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Se trata de un estudio retrospectivo, donde hemos descrito el contenido de miRNAs del líquido peritoneal de una cohorte de pacientes, por medio de dos procedimientos. Por una parte se han realizado arras de expresión para hacer un estudio previo y determinar que miRNAs se encontraban diferentemente expresados en el líquido peritoneal de las pacientes respecto de las controles. Posteriormente se ha llevado a cabo la segunda parte del estudio, validando por medio de la técnica RT-PCR aquellos micros diferentemente expresados. Hemos seleccionado los miRNAs que presentaban un particular interés en la etiopatogenia de la endometriosis. Así mismo también se han medido el contenido de diversos metabolismos angiogénicos y pro-inflamatorios en el líquido peritoneal, con el objetivo de evaluar el estado inflamatorio de la patología. Ambos conjuntos de datos se han correlacionado con el status de fertilidad de las pacientes. Siendo el objetivo final del trabajo que los miRNAs en el líquido peritoneal se pudieran convertir en un biomarcador mínimamente invasivo para poderlo utilizar (integrado con marcadores clásicos de la patología) como herramienta diagnóstica y/o pronostica de la endometriosis. Esta tesis por tanto ha dado como resultado por una parte, descriptivo, con el fin de poder incorporar el perfil de miRNAs como herramienta diagnóstica de la patología de la endometriosis, y por otra parte la relación de dicho contenido en miRNAs con el status de fertilidad de las pacientes.

La miocardiopatía arritmogénica (MCA) es una enfermedad cardíaca de origen genético caracterizada por la sustitución fibroadiposa del tejido miocárdico que predispone al desarrollo de arritmias ventriculares y muerte súbita cardíaca. Su diagnóstico resulta en ocasiones muy complicado, ya que los criterios vigentes Task Force 2010 presentan una baja sensibilidad para diagnosticar las formas con afectación predominante del ventrículo izquierdo (VI). El objetivo de esta tesis doctoral es caracterizar los rasgos de la MCA con afectación del VI tanto a nivel electrocardiográfico, estructural y molecular para así profundizar en el conocimiento de esta entidad de difícil diagnóstico y alta letalidad. Se incluyó una cohorte de pacientes vivos reclutados en la Unidad de Cardiopatías Familiares y Muerte Súbita del Hospital Universitario y Politécnico La Fe de Valencia y una cohorte de sujetos fallecidos del Instituto de Medicina Legal y Ciencias Forenses de Valencia y del Instituto Nacional de Toxicología y Ciencias Forenses de Madrid. Entre los principales resultados destaca: 1)La prevalencia estimada de MCA en la provincia de Valencia fue de al menos 7.6/100000 habitantes y la incidencia de muerte súbita cardiaca de al menos 0.9/100000 habitantes/año. 2)La forma predominante fue la MCA con afectación del VI tanto en la cohorte de sujetos fallecidos como en la de vivos. 3)Tanto en la cohorte de sujetos vivos como en la de fallecidos DSP fue el gen responsable mayoritario en las formas con afectación del VI y PKP2 en las formas derechas, siendo DSP el gen más frecuentemente mutado globalmente. 4)El realce tardío de gadolinio en la resonancia magnética cardiaca mostró una afectación predominante sobre la región inferior (69%) y lateral (50%) del VI. Exhibiendo los pacientes con más fibrosis una mayor carga arrítmica y una menor fracción de eyección del VI. 5) La inclusión en los criterios Task Force de la disincronía radial del VI y la fracción de eyección del VI con unos puntos de corte de 70 mseg y 48.5% respectivamente, reclasificaría al 30% de los pacientes de categorías límite o posible a un diagnóstico definitivo. 6) Los pacientes con MCA VI de nuestra serie muestran voltajes menores del QRS, mayor número de QRS fragmentados y ondas T negativas especialmente a nivel de la cara inferior. 7)El vectocardiograma de los pacientes con afectación del VI muestra una distancia respecto al plano mayor, una trayectoria más compleja y un menor nivel de ajuste al plano que los controles. 8)La amplitud de la señal del electrocardiograma se encuentra reducida en los pacientes con afectación del VI. 9)Los arrays de expresión de miRNAs realizados en plasma de pacientes y controles demostraron que tanto miRNAs maduros como precursores permitían diferenciar entre ambos grupos. El pre-miRNA mir-1271 permitió identificar los pacientes, así como diferenciar entre afectaciones izquierda y derecha.

