Introduction Endometriosis is a common gynaecological disease associated with pelvic pain and subfertility. There are no non-invasive diagnostic tests, medical management requires suppression of oestrogens and surgical removal is associated with risk. Endometriosis is a complex genetic disease with variants in at least 27 genetic regions associated with susceptibility. Previous research has implicated a variety of biological mechanisms in multiple cell types. Endometrial and endometriotic epithelial cells acquire somatic mutations at frequency higher than expected in normal tissue. Stromal cells have altered adhesive capacity and immune cells show altered cytotoxicity. Understanding the functional consequences of these genetic variants on each cell type requires the collection of patient symptoms, clinical and genetic data and disease-relevant tissue in an integrated program.
Methods and analysis The aims of this study are to collect tissue associated with endometriosis, chart the genetic architecture related to endometriosis in this tissue, isolate and propagate patient-specific cellular models, understand the functional consequence of these genetic variants and how they interact with environmental factors in pathogenesis and treatment response.
We will collect patient information from online questionnaires prior to surgery and at 6 and 12 months postsurgery. Treating physicians will document detailed surgical data. During surgery, we will collect blood, peritoneal fluid, endometrium and endometriotic tissue. Tissue will be used to isolate and propagate in vitro models of individual cells. Genome wide genotyping and gene expression data will be generated. Somatic mutations will be identified via whole genome sequencing.
Ethics and dissemination The study has been approved and will be monitored by the Metro North Human Research Ethics committee (HREC) and research activities at the University of Queensland (UQ) will be overseen by the UQ HREC with annual reports submitted. Research results will be published in peer-reviewed journals and presented at conferences were appropriate. This study involves human participants and was approved by RBWH Human Research Ethics Committee; HREC/2019/QRBW/56763.The University of Queensland; 2017002744. Participants gave informed consent to participate in the study before taking part.
- REPRODUCTIVE MEDICINE
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STRENGTHS AND LIMITATIONS OF THIS STUDY
Carefully worded patient questionnaires that ensure patients’ responses and the tissue collected corresponds to specific episodes of endometriosis.
Patient data collection is conducted online via email contact and with direct patient entry of data and will be collected both presurgery and postsurgery.
Patient samples are collected in line with harmonised World Endometriosis Research Foundation protocols.
In vitro models are underpinned with both genetic and phenotype data.
It is currently performed at a single tertiary centre, limiting sample size and heterogeneity.
Endometriosis affects approximately 10% of women of reproductive age, leading to significant pelvic pain and subfertility.1 It has been associated with an increased chance of developing ovarian cancer later in life.2 It is characterised by the growth of endometrial epithelial and stromal cells in lesions outside the uterine cavity currently defined in three categories; superficial peritoneal (SUP), ovarian endometrioma (OMA) and deeply infiltrating endometriosis (DIE) lesions, most severe form defined by infiltration of more than 5 mm into the underlying tissue. Endometriosis is currently managed either medically via hormonal modulation or surgically by laparoscopic excision. There are no non-invasive diagnostic tests. Hormonal modulation is inadequate for women wishing to conceive,3 and surgical excision can be difficult and accompanied by a high rate of recurrence, with up to 30% of patients having lesions return within 5 years.4 Inadequacies of medical management places extraordinary strain on healthcare systems.5 The critical need to improve endometriosis management has been recognised by the Australian Federal Government with the launch of a National Action Plan.6
Originally proposed in 1928, Sampson’s theory of retrograde menstruation, in which viable endometrial cells are refluxed back into the peritoneal cavity,7 is still the most commonly accepted theory for the origin of the lesions. However, up to 90% of women experience retrograde menstruation,8 thus additional factors must predispose some to an increased risk. Inherent differences have been observed in both the endometrial epithelial and stromal cells of women with endometriosis that leads to favourable growth profiles of these cells.9 Endometrial mesenchymal stem cells, precursor of the endometrial stromal cells10 are present in the menstrual efflux11 and may initiate lesions, as are epithelial progenitor cells.12 Macrophages13 and dendritic cells14 have altered phagocytosis, and natural killer cells show reduced cytotoxic function15 in women with endometriosis. Increased levels of neutrophils16 and cytotoxic T cells17 in women with endometriosis have also been reported. What underpins the aberrant activity of each cell type and how it contributes to endometriosis progression and how these interact is not yet clear.
