Sample collection and ethical approval
Pregnancies were recruited from multiple centers in China for chorionic-villus sampling, amniocentesis or percutaneous umbilical blood sampling, between January 2016 and October 2020. Each participant was subjected to low-pass GS and ultrasonography. Soft markers were categorized into 12 isolated types, while ultrasonographic anomalies were categorized into 12 isolated types based on Human Phenotype Ontology (HPO) terms and the site of anomalies detected by ultrasonography. Fetuses satisfying any of the following conditions were included in the study: 1) advanced maternal age (women aged ≥ 35 years) with normal ultrasonographic results, normal non-invasive prenatal screening results, normal Down syndrome biochemical screening tests, and no family/personal history of chromosomal abnormality; 2) ultrasonographic anomalies (including anomalies in cardiovascular, genitourinary, central nervous, gastrointestinal, skeletal, facial, or respiratory system, cystic hygroma, abnormal amniotic fluid, fetal growth restriction, fetal hydrops, and abdominal wall defect); 3) ultrasonographic soft markers (including increased nuchal translucency, choroid plexus cysts, absent or hypoplastic nasal bone, echogenic intracardiac focus, mild ventriculomegaly, single umbilical artery, mild pyelectasis, aberrant right subclavian artery, echogenic bowel, short femur/humerus length, thickened nuchal fold and enlarged cisterna). In total, 43,721 samples were included in this study. The cohort was divided into three groups by gestational week: First trimester (11w-13w+6d), Second trimester (14w-27w+6d), and Third trimester (28w-). Although there was some overlap between soft markers and anomalies in all trimesters, it had little influence on the result obtained. This study was approved by the Ethics Committee of Center for Medical Genetics, Central South University, Hunan, China. The ethics application reference numbers were 2015031002 (approval date: 2016.1-2019.12) and 2019-1-23 (approval date: 2019.05-2024.05). All subjects signed a written informed consent form for genetic investigation of pregnancy.