• Aitana Braza-Boïls, Salam Salloum-Asfar, Josep Marí-Alexandre, Ana Belén Arroyo, Rocío González-Conejero, Moisés Barceló-Molina, Javier García-Oms, Vicente Vicente, Amparo Estellés, Juan Gilabert-Estellés, and Constantino Martínez.
• Grupo de Hemostasia, Trombosis, Aterosclerosis y Biología Vascular, Instituto de Investigación Sanitaria La Fe, Valencia, Spain a.braza.boils@gmail.com.
• Hum. Reprod. 2015 Oct 1; 30 (10): 2292-302.

Study QuestionCould peritoneal fluid (PF) from patients with endometriosis alter the microRNA (miRNA) expression profile in endometrial and endometriotic cells from patients?Summary AnswerPF from patients with endometriosis modifies the miRNA expression profile in endometrial cells from patients.What Is Known AlreadyAngiogenesis 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.Study Design, Size, DurationPrimary 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.Participants/Materials, Setting, MethodsMiRNA 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.Main Results And The Role Of ChanceEPF 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.Limitations, Reasons For CautionThe 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.Wider Implications Of The FindingsThis 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.Study Funding/Competing InterestsThis 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.© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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