Endometriosis is a heritable disease with common genetic variants contributing approximately 51% to endometriosis risk.18 Genome wide association studies are mapping genetic risk factors for endometriosis19–21 and a recent study from 11 case–control data sets (17 045 endometriosis cases and 191 596 controls) replicated nine previously reported genomic regions and identified five novel regions.19 Acquired, rare mutations may also play a role in endometriosis pathogenesis. Using exome sequencing researchers identified somatic changes in cells from 79% of the 24 patients with DIE lesions22 with lesions from five patients (21%) harbouring known somatic cancer driver mutations in ARID1A, PIK3CA, KRAS and PPP2R1A. Importantly, even though lesions contain multiple cell types, separation of epithelial and stromal cells found the cancer driver mutations of KRAS, ARID1A and PPP2R1A were confined to the epithelial cells. Evidence supports a cell-specific influence of genetic variants23 on cellular behaviour and these studies underline the importance not only of inherited genetic risk but also acquired mutations and their potential for differing contributions in individual cell types.
Currently, medical management of endometriosis focuses on reducing circulating estrogen.24 This is often accompanied by unwanted side effects and in many cases, fails to alleviate primary symptoms, nor ablate lesions. Understanding the role of genetic variants in current hormonal treatments is warranted, as genetic architecture of individuals can be a predictive factor in treatment response. A genetic variant in a progesterone metabolising enzyme correlates with an increased progesterone breakdown and reduced effectiveness of progesterone-based treatments.25 While improved targeting of current therapeutics is a short-term goal, novel, targeted non-hormonal treatments are still needed to avoid systemic hormone modulation. A number of biological mechanisms, including the inflammatory response and the endocannabinoid system (ECS), play a role in endometriosis and could potentially be targets for non-hormonal treatments. The ECS affects mechanisms critical to endometriosis establishment and maintenance including cell migration, proliferation, survival and inflammation26 and is significantly influenced by genetic variants.27–29
The challenge now is to understand how these genetic variants, both common across the population and acquired rare mutations, influence cellular function and contribute to endometriosis pathogenesis, progression and response to treatment. Studying the consequences of genetic variants on biological and cellular behaviour requires well-characterised patient tissue that can be purified and studied in the laboratory. We have established tissue collection protocols, linked to the patient and surgical phenotyping data at the Royal Brisbane and Women’s Hospital (RBWH) to undertake this study designed to improve diagnosis, understand the pathogenesis and stratify treatment response in endometriosis, through the implementation of the aims and objectives outlined below.
Identify underlying genetic variants in women with and without endometriosis, determine their relationship to biological variation and functional activity in individual cells and identify their role in individual cells that lead to disease initiation, progression, symptoms and treatment response.
Collect sufficient, high-quality tissue involved in endometriosis pathogenesis from women with and without endometriosis for subsequent clinical and laboratory-based studies to understand pathogenesis and cellular response to specific compounds.
Characterise the genetic variants related to endometriosis pathogenesis and treatment response in the variety of cell types from relevant tissue.
Identify gene and protein expression differences, resulting from genetic variants in endometriosis-related biological material, which may relate to endometriosis pathogenesis and inform treatment approaches.
Isolate and propagate pure populations of endometriosis associated cells with known germline and somatic genetic variants.
Identify the consequence of germline and somatic genetic variations in endometriosis-related cells from women with and without endometriosis.
Understand how these genetic variants interact with environmental factors to influence the prevalence and progress of the disease and how it may respond to treatment.
It is expected that this project will establish a large, professionally processed and curated tissue resource with linked clinical and surgical information. These samples and information will greatly contribute to both current and future endometriosis projects. In the context of this project, it will allow investigation into the germline and somatic mutations in endometrial tissue. It will provide an understanding of:
How both somatic and germline genetic variants influence gene expression and the functional activity of individual cell types associated with endometriosis?
How these genetic, biological and functional variations contribute to endometriosis pathogenesis?
How these genetic, biological and functional variations lead to differences in symptoms and treatment response?