Introduction: Endometriosis is an oestrogen-dependent inflammatory disorder defined by the presence of endometrial-like tissue in ectopic locations, which limits the quality of life of affected women. This pathology affects 10% of reproductive-age women from all ethnic and social groups, although the prevalence in those patients experiencing pain, infertility or both is as high as 35%-50%, being the estimated prevalence of this condition around 176 million worldwide. Endometriosis is associated with an average diagnostic delay of 7 years, what could be partially explained by the lack of non-invasive biomarkers for diagnose, since the gold standard method is laparoscopy followed by histological confirmation of the presence of endometrial-like glands and stroma in biopsies. Although several aethiogenic theories have attempted to explain the disparity of manifestations of the disease, the most accepted theory is still the Sampson’s retrograde menstruation theory, proposed almost 100 years ago.According to Sampson’s proposal, endometrium shed during menstruation is capable of reaching the pelvic cavityby retrograde flow through the fallopian tubes, then being able to implant and proliferate. Albeit it has been documented that retrograde menstruation occurs in 90% of healthy women in reproductive age with patent fallopian tube, the fact that only a small percentage develops the disease suggests that there must be additional mechanisms that allow the migrated tissue to implant and survive. In this context, it has been proposed that endometrial and peritoneal factors could be involved in the pathophysiology of endometriosis. At tissue level, endometriotic implants require neovascularization to proliferate, invade the extracellular matrix (ECM) and establish the lesion, displaying an oncomimetic behaviour. Neovascularization is achieved by means of angiogenesis, or the process of formation of new blood vessels from pre-existing ones. Previous results from our group showed that protein levels of VEGF-A were significantly elevated in the eutopic endometrium of patients with respect to the endometrium of women without endometriosis. In addition, in the various endometriosis lesions, peritoneal implants had levels of VEGF-A significantly increased compared to ovarian endometriomas.Additionally, remodelling of ECM plays a critical important role in the establishment of endometriotic lesions. Abnormal expression of components of metalloproteinase system and proteolysis system has been reported in both the endometrium and endometriotic tissue of women affected by endometriosis, suggesting their involvement in the establishment of lesions. Thus, it seems reasonable to simultaneously study components of the angiogenic and proteolytic system in our patients. Furthermore, peritoneal fluid (PF) represents one of the most important peritoneal factors. This biofluid is defined as an ovarian exudation product originated mainly as a result of an increased vascular permeability, with cyclic variation in volume and steroid hormones. Because ectopic endometriotic lesions located in the pelvic peritoneum are in close relationship with this biofluid, their components have emerged as an important field of study. In PF, there are cell types such as erythrocytes, macrophages and endometrial cells. Endometrial cells secrete products such as glycodelin, the peritoneum substances such as CA-125 and macrophages secrete a large diversity of molecules, among which are cytokines, growth factors and angiogenic factors. Regarding angiogenesis, VEGF-A has been detected in higher concentrations in the PF of women with endometriosis (McLaren et al, 1996; Gilabert-Estellés et al, 2007, Marí-Alexandre et al, 2017b), being correlated with the disease stage (Shifren et al, 1996). It has been suggested that increased levels of VEGF-A in PF in patients with endometriosis are the consequence of combined expression by ectopic lesions and activated macrophages present in the fluid. In addition, some studies indicate that the PF of women with endometriosis induces cell proliferation in vitro, although the underlying mechanism is unknown. Epigenetics is defined as “the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states”. There exist four epigenetic mechanisms, whose marks are dynamic and reversible: non-coding RNAs (ncRNAs), DNA