Inclusion and exclusion criteria

Women aged > 18 years with a singleton pregnancy and a live fetus at the 11- to 13-week scan were included in the study. Women who were unconscious or severely ill at the time of recruitment, those with learning difficulties or serious mental illness and those with major fetal abnormality identified at the 11- to 13-week scan were excluded from the study.

Research ethics approval

Approval for the study was obtained from the London–Surrey Borders Research Ethics Committee. The study is registered with the ISRCTN registry, number 83611527.

Quality control of screening

Quality control of screening and verification of adherence to protocol were performed by the University College London Comprehensive Clinical Trials Unit (UCL-CCTU).

Procedures

All eligible women with singleton pregnancies attending their routine hospital visit at 11⁺⁰ to 13⁺⁶ weeks’ gestation were given written information about the study and those who agreed to participate provided written informed consent.

Gestational age was determined from the measurement of the fetal crown–rump length.³¹ Mat-CHs, medical, obstetric and drug history were recorded, and maternal weight and height measured. The MAP and UTA-PI were measured in accordance with standardised protocols;^12,32 the measurements of MAP were carried out by health-care assistants or research sonographers and measurements of UTA-PI were performed by research sonographers. Maternal serum concentrations of PAPP-A and PLGF were measured by one of two automated devices (DELFIA^® Xpress analyser, PerkinElmer Life and Analytical Sciences Ltd, Waltham, MA, USA, or BRAHMS KRYPTOR™ analyser, Thermo Fisher Scientific, Hennigsdorf, Germany). Quality control was applied to achieve consistency of measurement of biomarkers across different hospitals throughout the duration of the study.

Risks calculated using the competing risks model (see Chapter 2)^17,33 were not made available to the participants or their clinicians. The decision concerning administration of aspirin was made by the attending clinicians in accordance with routine standard of care at each site and the information was recorded in the research database both at the time of screening at 11–13 weeks’ gestation and during collection of data on pregnancy outcome.

All data on participant characteristics, biomarker values and outcome from each site were reported to UCL-CCTU. The data, blinded to outcome, were then provided to the study statistician who:

1. defined the screen-positive group in accordance with NICE criteria
2. computed risks for all pre-eclampsia and preterm pre-eclampsia for the prespecified combinations of biomarkers using the competing risks model^17,33
3. identified the group that was treated with aspirin (≥ 75 mg/day, starting at < 14 weeks’ gestation and ending at ≥ 36 weeks’ gestation or at the time of earlier birth)
4. examined associations between treatment using aspirin and baseline covariates, including the components of the NICE method and biomarkers.

Details of the imputation methodology for dealing with the effects of aspirin and summaries of Mat-CHs and treatment with aspirin were added to the statistical analysis plan. After the signed statistical analysis plan and the data file containing data fields (a)–(c) were received and approved by the UCL-CCTU research team, they provided data on pregnancy outcomes to the study statistician for linking for the unblinded analysis.

Diagnosis of pre-eclampsia

Data on pregnancy outcome were collected from the hospital maternity records or the women’s general medical practitioners. The obstetric records of all women with pre-existing or pregnancy-associated hypertension were examined to determine the diagnosis of pre-eclampsia. This was based on the finding of hypertension (i.e. systolic blood pressure of ≥ 140 mmHg or diastolic blood pressure of ≥ 90 mmHg on at least two occasions, 4 hours apart, developing after 20 weeks’ gestation in previously normotensive women) and at least one of the following: proteinuria (i.e. ≥ 300 mg/24 hours or protein-to-creatinine ratio of ≥ 30 mg/mmol or ≥ 2+ on dipstick testing), renal insufficiency (i.e. serum creatinine > 1.1 mg/dl or twofold increase in serum creatinine in the absence of underlying renal disease), liver involvement (i.e. blood concentration of transaminases to twice the normal level), neurological complications (e.g. cerebral or visual symptoms), thrombocytopenia (i.e. platelet count < 100,000/µl) or pulmonary oedema.^21,34

Outcome measures

The primary comparison was DR of risk assessment recommended by the NICE guidelines compared with screening by a mini-combined test (i.e. Mat-CHs, MAP and PAPP-A) in the prediction of pre-eclampsia occurring at any gestational age (i.e. all pre-eclampsia) after adjustment for the effect of aspirin, for the same SPR determined by the NICE method. This combination of biomarkers was selected because the test can be introduced without additional cost, as all NHS maternity hospitals in England offer first-trimester combined screening for trisomies, which includes measurement of PAPP-A.