Methods and analysis
Project design and study sites
The project commenced in September 2019 and is initially planned to run for 5 years with 4 years of sample and data collection finishing in December 2023 with an additional year of data analysis to be completed by December 2024. A decision on whether to extend the trial will be made if it is considered worthwhile and if can be secured. The trial will be run as a collaboration between the Department of Obstetrics & Gynaecology (RBWH) and the Genomics of Reproductive Disorders laboratory, Institute for Molecular Bioscience, the University of Queensland (IMB, UQ). Sample collection will be performed at the Department of Obstetrics & Gynaecology, RBWH. Tissue and data collected will be processed at the IMB, UQ site. Experimental procedures and data analysis will be performed at the UQ IMB laboratory. Tissue collected and processed will be stored in locked University sites accessible only through identifiable keyed entry.
Patient and public involvement
The Endometriosis Research Queensland Study was designed based on our previous experience and informal discussion with patients and patient advocacy groups. All women recruited have been provided the opportunity to provide feedback, both informally and through dedicated sections on the patient questionnaires, on their clinical experience, workflow of the study and data collection.
Approach to provision of information to participants and consent
The recruitment will be continuous until the close of the study and under the direction of the project leader. Once patients have been identified as eligible for the study, the main purpose and design of the study will be discussed directly with the patient by a research nurse or the treating physician at their original consultation. Once the project has been verbally explained by trained staff, the patient will also be provided with the participant information sheet and consent form that further outlines the details of the study and the procedures involved. If they choose to participate in the study, they will be asked to sign the consent form. They will be provided with a copy of the signed consent form for reference of the details of the study, the requirements of their involvement as well as the phone number of the Department of Obstetrics & Gynaecology that can be used if further information is sought.
Both the participant information sheet and the consent form will state that participation is voluntary, and consent can be withdrawn at any time and that a decision to not participate will not affect treatment in anyway. The information sheet will outline how their data will be managed to maintain confidentiality and also detail the risks and benefits of participation in the study. If at any stage, the patient withdraws consent any biological material and patient information collected will be destroyed. Subject data not included in the prior analysis will not be affected by the withdrawal from the study.
Inclusion criteria will be women already selected for laparoscopic surgery for suspected endometriosis. Women of all age, ethnicity and sociodemographic background will be eligible for inclusion. Inclusion criteria for the additional lifestyle and environmental factor (LERF) questions are any participant that has already consented to the study and completed the baseline questionnaire.
Patients will be considered either endometriosis positive or endometriosis negative based on either the surgical findings or histopathological examination of excised tissue. Both results will be collected and entered into the database. Subsequent analysis based on endometriosis status will be defined by the values in these fields and will be stated as such in the resulting data and outcomes.
Exclusion criteria will be patients suffering from another pre-existing inflammatory disease, pregnancy, malignancy or if surgery is performed due to an emergency.
Engagement for the patient will include time required to complete the initial questionnaire as well as the potential to contact the patient at least two times (at 6-month intervals) after the completion of surgery to provide details on the subsequent result of the treatment, as well as LERF, or additional details that might be required for this or other ethically approved projects. The participant commitment will include the completion of a series of online questionnaires both pre and postsurgery and the removal of biological material during laparoscopic surgery that is already planned for their treatment. Eligible patients will be requested to complete the LERF questions at 6 or 12-months follow-up.
Participant recruitment strategies and timeframes
Initial patient contact will occur during an initial consultation in which the treating physician and patients will become known to each other. After the initial consultation, the treating physician, under the supervision of the chief investigator, will identify women that match the inclusion criteria for the study and that have been indicated and scheduled for laparoscopic surgery at the Department of Obstetrics & Gynecology, RBWH.
Once potential patients have been identified, the treating physician will organise subsequent contact via a phone call, or other approved modes of telehealth contact by either the treating physician or a dedicated research nurse. This may occur as part of either the patients planned telehealth treatment or as an additional phone call. The treating physician or dedicated research nurse will explain the project in detail and will ask the patient whether they would like additional information.
A follow-up phone call between the study doctor and the patient will be arranged to discuss the information that was provided. During this phone call, the study doctor, or study nurse, can go through the patient information sheet and informed consent forms and allow the patient to ask any questions they may have. If they agree that the patient will be asked to sign the Participant Information and Consent Form (PICF) by a process considered acceptable to Queensland Health.