methylation, histone modifications and chromatin remodelling. microRNAs (miRNAs are small (19-22nt) non-coding RNAs that can act as post-transcriptional regulators of gene expression, reducing the expression of their target mRNAs either inhibiting its translation or promoting its degradation. miRNAs usually regulate gene expression by binding to the 3’ UTR (UnTranslatedRegion) of their target mRNAs. Importantly, several miRNAs can target a given mRNA and a single miRNA can target several mRNA, increasing the complexity of the regulatory mechanism mediated by these molecules. A growing body of literature, including papers from our research group supports the regulatory function of miRNAs in a myriad of processed involved in the pathophysiology of endometriosis, such as angiogenesis, proteolysis, extracellular matrix remodelling, inflammation, proliferation or apoptosis. Nevertheless, the lack of overlapping among different studies is noticeable (Marí-Alexandre et al., 2017). DNA methylation is a covalent modification defined by the addition of a methyl (-CH3) group at the 5’ carbon of cytosines, being by far the most widely studied epigenetic mark. Deviations in DNA methylation patterns can occur via gain (hypermethylation) or loss (hypomethylation) of methylation marks with respect to methylomes defined as normal. Whereas hypermethylation at CpG islands of gene promoters is associated with the repression of gene expression, hypomethylation has been linked to gene expression. Albeit largely studied in oncological malignancies, the role of DNA methylation in the pathophysiology of endometriosis alterations has just begun to be explored. Hypothesis and objectives: the overall hypothesis is that an aberrant epigenetic profile could modulate the molecular mechanisms underlying the aetiology and pathophysiology of endometriosis. Getting into detail, the first hypothesis is that endometrial factors, in terms of an aberrant miRNA and DNA methylation profile, are responsible for the altered survival, proliferation, angiogenesis and extracellular matrix remodelling observed in the disease. The second hypothesis is that peritoneal factors, in terms of altered composition of peritoneal fluid in patients with endometriosis, could mediate the deregulation of miRNAs in endometrial and endometriotic stromal cells, being responsible for the altered biological processes observed in this pathology. Finally, the third hypothesis is that the expression of VEGF-A in stromal cell cultures can be epigenetically modulated by means of selected miRNA mimics and of targeted DNA methylation at VEGFA promoter. With regards to the objectives, the first objective was to assess miRNA expression profiles in samples of eutopic and control endometria and ovarian endometrioma from women with endometriosis; and also, to validate the results in a higher cohort of samples, including paired peritoneal implants and rectovaginal nodules. Additionally, we aimed to evaluate the role of deregulated miRNAs on the expression of the main regulators of angiogenesis and fibrinolysis. The second objective was to validate our in vitro model in primary stromal cell cultures from endometrial samples and from ovarian endometrioma. Additionally, we wanted to evaluate the influence of peritoneal fluid on the expression of selected angiogenesis- and fibrinolysis-related miRNAs. The third objective was to assess the influence of peritoneal fluid on miRNA expression profiles in primary endometrial and endometriotic stromal cell cultures. Additionally, we wanted to validate VEGFA mRNA as a target of miR-16-5p, miR-29c-3p, miR-424-5p. The fourth objective was to define the DNA methylation profiles in endometrial samples from patients with endometriosis and from control women by employing the Illumina Infinium MethylationEPIC BeadChip. Finally, the fifth objective was to down-regulate the expression of VEGF-A in primary stromal cell cultures of patients with endometriosis by employing techniques for targeted DNA methylation (fused Zinc fingers proteins – Dnmt3a3L enzyme). Results: To allow a better understanding of the experimental results, these have been structured in 5 chapters. In Chapter 1 we investigate the implication of miRNA deregulation at tissue level in the pathophysiology of endometriosis. To this end, we established a case-control