A cohort as well as diagnostic study was conducted as a secondary analysis of a prospective database of the maternal-fetal medicine (MFM) unit of Chiang Mai University, Thailand. The database was developed under the “Prenatal Control of Down Syndrome Project”of the National Health Security Office, Thailand. Under the project, all pregnant women that attended our antenatal care clinic and our network hospitals were offered quad test as a screening test for fetal Down syndrome in the second trimester free of charge. This study was conducted with ethical approval by the Institutional Review Board (The Research Ethics Committee 4; Faculty of Medicine, Chiang Mai University; Study Code: OBG-2562-06069 / Research ID: 06069). This diagnostic study was conducted with adherence to the STARD and TRIPOD standards (18, 19). All the women, who participated in the project, provided written informed consent. The study population was pregnant women who attended antenatal care at Maharaj Nakorn Chiang Mai Hospital and the network hospitals in the northern part of Thailand and underwent second trimester quad test for fetal Down syndrome screening, with known pregnancy outcomes, between January 2016 and October 2019.
Database development: The primary project was undertaken to assess the efficacy of the maternal quadruple (quad) test in our population in the detection of fetal Down syndrome. All pregnancies were prospectively followed up for the pregnancy outcomes and fetal status of aneuploidy. All participants were of Thai ethnicity and were living in the North of Thailand. They participated in the project with informed consent after counseling by the project team. The baseline characteristics (age, parity, body weight, ethnicity, medical conditions, etc.) and laboratory analysis of the serum biomarkers were reviewed by the authors and prospectively obtained. The serum biomarker levels of all collected samples were determined at the same project laboratory (completely automated assay, DELFIA® Xpress system; Perkin Elmer, Waltham, MA, USA), with the standard immunoassay kits of AFP, b-hCG, uE3, and IHA. The quad screens were conducted free, as they were financially covered by the National Health Security Office, Thailand. The participants were followed-up for obstetric outcomes, such as birthweight, gestational weeks at birth, route of delivery, fetal anomalies, obstetric complications, etc. The newborns were assessed by pediatricians. Chromosome studies were performed only in women categorized as high risk by the quad test or newborns with suspicion of abnormalities after assessment by the neonatologists. The chromosome abnormalities were confirmed either by amniocentesis or cytogenetic studies after birth, whereas diagnoses of normal chromosomes were confirmed by cytogenetic work-up or the conclusion by the neonatologists in cases where cytogenetic study was not done.
Data Retrieval: The project database, which was developed between 2016 and 2019, was accessed to obtain the records meeting the inclusion criteria as well as having complete information of baseline characteristics and obstetric data, such as maternal age and weight, underlying medical diseases, smoking history, gestational week of blood sampling for maternal serum biomarkers, gestational week at birth, baby weight and anomalies. The inclusion criteria for retrieval of the records are as follows: 1) single gestation, 2) underwent serum biomarker test (quad test) at gestational age of 15-21 weeks, and 3) availability of final obstetric outcomes. The cases with the following criteria were excluded: 1) multifetal gestation, 2) fetal anomaly or aneuploidy, 3) unavailability of obstetric outcomes, 4) pregnancy termination before 20 weeks of pregnancy, and 5) significant medical complications, for examples, uncorrected cyanotic heart disease, uncontrolled hyperthyroidism, renal impairment, etc.
Data Processing: The women who met the inclusion criteria were divided into two groups: pregnancies without SGA (control group) and pregnancies with SGA (study group). All records, including baseline characteristics, serum biomarker levels, obstetric and neonatal outcomes, were reviewed and validated. The definitions used in this study are as follows: 1) Gestational age: Gestational age was based on crown-rump length (CRL) in the first trimester or biparietal diameter (BPD) in the second trimester. 2) Second trimester serum screening: Screening test for fetal aneuploidy in the second trimester (quad screen) using four serum biomarkers, including maternal serum AFP, b-hCG, uE3, and IHA, collected at gestational age of 15-21 weeks. Abnormal levels of the biomarkers were defined as the levels of greater than 2 MoM for AFP, b-hCG and IHA and less than 0.5 MoM for uE3 (based on previous studies). 3) Fetal growth restriction: A fetus with birth weight lower than the 10th percentile of the gestational date (20).
Sample size estimation: Based on previous studies, the relative risk of SGA among pregnant women with abnormal quad screen (elevated AFP) is approximately 1.6-4.0 (7). To estimate the sample size, a cohort study with an estimated relative risk of 1.8 and a prevalence of SGA in the control group of approximately 7% needs a sample size of at least 536 affected cases, at 95% confidence and 80% power of test.
Primary outcome: Incidences of SGA among pregnant women with normal and abnormal concentrations of the four serum biochemical markers: AFP, b-hCG, uE3, and IHA.
Statistical analysis: The statistical procedures were undertaken using the SPSS software (IBM Corp. Released 2012; IBM SPSS Statistics for Windows, Version 21.0. Armonk, New York). The statistical techniques are the same as those used in determining the fetal risk of aneuploidy, summarized as the followings. The multiples of the median (MoMs) of the four biochemical markers (AFP, b-hCG, uE3 and IHA) were obtained by the following steps: (1) performing regression analysis, using a stepwise technique, of the log10 levels of the four serum markers as a dependent variable against potential independent factors, for examples gestational week of blood sampling, maternal age and weight, history of smoking, etc.; (2) determining the predicted log10 levels of each serum marker of quad test for individual women by using the constructed model in Step 1; (3) transforming the obtained log10 from the prior step to the predicted level of the serum markers; (4) calculating the MoMs by dividing the actual measured values of the serum markers of all subjects by their predicted values. The models for estimating the risk of small for gestational age (SGA) were constructed using a binary logistic regression method, with serum markers as dependent parameters and SGA as an independent parameter. Using the adjusted MoMs, the log-Gaussian distributions of each serum marker for SGA was derived. The effectiveness of the created models was determined by receiver-operated characteristic (ROC) curves, together with the sensitivity and false positive rate in the risk estimation for SGA. The effectiveness of each serum marker and their combination was assessed by comparing the ROC area under the curve of the serum markers. The positive likelihood ratio of SGA for individual women was also determined by dividing the sensitivity by the false positive rate of each serum marker. The final risk of SGA was obtained by multiplying the positive likelihood ratio by the background risk.