Any electronically signed consent forms must be printed out on receipt by the study doctor or research nurse. The study doctor or research nurse is to write a file note on the consent form on the process used to obtain consent, sign and date the form. A copy of the completed consent form will be returned electronically to the patient for their records.
Recruited and consented participants will be sent either a link to an online questionnaire, or, if requested, a hardcopy version that has been based on the World Endometriosis Research Foundation (WERF) questionnaire. The approach used is based on the WERF questionnaire, although minor variations the language of the questions ensure participants answer questions relevant to their current episode of endometriosis. As endometriosis is a recurrent disease, we wish to ensure that the data collected are linked to the tissues collected. If the patient reports a previous episode of endometriosis, they are given the opportunity to answer the same set of questions again, describing and comparing their experience to their previous episode. The patients are also afforded the opportunity to complete a free form section describing their journey through endometriosis.
The online Questionnaire data will be collected using a ‘LimeSurvey’ interface. This software is freeware but allows a user to instal a version behind a secure firewall and does not rely on cloud-based storage. All data will be held independently within the Human Study Unit, endometriosis database at the University of Queensland with the local site investigator approving access to site-specific staff. Access is through personalised logins, passwords and mutifactor authentification for the research team. Collection of this data is done under informed consent allowing indefinite storage and use with other information collected and/or generated. Withdrawal of a participant may necessitate destruction of the data. This can be done although summary data may already have been used in previous publications. This questionnaire is to be completed in the participants own time and prior to surgery.
Day of surgery
The women will arrive at the Department of Obstetrics & Gynaecology, RBWH for the scheduled surgical procedure. If a paper copy of the presurgical questionnaire was completed, patients will hand in their completed questionnaire. During surgical preparation and prior to the administration of anaesthesia, peripheral blood will be collected. Remaining biological samples, including eutopic endometrial biopsies, ectopic endometriotic lesions and peritoneal fluid/washing, will be collected when clinically indicated during surgery and processed as described. Patients will receive the required standard of care and involvement in this study will not have an influence on the quality of treatment provided.
Collection of relevant patient symptom and phenotypical data
Patients who agree to the study and have provided informed consent will be asked to complete the background questionnaire that will capture data relevant for the study and include information on symptoms related to endometriosis based on the previously tabulated adapted version of the WERF Endometriosis phenome and biobanking harmonisation project (EPhect) questionnaire. Modifications have been made to this questionnaire that does not change the content but does clarify the language to ensure the participant answers questions relevant to their current episode of endometriosis. The questionnaire contains the possibility to answer the same set of question, but in relation to a previous episode of endometriosis if the patient is willing. Additional questionnaires 6 and 12 months after treatment will elicit information regarding treatment response.
Collection of surgical and clinical data
Clinical data relevant to endometriosis pathology and symptoms, including but not limited to age, weight, Body Mass Index (BMI), gravidity and parity, ethnicity, previous use of medication and other gynaecological disorders, will be collected through the completion of the WERF EPHect surgical phenotype data collection form that is to be completed by the surgeon at the time of surgery. To assist in entry of surgical and clinical data into the patient database, an electronic interface has been built that mirrors the questions of the WERF surgical forms that allows streamlined data entry directly into the database and appended to the patient data.
Baseline data collection
Clinical and phenotypic data collection
Demographic characteristics—name, date of birth, Medicare number, healthcare identifier, residential postcode, gender, ethnicity, language spoken at home, education and employment, marital status and history of smoking and alcohol use.
Clinical presentation characteristics—Presenting symptoms (cyclical and non-cyclical signs and symptoms: dysmenorrhea, deep dyspareunia, dysuria, dyschezia, painful rectal bleeding or haematuria, shoulder tip pain, catamenial pneumothorax, cyclical cough/haemoptysis/chest pain, cyclical scar swelling and pain, fatigue), pain history and symptoms, endometriosis history.
Pain-related questionnaires—Visual analogue scale scores for dysmenorrhoea, non-menstrual pelvic pain, dyspareunia and dyschezia.
Patient-reported outcome measure characteristics—depression, anxiety and health-related quality of life.
Menstrual history including age at menarche, menstrual irregularities, last date of menstrual period, frequency, duration of flow and menstrual cycle length will be recorded.