study involving specimens from 51 women with endometriosis and 32 women without the disease. miRNA expression profiles (GeneChip miRNA2.0; Affymetrix) were performed in samples of control (CNT) and eutopic (EUT)endometria and ovarian endometrioma (OMA) from women with endometriosis.After statistical treatment of microarray results, in silico analysis allowed us to select 12 differentially expressed miRNAs (namely (miR-16-5p, -29c-3p, -138-5p, 202-3p, -373-3p, -411-5p, -411-3p, -424-5p, -449b-3p, -556-3p, -636, -935), which targets were involved in angiogenesis, fibrinolysis, or that were implicated in endometriosis. These miRNAs were validated in a larger cohort of samples, including rectovaginal nodules and peritoneal implants. Additionally, levels of vascular endothelial growth factor(VEGF-A), thrombospondin-1 (TSP-1), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) were measured by ELISA. We observed that EUT 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 CNT endometrium. However, OMA showed significantly lower expression of miR-449b-3p than endometrium from both controls and patients, and higher levels ofPAI-1 and the angiogenic inhibitor TSP-1.Asignificant inverse correlation between miR-424-5p and VEGF-A protein levels was observed in EUT, and an inverse correlation between miR-449b-3p and TSP-1 protein levels was observed in OMA. Peritonealimplants had significantly higher levels of VEGF-A than ovarian endometrioma samples. Hence, differences in miRNA levels could modulate the expression of VEGF-A and TSP-1, whichmay play an important role in the pathogenesis of endometriosis. The higher angiogenic and proteolytic activities observed in EUTfrom patients might facilitate the implantation of endometrial cells at ectopic sites. In Chapter 2 and Chapter 3 we investigate the influence of peritoneal fluid from patients with endometriosis on the deregulation of miRNAs in endometrial and endometriotic stromal cells, being responsible for the altered biological processes observed in this pathology. Specifically, in Chapter 2 we validated an in vitro model of endometriosis,previously established by our research group,in which primary stromal cell cultures from CNT (n=8), EUT (n=11) and from OMA (n=11) were exposed to PF pools from patients (EPF) and control women (CPF) in order to simulate the peritoneal microenvironment. Afterwards, we quantified the expression of six miRNAs (miR-16, -17-5p, -20a, -125a, -221, and -222) by qRT-PCR and protein and mRNA levels of VEGF-A, TSP-1, uPA and PAI-1 by ELISA and qRT-PCR, respectively. We observed that EPF and CPF pools induced a significant reduction of the levels of all studied miRNAs in endometrial and endometriotic cell cultures. Moreover, both peritoneal fluids (CPF and EPF) induced a significant increase in VEGF-A, uPA and PAI-1 protein levels in all cell cultures without significant increase in mRNA levels, suggesting a post-transcriptional mechanism of gene expression regulation. Endometrial cell cultures from patients treated with EPF showed lower expression of miRNAs and higher expression of VEGF-A protein levels than cultures from controls. All in all, this in vitro study allowed us to validate previous results from our research group, alsoindicating that EPF modulates the expression of miRNAs that could contribute to the angiogenic and proteolytic disequilibriumobserved in this disease. In Chapter 3, we broaden the scope of our interest to assess the influence of peritoneal fluid on miRNA expression profiles in primary endometrial and endometriotic stromal cell cultures. Thus, we performed miRNA expression arrays (Affymetrix, GeneChip 4.0) in cells stimulated with the experimental condition described in Chapter 2. We observed that EPF modified themiRNAexpression profile in eutopic cells. A total of 267miRNAweremodified in response to EPF compared with eutopic cellswithout PF stimulation (ØPF). 