Prove—pre‐eclampsia obstetric adverse events: Establishment of a biobank and database for pre‐eclampsia

Pre‐eclampsia is a leading cause of maternal and perinatal morbidity and mortality. The burden of disease lies mainly in low‐middle income countries. The aim of this project is to establish a pre‐eclampsia biobank in South Africa to facilitate research in the field of pre‐eclampsia with a focus on phenotyping severe disease.The approach of our biobank is to collect biological specimens, detailed clinical data, tests, and biophysical examinations, including magnetic resonance imaging (MRI) of the brain, MRI of the heart, transcranial Doppler, echocardiography, and cognitive function tests.Women diagnosed with pre‐eclampsia and normotensive controls are enrolled in the biobank at admission to Tygerberg University Hospital (Cape Town, South Africa). Biological samples and clinical data are collected at inclusion/delivery and during the hospital stay. Special investigations as per above are performed in a subset of women. After two months, women are followed up by telephonic interviews. This project aims to establish a biobank and database for severe organ complications of pre‐eclampsia in a low‐middle income country where the incidence of pre‐eclampsia with organ complications is high. The study integrates different methods to investigate pre‐eclampsia, focusing on improved understanding of pathophysiology, prediction of organ complications, and potentially future drug evaluation and discovery. The study is registered at the International Standard Randomised Controlled Trial Number (ISCRTN) with trial registration number ISRCTN10623443. We include women who are admitted to Tygerberg Hospital for the birth of their baby. Cases (women with pre-eclampsia) are included before or after delivery depending on presentation. Controls (women with normotensive pregnancies) are recruited when admitted for delivery or at a visit to the outpatient clinic. For the controls, samples are collected at inclusion and delivery. Cases are seen daily and clinical information and samples are collected serially until discharge. All participants are contacted telephonically in the postpartum period. Data are collected prospectively and from the medical charts on maternal health and neonatal outcomes (Figure 1). Flow chart of the study. All women with pre-eclampsia who are admitted to Tygerberg hospital are eligible for inclusion. Our controls are women who deliver at Tygerberg without the hypertensive disease. Pre-eclampsia is defined according to the International Society for the Study of Hypertension in Pregnancy (ISSHP) [18]. Both women with pre-eclampsia without severe features and pre-eclampsia with end-organ complications are eligible for inclusion. End-organ complications are defined according to the ISSHP classification system and include haemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome, eclampsia, pulmonary oedema, renal impairment, and acute severe hypertension [18]. All parameters are included in the online database described below, and women can thus be classified according to different classification systems. All participants must be competent to provide informed consent before enrolment. If they are minors, they need to read and sign the assent form, and a parent or guardian must sign consent. All individual studies originating from the biobank require a unique ethical approval. Clinical information is collected on datasheets and entered into an online secure password-protected Research Electronic Data Capture (REDCap) database. The data collection includes information on the participant’s age, gravidity, parity, medical history, current pregnancy information, and outcome of the pregnancy. Only the principal investigators have access to patient identifying information and each participant is allocated a study number. The variables collected in the database correspond to the recommended core demographics, predictors, and outcomes listed in “Strategy for standardization for pre-eclampsia research design” by the international group Co-Lab for harmonisation of pre-eclampsia research (Table 1) [17]. The women are also interviewed at inclusion with a short questionnaire regarding signs and symptoms specific for pre-eclampsia (Table 1). Clinical information collected in the database RedCap. APGAR; appearance, pulse, grimace, activity, respiration, AST; aspartate transaminase, CFQ; Cognitive Failure Questionnaire, CPAP; continuous positive airway pressure, GCS; Glasgow Coma Scale, HELLP; haemolysis, elevated liver enzymes, low platelets, HIV; human immune deficiency virus, ICU; intensive care unit, LV; left ventricle, MoCA; Montreal Cognitive Assessment, MRI; magnetic resonance imaging, PPH; postpartum haemorrhage, PRES; posterior reversible encephalopathy syndrome. Nominal (n); binary (b); ordinal (o); continuous (c). Samples are collected by research midwives. After a blood sample is drawn, it is centrifuged, aliquoted, and frozen within two hours of collection. The same procedure is performed for cerebrospinal fluid and umbilical cord blood. Saliva and urine are frozen directly and are not centrifuged. Placental samples are taken at four sites on the maternal and foetal surfaces of the placenta. They are stored in RNAlater for at least 48 h and then frozen. All samples are stored in a minus 70 °C freezer which has an electronic temperature monitoring system. All samples are labelled only with the study number, sample number, and date. Samples collected for the biobank are presented in Table 2. Biological samples collected for the PROVE biobank. EDTA; ethylenediaminetetraacetic acid, RNA; ribonucleic acid. Cognitive function is assessed subjectively and objectively as close to discharge as possible. The subjective function is assessed using the Cognitive Failure Questionnaire (Supplemental Figure S1). Cognitive function is also assessed objectively using the Montreal Cognitive Assessment (MoCA) (Supplemental Figure S2). A detailed description of the cognitive function tests can be found in the Supplementary Methods Section. For women with eclampsia and women with pre-eclampsia with severe features without eclampsia as a control group, we perform MRI examinations on entry into the study. We use a 1.5 Tesla scanner, used in clinical practice, at the Department of Radiology at Tygerberg University Hospital. The MRI protocol includes sequences for evaluation of brain morphology including infarcts, oedema, and haemorrhages, as well as assessment of arterial spasm and cerebral perfusion. Imaging sequence information can be found in the Supplementary Methods Section. Transcranial Doppler examination is performed in a subgroup of women to evaluate the cerebral perfusion pressure and dynamic cerebral autoregulation on entry into the study and for some women also before discharge from the hospital and in addition before and after vasoactive medication. Both women with normotensive pregnancies, pre-eclampsia without severe features, and pre-eclampsia with severe features are eligible for transcranial Doppler examination. This is performed by locating the middle cerebral artery bilaterally by the Doppler technique and simultaneously recording continuous blood pressure and end-tidal CO2. A detailed description of the methodology can be found in the Supplementary Methods Section. A subgroup of cases (women with pulmonary oedema) and controls (women with pre-eclampsia without pulmonary oedema and women with normotensive pregnancies) have echocardiograms and cardiac MRI on entry into the study. Investigations are performed by two cardiologists according to a predefined protocol that can be found in the Supplementary Methods Section. Results from echocardiography, MRI, and cerebral Doppler will be calculated by two independent interpreters blinded to groups and entered manually into the database. MR data will be analysed in collaboration with Uppsala University by neuroradiologists, blinded to groups. Blood samples will be analysed for placental, cardiac-, renal-, neurological- and endothelial biomarkers, using standardised platforms when available and for the remaining analyses through manual enzyme-linked immunosorbent assay (ELISA) analyses in duplicates. Inter- and intra-assay coefficients of variation will be aimed at below 10%. Demographics will be presented as medians or means as appropriate by distribution. Comparison between groups will be analysed by Student’s t-test or Mann–Whitney u-test with means or medians and confidence intervals or interquartile range, as appropriate according to the distribution of the variables. When comparing multiple groups, the Kruskal–Wallis test or one-way ANOVA will be used as appropriate according to the distribution of the variables. Correlations will be analysed by Pearson’s r or Spearman’s rho, as appropriate by the distribution of the variable. Regression analyses, unadjusted and adjusted, will be performed to adjust for known confounding variables. A total of 10 cases per variable at the lowest will be considered appropriate to avoid overfitting of the model. All statistical analyses will be performed in SPSS or R. Tygerberg University hospital has approximately 8000 high-risk deliveries yearly. Pre-eclampsia affects a large proportion of these deliveries but the exact numbers are not known. Prospective power calculations for some investigations will not be possible since no reference values or results exist. In addition, the biobank is designed to be open for future research on pre-eclampsia where the research question to date might not be known. Some examples of power calculations for planned analyses are given below. Cerebral blood flow regulation: To detect a difference in dynamic cerebral autoregulation index of 1, 2 with a standard deviation (SD) of 1.5, 25 women are required in each group [19]. Cerebral biomarkers: To detect a difference of 4 pg/mL between cases and controls for NfL with an SD of 5, 25 women are required per group [20]. In order to extend the comparisons to correlations and sub-analyses, the initial sample size for PROVE was set to 100 women with eclampsia, 50 women with pulmonary oedema, and 50 women in each control group before the first round of analyses are initiated. No input from patients has been solicited in the creation of the database and biobank. Due to the frequency, morbidity, and mortality associated with pre-eclampsia, there is a great scientific and social value to undergird this research. Blanket and broad consent have been avoided, and only research linked to the topic of pre-eclampsia will be conducted. The risk of adverse events is very low. Peripheral blood collection is carried out by an experienced midwife/doctor at the time of routine blood collection. Cerebrospinal fluid is collected from a discarded sample from the anaesthetist during the administration of spinal anaesthesia. MRI imaging in selected participants with eclampsia or cerebral signs has fewer side effects than conventional imaging. Performing transcranial Doppler measurements holds no risks. Participant information databases will be routinely backed up to prevent loss due to technical issues, and samples within the biobank will be secured via routine laboratory safety nets (e.g., the availability of backup freezer space, documentation of usage, etc.). Blood samples are collected at the same time as routine blood testing, and thus, no additional discomfort is inflicted. Other samples are collected from tissue and fluid that are usually discarded. No publications will come directly from the biobank. The biobank will provide a source of material for further studies that will each need individual ethics approval.