Fertility history, age at the start of each pregnancy, type of fertility treatment used for each pregnancy and pregnancy outcome (if applicable), live births, type of delivery and pregnancy complications will be captured.
Medical and surgical history—a detailed history of medical and surgical treatments, medication history and primary diagnosis.
Surgical data collection
The surgical data will be collected using the WERF-EPHect Standard Surgical Form (SSF)30 designed to capture all relevant visual information of the endometriosis lesion phenotype and the surgical treatment including:
Current menstrual cycle, current hormonal treatment, pain or other medications used, dose and duration, side effects and other types of complementary interventions used. History of previous surgery for endometriosis.
Imaging (ultrasound and/or MRI) detection or presurgical mapping of endometriosis.
Surgical management—type of surgery performed, duration of the procedure, complications, additional conditions identified at surgery, confirmation of endometriosis.
Detailed visual information on intraoperative findings such as extent, exact location and colour of endometriotic lesions, with a specific focus on endometrioma dimensions and endometriotic nodules. It also includes information on the endometriosis fertility index.
Based on the EPHect SSF data, the phenotype characterisation of endometriosis will be carried out into SUP endometriosis, cystic ovarian endometriosis or endometrioma and deep infiltrating endometriosis.
From the EPHect SSF data, Revised American Fertility Society score will be calculated and endometriosis cases will be grouped into stage I/II or stage III/IV.
Documentation of histopathology reports from samples excised at time of surgery for histopathological diagnosis and confirmation of endometriosis.
The soft copies of the labelled laparoscopic images will be uploaded to the electronic database.
Collection of biological tissue
Relevant biological samples from peripheral blood, peritoneal fluid/washing and tissue implicated in endometriosis pathogenesis, including endometrium and endometriotic lesions, will be collected from women undergoing laparoscopic surgery and endometrial biopsies indicated as part of their designated treatment regime.
Blood samples will be collected from every participant on the day of surgery when intravenous access is sited. The remaining biological tissue, including peritoneal fluid, endometrial biopsies and ectopic lesions, will be collected as clinically indicated during surgery. Endometrial biopsies will be performed only when clinically indicated. Ectopic lesions will be removed to surgically treat endometriosis, peritoneal fluid is removed and peritoneal cavity is washed with normal saline as a routine procedure. In the context of the surgery, no additional surgical action is performed and, therefore, there is no additional risk to the patient. The treatment received is not altered by participation in the study. Biological material collected will be stored securely at appropriate storage facilities at the IMB UQ laboratory. In all cases, only approved research personnel will have access to the specimens.
Whole blood: whole blood to a total of 20 mL will be collected in evacuated tubes per patient and separated into plasma, serum and buffy coat. Buffy coats will be used either for immune cell isolation and culture or DNA isolation for genomic analysis.
Endometrial biopsy: primary samples will be saved in RNAlater for subsequent analysis of somatic mutations, gene or protein expression. Any remaining sample will be flash frozen in liquid nitrogen or stored in cyroprotective media and used for gene or protein expression and the isolation and culture of endometrial and immune cells contained within endometrial biopsies. Any remaining sample(s) will be stored in tissue tek for the potential use of frozen sections.
Peritoneal fluid/washing: on arrival in the laboratory peritoneal fluid/washing will be immediately centrifuged to remove any immune cell content. The immune cell pellet will be kept in cyroprotective media for subsequent culture of immune cells, and the supernatant aliquoted for the analysis of pure peritoneal fluid. The aliquoted fluid will be stored at −70°C to allow batch analysis of immune, hormonal, endocannabinoids or metabolomic components related to endometriosis pathogenesis and progression.
Ectopic lesions: endometrial ectopic tissue collected from either the SUP, OMA or DIE lesions will be carefully dissected from any surrounding tissue and both the lesion and surrounding tissue collected in either RNAlater for somatic mutation, gene or protein expression analysis or either flash frozen or stored in cyroprotective media for subsequent isolation and culture of ectopic endometrial cells or formalin fixed for production of slides that will be used for H&E staining, histological confirmation of endometriosis and protein analysis through immunohistology.
