Serum samples were collected and aliquoted as part of the Maternal-Fetal Medicine Units Network multicenter randomized controlled trial of low-dose aspirin for the prevention of preeclampsia in high-risk women [4]. Samples were collected between June 1992 and December 1995, aliquoted, frozen and stored at −80°C. Samples were thawed one time (74%) or thawed two times (26%), and the storage time and handling of samples was similar to that of other similar studies including the CPEP cohort. Serum samples were assayed concurrently for sFlt1, sEng and PlGF using commercially available immunoassay kits purchased from R&D Systems (Minneapolis, MN). All kits utilized were from the same manufacturing lot for each specific analyte, a policy designed to minimize variability. ELISAs were validated by performing dilutional parallelism and spike-recovery tests. Correlations between the degree of sample dilution and measured analytes were linear (r2 >0.99, all). Calculated recoveries of excess analytes were >94% for all. Measurements of each angiogenic factor were performed in duplicate according to the manufacturer's protocol. All laboratory analyses were performed by personnel unaware of either pregnancy outcome or the high-risk group from which the sera were obtained. In general, samples were diluted 1 to 5 for sFlt1 and sEng, and 1 to 2 for PlGF in order for the samples to fall within the measurable range of the kit's standard curves. A minimal number of samples required re-analysis because the results were outside of the range of the standard curve. Of the total number of serum samples, 135 (5%) required re-assay for sFlt1, 29 (0.9%) for sEng, and 33 (1.2%) for PlGF. The inter-assay variability for each analyte was 10% for sFlt1, 11% for sEng and 7% for PlGF.