MEMORIA DE LA INSTALACIÓN DE CLIMATIZACIÓN 1 OBJETO DEL PROYECTO

Endometriosis is a gynaecological disorder that affects 6-10% of women of reproductive age. It is characterised by the presence of endometrial glands and stroma in ectopic locations such as the ovaries, fallopian tubes and rectovaginal septum (Fassbender et al., 2013). Common symptoms of endometriosis include severe dysmenorrhoea, non-menstrual pelvic pain, dyspareunia, dysuria, dyschezia and infertility (Meehan et al., 2010). Pain associated with this disease is a result of inflammation in the peritoneum, presence of adhesions and innervation of endometriotic lesions. (Bulun 2009 (Ballard et al., 2006).

Endometriosis is not a life-threatening disease, but it impacts greatly on the quality of life of affected women (Chapron et al., 2003, Chapron et al., 2006). However, this impact has been poorly researched with available reports focusing on selected populations; mainly western nations; (Simoens et al., 2007) with small sample sizes, poorly selected control subjects and inadequate validation tools (Gao et al., 2006b). Despite this, it is recognised as a major cause of severe morbidity in women and impacts on their physical and emotional wellbeing. Psychologically, endometriosis and related symptoms may cause anxiety, depression and feelings of uncertainty, which in turn can interfere with a woman’s perceived sense of control, handling of adverse situations and resourcefulness.

Physically, endometriosis pain can impair work-related and daily activities e.g. ability of the affected women to maintain a career (Gao et al., 2006a). It has been reported that 50% of women with endometriosis are bed-ridden several times each year, interfering with education, work and day to day living (Kjerulff et al., 1996). Loss of productivity has been estimated at 10.8 hours a week owing to reduced effectiveness while working (Nnoaham et al., 2011). Associated costs to society including those of delayed diagnosis, mistreatments and individual costs incurred when symptoms associated with this disease interfere with daily life are considerable, but poorly characterised (Gao et al., 2006a). The annual cost estimates in the US was reported as $22 billion in 2002. This was calculated from the estimated cost per patient; $1023-

77

$2801 per year at a prevalence of 10% among women of reproductive age (Simoens et al., 2007). Indirect costs of the disease were not calculated in this study. Despite this significant health burden the disease is still poorly researched, diagnosed and treated. Infertility/subfertility which is a major consequence of the disease causes an extra burden to the patient due to the uncertainty of ever having a family. Sexual dysfunction due to dyspareunia can disrupt the relationship between a man and a woman (Gao et al., 2006b). This comes with an extra burden of social stigma especially in settings whereby infertility is considered shameful (Somigliana et al., 2010). Efforts to assess the societal cost effects of endometriosis have been reported. The World Endometriosis Research Foundation (WERF) EndoCost study is the first prospective study from 12 centres in 10 countries to examine the direct and indirect costs of endometriosis (Simoens et al., 2011). The study measures direct health care costs like cost of medication and physician visits, direct non-health care costs and indirect costs associated with loss of productivity. The average costs of endometriosis were reported as €9579 per woman per year which equates to €3113 for direct health care costs. The inabilty to work due to symtoms is therefore twice the direct health costs. These cost estimates may be used to raise awareness of endometriosis with policy makers, health professionals and researchers inorder to emphasise the importance of early diagnosis and treatment.

The gold standard of diagnosis is laparoscopy together with histological confirmation. Laparoscopy is an invasive procedure which is expensive and bears significant risks (Kennedy et al., 2005, Bulun, 2009). Efforts aimed at early diagnosis and treatment of endometriosis have been hindered by a lack of proper methods to study and manage the disease. The mean interval between first symptom appearance and diagnosis has been reported to be 7-10 years (Hadfield et al., 1996, Dmowski, 1984, Husby et al., 2003, Ballard et al., 2006). Patients who present with severe pelvic pain that has not been relieved by pain medication or oral contraceptives and those seeking pregnancy for more than one year are the most common patients to whom laparoscopy is recommended in order to guide therapeutic interventions. The availability of a non- invasive diagnostic test would therefore be important to establish endometriosis

78

instead of subjecting these women to uneccessary surgery whose outcomes might be negative for endometriosis.

Development of a non-invasive test for diagnosis and follow-up has been identified as a top research priority (Rogers et al., 2009b, Rogers et al., 2013). The need for a non- invasive test for asymptomatic women (i.e. screening) is still debatable because this will mean subjecting women to unnecessary and potentially harmful procedures. It is important to note that most subfertile women with or without pelvic pain, having regular cycles, a partner with a normal sperm count and quality, and normal pelvis on ultrasound imaging, may have endometriosis (Meuleman et al., 2009). A non-invasive test would therefore be important for such women. These women could be those with early stage disease and some cases of late stage disease not picked up by imaging methods and those with pelvic adhesions and/or other pelvic pathology who on diagnosis would benefit from laparoscopic treatment.

Serum, plasma, urine, endometrial fluid, menstrual fluid, tissue biopsy and peritoneal fluid are samples that can be studied in the search for a non-invasive or minimally invasive biomarker (Vodolazkaia et al., 2012, Vodolazkaia et al., 2010, Casado-Vela et al., 2009, Kyama et al., 2007). The vital aim is therefore to develop a test in which no woman with endometriosis and/or any other pelvic pathology that would benefit from laparoscopic treatment are missed. A test with high sensitivity and specificity would be ideal for detecting or ruling out endometriosis in patients presenting with symptoms. At present no such test exists.