Laboratory studies and data generation
Cell isolation and culture: the eutopic, ectopic and immune cells isolated from women undergoing surgery will be used for the isolation and culture of cells to produce in vitro models. Standard isolation procedures including size exclusion and fluorescence-activated cell sorting will be used to isolate cells of interest and establish in vitro models.
Genetic assays: peripheral blood will be separated into serum and plasma. To determine genetic variants related to endometriosis risk, DNA will be extracted from buffy coats and genotyped on Illumina GSA chips using standard protocols assessed on genomic arrays to determine genomewide genetic variants. These arrays are developed to identify single-nucleotide polymorphisms (SNPs) that commonly occur randomly across the population. To increase the number of assayed SNP imputation using data derived from the 1000 genomes project will be used to infer SNPs in linkage disequilibrium.
Somatic mutational analysis: selected tissue or isolated cells will be used for somatic mutational screening via either targeted sequencing assays, whole exome or whole genome sequencing.
Induced genetic mutations for in vitro analysis of somatic cells: to assess the influence of individual or multiple SNPs associated with endometriosis in the context of consistent genetic backgrounds in isolated endometrial cell-specific alleles can be induced in cells via CRISPR-cas9 technology. We will introduce specific allelic variants in the in vitro cell models generated from isolated endometrial cells. We will determine their influence on gene expression and cell function using methods described below. These somatic cells will be used for in vitro experiments only.
Gene expression: RNA will be isolated using standard procedures and either targeted gene expression via real-time PCR, multiplexed targeting via Nanostring technology or genomewide gene expression via RNA sequencing or single-cell RNA sequencing will be performed to determine gene and transcript expression levels.
Protein expression: cellular protein concentrations of the isolated tissue will be performed by both quantitative and semiquantitative methods, including Western blot analysis and immunohistochemistry. Secreted proteins will be measured via enzyme-linked immune-absorbent assay using the standard manufacturers’ protocol.
Metabolomic profiling: a non-targeted metabolomic profiling will be performed with ultra high-performance liquid chromatography high-resolution mass spectrometry.
Functional assay: the influence of observed biological variations on the cellular functional behaviour either as individual or as dual cell cultures will be determined by numerous cellular assays that will include, but not be limited to viability, proliferation, apoptosis, migration and invasion.
Postsurgical treatment patients will be followed up to determine their response to treatment, including the reduction in symptoms as well as the presence of side effects. Follow-up questionnaires are to be provided to participants at 6-month and 12-month postsurgery. These questionnaire capture details of the individual’s response to treatment and will be either emailed via a personalised link to the questionnaire or mailed to individual participants by personnel authorised to access the address of the patients.
Collection of LERF
Participants who had surgery and completed the baseline questionnaire will be invited to answer LERF questions at the 6 or 12-month follow-up. An initial assessment will be conducted with 500 participants invited to complete LERF questions.
The LERF questions collect information in relation to:
Lifetime occupations (formatted to match Australian census data).
General health (including mental health).
Exposures to potential environmental toxins.
There are some questions regarding use of prescription and illicit drug as well as medical history information including questions pertaining to sexually transmitted diseases. For female participants, there are specific questions relating to menstruation and pregnancy history. This will be administered to all consented individuals and will take approximately 40 min to complete. Participants are informed that they are not obliged to answer any questions that make them feel uncomfortable or they can skip questions they do not wish to answer.
Patients may withdraw their consent at any time during the project by contacting either the Department of Obstetrics and Gynaecology or their treating physician whose details are provided on the patient information sheet. Any biological sample already collected as well as health-related data will be removed from the project and the laboratory. Subject data included in the prior analysis will not be affected by the withdrawal from the study.
Each patient identified for inclusion in the study will be assigned a unique study identification number (study ID). This study number will be assigned on collection of the signed consent at the Department of Obstetrics & Gynaecology, RBWH and matched with a hospital-derived patient identification number (PID). This file will represent the only link between the study number and PID number and will remain accessible only to authorised members of the RBWH clinical team in a secure database with an audit trail function and accessible only via a username and password. Clinical and surgical data collected through the completion of the surgeons reporting questionnaire and the patient questionnaire, respectively, will be entered into a secure database by authorised staff.