Key secondary comparisons were DR for preterm pre-eclampsia using NICE guidelines compared with the competing risk model with the following marker combinations:

• Mat-CHs, MAP and PAPP-A
• Mat-CHs, MAP and PLGF
• Mat-CHs, MAP, UTA-PI and PLGF.

The combination of Mat-CHs and PLGF was selected because PLGF can be measured in the same sample on the same machines used in screening for trisomies. In addition, in previous studies this was found to be more effective than PAPP-A in the prediction of pre-eclampsia.³⁵ The combination of Mat-CHs, MAP, UTA-PI and PLGF was selected because in previous studies it was found to be the most effective method of screening.

Statistical analysis

We proposed to recruit 16,850 women. On the assumptions of an incidence of pre-eclampsia of 2.6% and a loss to follow-up rate of 5% there would be 16,000 women for evaluation. On the extreme assumption that 90% of NICE screen-positive patients and 10% of NICE screen-negative patients would be treated with aspirin and that aspirin reduces the incidence of all pre-eclampsia by 50%, the power to detect a 10% difference in DR between the NICE method and the mini-combined test in the prediction of all pre-eclampsia at the one-sided 2.5% level would be > 80%.

We used McNemar’s test to compare the DR of the NICE method with that of the Bayes’ theorem-based method. However, as some of the women who were screen positive according to NICE guidelines were prescribed aspirin, which could have reduced the risk of pre-eclampsia, some of the patients in the screen-positive group would have effectively been converted to false positives. Consequently, treating NICE screen-positive women with aspirin would reduce the DR and bias the McNemar’s test against the NICE method. Our approach to dealing with this was to apply multiple imputation of data on the incidence of pre-eclampsia that would have occurred had it not been for the effect of treatment. Markov chain Monte Carlo was used to impute incidence data and generate 10 complete data sets for analysis.³⁶ Estimates of DR were then pooled across data. The incidence of pre-eclampsia that would have occurred had it not been for the effect of treatment was determined from a logistic regression model dependent on NICE and centre, and that aspirin reduced the incidence with a prespecified probability of 0.3 for all pre-eclampsia and 0.6 for preterm pre-eclampsia.⁷ Although these probabilities were based on the results of the ASPRE trial, in which the daily dose of aspirin was 150 mg rather than 75 mg (as recommended by NICE),⁸ we wanted to avoid any potential criticism of bias against the NICE method by assuming that the effect of 75 mg was similar to that of higher doses of the drug. The method of imputation and the choice of treatment effects were prespecified and documented prior to receipt of the outcome data.

Additional evaluation of performance of the Bayes’ theorem-based method involved estimation of DRs of pre-eclampsia at a fixed SPR of 10% for all 16 combinations of biomarkers. McNemar’s test was applied to the effect of adding markers. No adjustments were made for the effects of aspirin in this additional evaluation.

Markov chain Monte Carlo was implemented using the WinBUGS 1.4.3 software (MRC Biostatistics Unit, Cambridge, UK). The WinBUGS model and a description of the methodology are given in Appendix 2. The statistical software package R (The R Foundation for Statistical Computing, Vienna, Austria) was used for data analyses with the MICE package, pooling estimates across the 10 complete data sets using the function pool.scalar. Results were reported according to standards for the reporting of diagnostic accuracy studies guidelines.³⁷

Public and patient involvement

Public and patient involvement input into this study was particularly important to (1) identify possible barriers to recruitment, (2) evaluate acceptability of early screening, (3) understand the implications of classifying somebody as screen positive and (4) ensure that findings are disseminated and implemented appropriately.

Melissa Green, Chief Executive Officer of Bliss, and Jane Fisher, Director of Antenatal Results and Choices, were fully involved with our study and were full, independent members of the Study Steering Group. They contributed to the development of the research question, to the application and provided input into the study from funding through to reporting of the findings.