9differently expressed miRNAs (namely 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) with potential targetsinvolved in angiogenesis, proteolysis or endometriosis, were validated in further experiments. Except for miR-149-5p, all validated miRNAs showed significantly lower levels after EPF treatment in primary cell cultures from EUT in comparison with ØPF. Additionally, functional experiments were performed in primary cell cultures using mimics for miRNAs miR-16-5p, miR-29c-3p and miR-424-5p in order to assess their effect as VEGF-A expressionregulators. We observed a significant down-regulation of VEGF-A protein expression after transfection of stromal cells with miRNAs mimics for miR-16-5p, miR-29c-3p and miR-424-5p, without significantly modifying VEGFAmRNA. One step further, we performed luciferase expression assays to confirm a repressive action of miR-29c-3p through forming miRNA:VEGFA duplexes. In Chapter 4, we defined the DNA methylation profiles in endometrial samples from patients with endometriosis and from control women by employing, for the first time in endometriosis, the Infinium MethylationEPIC BeadChip(Illumina). Arrays were performed in 13EUT from women with endometriosis (III-IV stage) and 11CNT from women without the disease, paired by age and phase of menstrual cycle. Unsupervised hierarchical clustering of methylation levels of 5000 random selected CpGs showed no segregation of endometrial tissues neither by disease status (patients vs control) nor by cycle phase (proliferative, secretory or menstrual). This was also confirmed by scatter plots of β-values from patients’ and controls’ samples. To define the existence of potential specific elements of distinction by disease status we used a supervised elasticnet-penalized logistic regression model. We determined 7 CpGs (4 CpGs related to LRP5, SEMA4F, MYL12B and PIK3AP and 3CpGs not assigned to any known gene) which together provided the highest discrimination power between patient and control women’s samples. However, Δβ were rather small, regardless of the phase of the menstrual cycle considered. Due to the importance of the endometrium in the implantation process and the singularity of the tissue in terms of cyclical changes in gene expression under hormonal influence, we aimed to provide a detailed description of genome wide DNA methylation profile of this tissue. We observed a bimodal distribution of CpGs, with 23.3% of them hypomethylated(β ≤ 0.2), whereas 24.8% of the CpGs were hypermethylated (β≥0.8). Additionally, when looking at functional distribution of CpGs, we observed an enrichment of promoters in hypomethylated regions, whereas hypermethylated CpGs were enriched in gene body regions. This was also extended to the main regulators of angiogenesis (VEGF-A, TSP-1) and fibrinolysis (uPA, PAI-1), in agreement with an active state of transcription for these genes in the endometrium and a notable gene stability. This supports the potential involvement of a post-transcriptional mechanism of regulation, highlighting the importance of miRNA regulation of these proteins partially deciphered in this work (Chapter 1 and Chapter 3). In Chapter 5 we wished to down-regulate the expression of VEGFA in primary stromal cell cultures from EUT by employing breakthrough techniques for targeted DNA methylation (Zinc finger proteins and the CRISPR-dCas9 system). Firstly, we employed bisulphite sequencing to validate results from methylation arrays, confirming a hypomethylated state at VEGF-A promoter.Secondly, we performed experiments to establish passage 3 as the optimum in which experiments would be performed. Finally, we transfected primary endometrial stromal cells from EUT with plasmids encoding Dnmt3a, GFP and either a specific ZFP or a dCas9/gRNA. Although multiple transfection conditions were employed (FuGene® HD, Lipofectamine LTX and Lipofectamine 3000 transfection reagents), we were unable to maintain cells alive for 120h with a reasonable transfection efficiency. In conclusion, the combined toxicity of transfection reagents and the multiple plasmids co-transfected is not a suitable strategy for primary endometrial stromal cell cultures, albeit it has shown to be successful for easily transfectable cells (e.g. HEK293 cell lines) and resistant tumour cells (e.g. ovarian cancer SKOV3 cell lines). In conclusion, our results corroborate the importance of both endometrial and peritoneal components in the pathophysiology of endometriosis, through a deregulation of angiogenesis and proteolysis. The lack of significant differences in DNA methylation in EUT vs CNT endometrium highlights the importance of other epigenetic mechanisms, such as miRNAs, in the survival of migrated endometrial tissues and establishment of endometriotic lesions.