Patient and surgical data, lifestyle and environmental data collected will be stored in locked, linked databases housed behind University/Hospital firewalls at IMB/UQ. Experimental procedures and data analysis will be performed at the UQ, IMB Laboratory. The biological material and the health-related data required for analysis will be provided to the UQ laboratory attached to the study ID only and all subsequent experimental data generated during the project will be stored attached to the study ID on secure servers behind UQ University firewall. This data will be considered anonymous to laboratory staff. Only authorised staff for the project will have access to the data. Transfer of any data will be via secure transfer protocols. Data storage will be for the duration of the project as well as and for subsequent projects based on information or material derived from this project and retained for a minimum of 5 years.
A dedicated database has been developed for the project using the open-source Drupal software. The data will be housed on a University server behind the University firewall. Treating physicians or research nurses who consent the patients will enter the patient data directly into the electronic database that will generate an ID. Subsequent patient, surgical and biological collections will linked to the study ID. Surgical data collected from the WERF-EPHect surgical form will be assessed for consistency by key clinical surgical project leaders and the data entered directly into the Drupal database via a built in electronic interface via the relevant physician, study nurse or project leader.
The study ID will also be used to generate personalised links to the patient’s questionnaire, both before and after surgery, that will allow the patient answers to be directly entered into the Lime Survey database. The Drupal database and Lime Survey interface will function as a central repository that will allow real-time data entry and reduce input error. Access to data will only be for authorised personnel.
Data analysis plan
Data analysis will be performed using a combination of SPSS, GraphPad Prism, plink and R statistical and genomic packages. Analysis of patient and surgical data will be used to identify presurgical, risk factors that may facilitate an early diagnosis of endometriosis. Patient data collection matched to the current episode of endometriosis will be used in linear regression models to establish correlation between patient symptoms and lesion characteristics observed during surgery and facilitate subgroup discovery. This will include statistical comparisons between patient-reported symptoms prior to surgery as well as phenotypic descriptions of lesions observed during surgery. Follow-up questionnaires will be used to assess patient response to their individualised treatments and whether linked presurgical data and surgical observation can subcategorise patients into groups that predict treatment response and disease progression.
Biological samples of peripheral blood, peritoneal fluid and eutopic and ectopic endometrium will be explored to identify biological mechanisms that underlie molecular signatures, including gene and transcriptomic expression, alternative splicing and common and acquired genetic mutations, proteomic and metabolic profiles. Molecular signatures generated with collected biological samples will be matched to presurgery patient data, surgical data, follow-up data and LERF data to discover correlations between molecular signature and clinically relevant patient outcomes.
In vitro models will be used to assess the functional influence of genotypes on individual cellular function, including gene and transcriptome expression, alternative splicing, common and rare genetic mutations, proteomic and metabolomic profiles. The interaction between different cell types and their influence on clinically relevant molecular signatures will be assessed. These molecular signatures will also be identified, or induced, in individual in vitro models and used to determine potential personalisation of current treatments to individual molecular profiles or identify novel treatments.
Additional genotyping data and functional genomics data sets aimed at identifying the causal genetic variants and biological mechanism that are associated with endometriosis susceptibility will be generated to potentially contribute to consortium meta-analysis in collaboration with other groups worldwide.
Patient samples with accompanying missing data relevant for an individual analysis will be removed from the analysis. Similarly, patients who have been lost to follow-up will be excluded only from analyses that require a complete dataset.
It is assumed that comparisons will be performed predominantly between two groups. This group could be defined as either women with and without endometriosis, or as either positive or negative response to treatment, as well as many other possibilities. Analysis between two independent means from patient-derived variables (including patient response and experimentally measured) will aim to achieve a moderate power of 80% to target small effect sizes of 0.2. It is estimated to achieve a significant difference a sample size of 394 samples per group, or 786 total would be required. It is intended for recruitment procedures to continue for 4 years to establish a database of sufficient samples that will satisfy power requirements. Over a 4-year period, we plan to recruit approximately 300 patients per year to reach up to 1200 patients by the end of the period. Based on first-year response rates, we predict an incomplete protocol from approximately 15% of patients each year, leaving approximately 100–1100 patients over the 4-year collection period, providing the required sample numbers.
Research team training and quality controls
Research staff are all professionally trained in their relevant positions. They have also received study-specific training. This included training for treating physicians and study nurse on the appropriate recruitment of study participants, how to obtain informed consent, counselling of the study participant, data collection, interacting with the database interface and the follow-up of patients with incomplete study protocols. Specific training has been provided on correct data entry, data management, data analysis and the maintenance of data sets.
