Methods
This study was a secondary analysis of the Stillbirth Collaborative Research Network (SCRN) study. Briefly, the SCRN study was a racially, ethnically, and geographically diverse, multicenter case-control study of stillbirth and selected live births with enrollment at the time of delivery. Recruitment occurred in 59 hospitals in 5 geographic regions throughout the US. Details about the participating hospitals and study population have been described previously.11, 14 The study was approved by the institutional review board at each clinical site and the data coordinating center. An advisory board reviewed the progress and safety of the study and written informed consent was obtained from each participant.
This work was supported by grant funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development: U10-HD045953 Brown University, Rhode Island; U10-HD045925 Emory University, Georgia; U10-HD045952 University of Texas Medical Branch at Galveston, Texas; U10-HDO45955 University of Texas Health Sciences Center at San Antonio, Texas; U10-HD045944 University of Utah Health Sciences Center, Utah; and U01-HD045954 RTI International, RTP. Secondary analysis of the primary research was supported in part by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number 1UL01TR002538. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Postmortem examinations and placental histologic examinations were performed by perinatal pathologists who underwent centralized training using a standardized format. Details of the placental pathologic evaluation have been previously reported.9 The INCODE cause of death classification tool was used across sites to best identify cases where a fetal or placental condition significantly contributed to the fetal death.15, 16 In the present analysis, we included singleton stillbirth deliveries with chromosomal microarray and postmortem examinations of the fetus and placenta (n=387). Of note, 98.6% of stillbirth cases in the SCRN study had placental examination completed.9.
A consensus-determined protocol was implemented to define placental pathological lesions types as maternal vascular, fetal vascular, maternal inflammatory, fetal inflammatory and immune/idiopathic lesions.17, 18 The specific pathologic placental lesions included in each category are listed in Table S1 .
Biospecimens collected as part of the SCRN protocol included placental tissue, fetal liver, muscle, and cord blood. Sizes of placental biopsies varied, but they were as large as 1 cm3 and were stored at – 200C from 2 – 5 years prior to DNA extraction. Microarray analysis was performed at a single laboratory (Columbia University Medical Center) in 2012. Samples were analyzed using the Affymetrix Genome Wide Human SNP Array 6.0 and the Chromosome Analysis Suite, version 1.0.1, and the NetAffx annotation database, version 28 with data aligned to the Human Genome release 18. CNVs of ≥500 kb in size were detected using the SNP array. Analysis of the array data was conducted to determine aneuploidy, potential maternal-fetal contamination, and sex discordance. Classification of CNVs was based on the American College of Medical Genetics (ACMG) standards and guidelines for interpretation and reporting, with modifications as described previously.19 Due to improving resolution for determination of pathogenicity of CNVs, the number of novel structural variants is constantly increasing.20 Therefore, we implemented the latest ACMG guidelines21 in high-throughput CNV analysis to classify and update pathogenicity of CNVs previously categorized as variants of unknown clinical significance (VOUS) by using ClassifyCNV tool.22 Since VOUS CNVs should not be considered benign, we classified CNVs into two groups: abnormal CNVs (abnormal CNVs), defined as pathogenic CNVs (including aneuploidy) or VOUS, and normal CNVs (normal CNVs), defined as no CNVs > 500 kb or benign CNVs.23-25. As such, the abnormal CNVs and normal CNVs groups were compared in statistical analysis. In addition, study characteristics were described separately for pathogenic, VOUS and normal CNVs. We discussed the implications of the findings based on only pathogenic CNVs (excluding trisomy, monosomy, sex-chromosome and VOUS CNVs).
To test for associations of placental pathological lesions with CNV categorization, frequency and percentages were calculated within category and compared using chi-square. Other categorical measures were similarly compared by CNV category. To compare continuous measures, ANOVA was used. Data were analyzed with the use of statistical software programs: SAS version 9.4 (SAS Institute Inc), R and STATA version 15.0 (StataCorp), and ClassifyCNV tool.22