INTRODUCCIÓN: la endometriosis es una enfermedad compleja de etiología aún desconocida, no presentando por tanto, tratamiento etiológico y es conocida a su vez, su tendencia a la recidiva. El diagnóstico de elección sigue siendo un procedimiento invasivo como la laparoscopia. Su diagnóstico no invasivo es difícil, especialmente en su variante de endometriosis profunda, causante del habitual retraso diagnóstico en esta enfermedad. Surge la necesidad de plantear modelos predictores no invasivos tanto en la detección de la enfermedad profunda como en su potencial recidiva. HIPÓTESIS: El empleo de marcadores clínicos y de imagen en asociación a biomarcadores (miRNAs) en mujeres con endometriosis, tienen un valor en el establecimiento del diagnóstico y pronóstico de la enfermedad. MATERIAL Y MÉTODOS: El diseño del presente proyecto está fundamentado en dos partes: 1.Serie Prospectiva: Estudio de cohortes de pacientes y controles aún no intervenidas en las que se analizarán previo a la cirugía una serie de marcadores clínicos (cuestionarios validados de dolor y de calidad de vida EHP-30) y de imagen, mediante ecografía y resonancia nuclear magnética. Tras obtención de muestra, se analizarán marcadores biológicos clásicos en suero (CA125 y CA19.9) y nuevos biomarcadores en tejidos obtenidos de la cirugía (endometrio eutópico, endometrioma ovárico, implante peritoneal y TRV) como miRNAs y factores angiogénicos, con el objeto de establecer una correlación entre todos estos parámetros biológicos, clínicos y de imagen con el tipo y extensión de la enfermedad, especialmente los casos de endometriosis profunda. 2.Serie Retrospectiva: cohorte de pacientes intervenidas por endometriosis de las que se tiene registro clínico de entre 3 a 9 años tras la cirugía y muestra congelada extraída en dicha cirugía previa. Se estudiaran las mismos marcadores clínicos y biológicos que en la parte anterior. Posteriormente se realizará una entrevista y una exploración con las que se pretende buscar una asociación entre dichos marcadores y el pronóstico de la enfermedad, en lo relativo al riesgo de recidiva. CONCLUSIONES: 1.MARCADORES CLINICOS: La escala analógica visual de dolor (VAS) y el cuestionario de calidad de vida EHP-30 son útiles en la predicción clínica de endometriosis profunda, especialmente la utilización del sumatorio de todos los valores de dolor y los valores del cuestionario central del EHP-30. Un valor del sumatorio VAS mayor de 90 y un sumatorio EHP-30 central mayor de 120 pueden utilizarse como predictores de enfermedad profunda. Un valor del Sumatorio VAS mayor de 285 y un sumatorio del EHP-30 central mayor de 280 son buenos predictores de recidiva en pacientes con endometriosis. 2.MARCADORES DE IMAGEN: La ecografía transvaginal de alta resolución (ETV) tiene una alta especificidad para la detección de enfermedad profunda pero una moderada sensibilidad. Es además la mejor técnica para la evaluación de la endometriosis rectal y debería ser considerada la técnica de elección inicial. La RNM convencional no mejora los resultados de la ETV. La RNM con gel mejora la sensibilidad de la técnica, especialmente para la localización retrocervical, rectovaginal y vaginal. Esta técnica debería reservarse para los casos dudosos o negativos por ecografía, en los que hay alta sospecha de endometriosis profunda por la exploración clínica y/o por los biomarcadores de enfermedad. Entre los todos los signos ecográficos indirectos estudiados, el signo de deslizamiento es el mejor como método de cribaje de endometriosis profunda y severa del compartimento posterior, por su sencillez y elevada sensibilidad. 3.BIOMARCADORES: La determinacion sérica de CA-125 puede emplearse como marcador de respuesta inicial al tratamiento quirúrgico y se asocia a una mayor posibilidad de recidiva en las pacientes que presentan valores prequirúrgicos aumentados. Existe una sobreexpresión de miRNAs relacionados con la angiogénesis en el endometrio eutópico de pacientes con endometriosis profunda, lo que indica su potencial como biomarcadores de enfermedad. Se ha encontrado correlación de diversos miRNAs con la recidiva de enfermedad (miR-21, miR-424-5p). Además, el miR-424-5p presenta una alta correlación positiva con el valor sérico de CA-125 y los marcadores clínicos analizados en el presente estudio.En los nódulos de endometriosis profunda de las pacientes que posteriormente recidivan, se encuentra un perfil diferencial en la expresión de angiomiRs (miR-373-3p, miR-556-3p). La combinación de marcadores clínicos (sumatorio de VAS y EHP-30 central), biomarcadores en sangre periférica (CA-125) y biomarcadores