Specific training has been provided for the collection and processing of biological samples, including blood and its separation into constituents, aliquoting and recording. Standard operating procedures based on best practice and the WERF biological processing protocols,31 32 including the storage of endometrial eutopic and ectopic lesions for either fresh frozen or cryopreserved process as well as the separation of individual cells for establishing in vitro models have been developed.
Ethics and dissemination
The study has been approved and will continue to be monitored by the Metro North Human Research Ethics committee (HREC) and has received site-specific approval for the conduct of the study at the RBWH. Research activities at the University of Queensland with the data and samples generated also overseen by the UQ HREC. Annual reports will be submitted to both the Metro North and UQ HREC for regular updates on the study. Research results will be published in peer-reviewed journals and the results presented at conferences where appropriate. If requested by the journals, deidentified results may be submitted to recognised data repositories with controlled access policies to provide open access for research purposes.
Project closure procedure
The total duration of the project is 5 years. Four years of tissue collection followed by an additional year to complete tissue and data analysis. There will be the potential to extend the project if deemed beneficial and ethics requirements are met. The project will include the production of in vitro models potentially useful to future projects and other researchers and samples will not be destroyed at the completion of the project. These models may be made conditionally available, subjected to the receipt of appropriate ethical approval. Third-party researchers will only have access to non-identifiable data and will not be able to match these cell models with patients.
The present study was conceptualised and designed in 2020. The first participant was enrolled in September 2020 and the study is currently ongoing with the number of collected sample reaching the desired number expected after the first year of recruitment. Baseline characteristics show positive recruitment and protocol completion rates. Characteristics of the endometriosis patients, patients identified to have another non-endometrial pathology and no discernible endometrial pathology collected in the first year are shown in table 1.
This protocol outlines the framework for a single site study investigating endometriosis and the development of the infrastructure that supports the collection of baseline data that will underpin the research. The infrastructure developed has the potential to be applied to additional sites if the study protocol was to be expanded. The study protocol has been designed to generate baseline data for each patient, including data on patient-reported symptoms, menstrual and fertility history and their previous treatment experience. It will also include surgical observations recorded during treatment and collected by physicians. The research infrastructure will ensure that the high-quality baseline data are both generated, entered and stored in purpose-built database that increase ease of entry and security of the data and decrease user input error.
Collection of baseline patient and surgical data will be built on with genotyping data from DNA isolated from peripheral blood that will allow functional investigation of genetic risk variants associated with endometriosis susceptibility. The collection of endometriosis relevant tissue will allow the generation of omics databases to investigate the functional influence of the genetic variants. It will also allow the creation of in vitro models of individual cell types with the accompanying genomics background.
This study represents a significant opportunity to collect deeply phenotyped endometriosis samples to generate cellular models and detailed integrated molecular data sets. It will be the foundation of endometriosis biorepositories of multiple tissue types including serum, plasma DNA, RNA as well as the establishment of personalised in vitro models that can be screened for responses to treatments and compounds. The results from the analysis will improve our understanding of endometriosis pathogenesis, the clinical symptoms associated with subtypes and how these respond to various treatments. It will provide valuable data sets to contribute to global networks and International Consortia studying endometriosis and improve outcomes for endometriosis patients.
Patient consent for publication
Contributors BM, AA and KT conceived the study and wrote the protocol with assistance from GWM. AA and KT consented patients. AA, KT, BS, PG, AK and DB, MS and DG collected patient data. BM, SS, SA, KT, LMW collected and processed patient samples. MBL and PL build and maintained database and AKH oversaw database and sample processing. SM assisted with power calculations and performed data analysis.
Funding GWM is supported by the National Health and Medical Research Council (NHMRC) fellowship (GNT1078399 and GNT1177194). GWM, AA, KT and SM were supported by the Metro North Hospital and Health Service (CRG-176-2019). KT is supported by RBWH and RBWH Foundation Post Graduate Scholarship. BM is supported by Endometriosis Australia (N/A).
Competing interests None declared.
Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details.
Provenance and peer review Not commissioned; externally peer reviewed.