The WERF Endometriosis Phenome and Biobanking Harmonisation Project (EPHECT) is a global initiative involving 34 clinical/academic and 3 industrial collaborators from 16 countries with a mission to develop a consensus on standardisation and harmonisation of phenotypic surgical, clinical data and biological sample collection methods in endometriosis research mainly to address large scale, cross centre, epidemiologically robust, translational biomarker and treatment target discovery research in endometriosis (Becker et al., 2014, Fassbender et al., 2014, Rahmioglu et al., 2014). This initiative outlines detailed international guildelines for

79

standardised clinical and personal phenotyping (phenome) data to be collected from women with endometriosis and controls to improve patient disease characterisation and standard operating procedures (SOPs) for biobanking of biological samples from women with endometriosis and controls with respect to collection, transport, processing and long term storage of samples collected from these women (Becker et al., 2014).

High-throughput proteomic methods have been developed over the years and are now being used to study various diseases of the female reproductive system (Meehan et al., 2010). These approaches have the potential to identify new disease biomarkers by comparing the abundance of hundreds or thousands of proteins simultaneously across cohorts of patients and controls. This has been the focus of many proteomic studies. Endometriosis is a complex disease, therefore it is may not be possible that a single marker will have sufficient diagnostic accuracy. A marker panel however could provide better diagnostic and/or prognostic power.

Biological sampling is an important step for many biomarker studies. Different types of samples can be used depending on the type of study and subsequent downstream application. Sample quality is a critical factor in proteomic analyses to ensure reproducibility of data. Standardised techniques for sample collection, processing and storage are therefore important in any biomarker study design. Research into the human endometrium may be complex and challenging. This tissue is composed of many cell types (epithelial cells, stromal cells, fibroblasts, pre-decidual cells, leucocytes and cells of the vasculature. The endometrium is also regulated by cyclic hormones and other paracrine and autocrine factors which when combined with the individuals’ genetic and environmental background may result in alterations to biological processes. Endometrial tissue is inherently heterogeneous with respect to developmental, temporal and biological composition, therefore cell types within a single tissue can be highly variable e.g. ectopic endometriotic lesions contain relatively few endometrial cells that are often dispersed along with leucocytes among the cells of their recipient surface. Biological variability may therefore arise through differences in tissue composition of the collected samples and heterogeneity of

80

cellular compositions due to phase of menstrual cycle. This inherent variability needs to be considered when designing studies and analysing data. Standard operating procedures for sample collection should therefore be implemented and must be highly standardised and robust to ensure proper tissue acquisition, processing, utilisation, storage and distribution. Laparoscopy together with histological dating is used to definitively diagnose endometriosis. Eutopic endometrial tissue, ectopic endometriosis tissue collected surgically and blood samples were collected for the purposes of this study. Tissue samples were used for the discovery of candidate biomarkers and selected markers were then tested in serum as potential non-invasive diagnostic markers of endometriosis.

The endometrium can be obtained in five different ways; by use of an endometrial sampling device (Pipelle®), endometrial curettage, hysteroscopy resection, post- hysterectomy excision and brushing. An endometrial sampling device is a thin plastic tube that is inserted into the uterus and used to aspirate the tissue. Curettage involves scraping ‘strips’ of the endometrium from the uterine lining with the use of a curette. In post-hysterectomy collection, the endometrium can be scraped off using a curette or scissors or aspirated using a Pipelle®. Endometrial brushing involves insertion of a disposable brush into the uterus that is used to collect the sample. The collection method varies with the type of study, tissue of interest and available expertise. In this study, eutopic endometrium tissue samples were collected from women with and without disease by curettage during laparoscopy or after hysterectomy. Ectopic tissue was excised during surgery.

Defining the phenotype of the study population is important to ensure that representative disease and sample types are being used. Heterogeneity resulting from improper classification of study participants may decrease both the sensitivity and power of the study. The impact of other pathological conditions affecting the endometrium must also be considered when defining phenotype. The presence of structural alterations (e.g. fibroids), cancer and immune changes may will affect the phenotype of the endometrium. Exposure to different medications e.g. those used to shrink the lesions before surgery, contraceptives that are also used to manage pain and

81

environmental toxins may also impact on tissue phenotype resulting in biological variability. The level of phenotypic heterogeneity between studies poses a challenge when it comes to reproducibility and replication of study findings.

Many protein extraction protocols have been developed but selecting a suitable protocol mainly depends on the nature of the starting material and on the downstream applications. Protein analysis involves a number of processing steps; homogenisation for protein extraction, denaturation, reduction of disulphide bonds, alkylation of cysteine residues, enzymatic digestion, protein fractionation/separation, analysis by MS and data analysis to identify and quantify peptides. Due to the diverse biochemical properties of cellular proteins e.g. their charge, size, hydrophobicity, susceptibility to proteolysis, ligand interactions and sub-cellular localisation, no single protein extraction method can capture the full proteome. Comprehensive, uncontaminated and representative protein populations can be difficult to extract from tissue samples partly because of the presence of structural proteins and due to contamination with blood proteins arising during tissue sampling.