en endometrio eutópico (VEGF-A, PAI-1, miR-21 y -424-5p) podrían utilizarse como modelo no invasivo predictor de recidiva en endometriosis. La obtención de un perfil no invasivo de recidiva individualizado para cada paciente sería útil en la toma de ciertas decisiones clínicas. INTRODUCTION: Endometriosis is a complex disease and the etiology is still unknown, not presenting therefore etiological treatment and is known to turn their tendency to recur. The diagnosis of choice remains an invasive procedure such as laparoscopy. Noninvasive diagnosis is difficult, especially in its variant of deep endometriosis, causing the usual diagnostic delay in this disease. The need arises to raise noninvasive predictors models both detecting deep disease and its potential relapse. SCENARIO: The use of clinical markers and image in association with biomarkers (miRNAs) in women with endometriosis have a value in establishing the diagnosis and prognosis of the disease. MATERIALS AND METHODS: The design of this project is based on two parts: 1. Prospective Serie: Study of cohorts of patients and controls has not intervened in to be analyzed prior to surgery a number of clinical markers (validated questionnaires of pain and quality of life EHP-30) and imaging, with ultrasound and magnetic resonance imaging. After sample collection, classic serum (CA125 and CA19.9) and new biomarkers in tissues obtained from surgery (eutopic endometrial, ovarian endometrioma, peritoneal implant and TRV) miRNAs and angiogenic factors as biological markers are analyzed, in order to correlate all these biological parameters, clinical and imaging with the type and extent of disease, especially cases of deep endometriosis. 2. Retrospective Serie: cohort of patients undergoing surgery for endometriosis which has clinical record of 3 to 9 years after surgery and frozen sample drawn in said previous surgery. the same as in the previous part clinical and biological markers are studied. Subsequently an interview and a scan which aims to find an association between these markers and prognosis of the disease, with regard to the risk of recurrence is performed. CONCLUSIONS: 1.CLINICAL MARKERS: The visual analog pain scale (VAS) and quality of life questionnaire EHP-30 are useful in the clinical prediction of deep endometriosis, especially the use of the sum of all values of pain and values of Central EHP-30 questionnaire. A summation value VAS greater than 90 and summation value core EHP-30 more than 120 can be used as predictors of deep disease. A summation value VAS greater than 285 and a summation of the core EHP-30 greater than 280 are good predictors of recurrence in patients with endometriosis. 2.IMAGING MRKERS: The high-resolution transvaginal sonography (TVS) has a high specificity for detection of deep disease but moderate sensitivity. It is also the best technique for assessing rectal endometriosis and should be considered the initial election technique. Conventional MRI does not improve the results of the ETV. The RNM gel improves the sensitivity of the technique, especially for retrocervical, rectovaginal and vaginal location. This technique should be reserved for equivocal or negative ultrasound cases in which there is high suspicion of deep endometriosis by clinical examination and / or biomarkers of disease. Among all indirect sonographic signs studied, the sliding the sign is the best method of screening as deep and severe endometriosis of the posterior compartment, for its simplicity and high sensitivity. 3.BIOMARKERS: The determination of serum CA-125 can be used as a marker of initial response to surgical treatment and is associated with a greater chance of recurrence in patients with preoperative values increased. There is overexpression of angiogenesis-related miRNAs in eutopic endometrium from patients the deep endometriosis, indicating its potential as disease biomarkers. Found correlation of various miRNAs with disease recurrence (miR-21, miR-424-5p). In addition, miR-424-5p has a high positive correlation with serum CA-125 value and clinical markers analyzed in this estudio. In deep endometriosis nodules of patients who later relapse, is a differential profile angiomiRs expression (miR-373-3p, miR-556-3p). The combination of clinical markers (VAS summation and core EHP-30), biomarkers in peripheral blood (CA-125) and eutopic endometrium biomarkers (VEGF-A, PAI-1, miR-21 and -424-5p) could be used as a model non-invasive predictor of recurrence in endometriosis. Obtaining a profile noninvasive recurrence individualized for each patient would be useful in making certain clinical decisions.