0005. There are no conflicts of interest to declare.