Methods
We conducted a secondary analysis of the Stillbirth Collaborative
Research Network (SCRN) study, a racially/ethnically diverse,
population-based case-control study of stillbirth with enrollment at the
time of delivery. The SCRN study recruitment sites and study population
details have been previously described.4, 12 The study
was supported by grant funding from the Eunice Kennedy ShriverNational 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. An advisory board reviewed the progress
and safety of the study and written informed consent was obtained from
each participant. Study participants were not involved in the
development of the research and a core outcome set has not been used in
the research.
In the present analysis, we excluded cases with multifetal gestations
and without perinatal postmortem examination data. Among eligible cases,
we included those with and without major fetal anomalies as described
previously.13 Women were enrolled at the time of
diagnosis of stillbirth, diagnosed as fetal death that occurred at 20
weeks’ gestation or more. Gestational age was determined by the best
clinical estimate from multiple sources including information from
assisted reproductive technology, last menstrual period and/or obstetric
ultrasound.14 Women underwent a standardized maternal
interview, medical record abstraction, biospecimen collection, an
obstetric ultrasound exam, and postmortem examinations of the fetus and
placenta. The standardized postmortem protocol ensured evaluation of
stillbirth consistently across study geographic regions to best identify
stillbirth where a fetal or placental condition caused or significantly
contributed to the fetal death.15 To avoid inadvertent
exclusion of fetuses that may have been ≥20 weeks of gestation, fetal
deaths occurring at 18-19 weeks and 6 days estimated gestational age
with poor gestational dating criteria were included as stillbirth
cases.12, 13 Fetal autopsy and placental examinations
of stillbirth cases were performed by perinatal pathologists using
study-specific standardized protocols, that included centralized
training.15, 16
Fetal structural malformations were prenatally diagnosed and grouped as
major malformations by anatomic system and specific malformation type as
described previously.13 The groupings include cystic
hygroma, central nervous system (open neural tube defect, anencephaly,
hydranencephaly, hydrocephalus, holoprosencephaly, other) using a
protocol for neuropathologic examination,16 thoracic
(congenital diaphragmatic hernia, cystic adenomatoid malformation,
pulmonary sequestration, other), cardiac (anteroseptal defect,
ventricular septal defect, atrioventricular canal defect, transposition
of the great vessels, tetralogy of Fallot, other), gastrointestinal
(gastroschisis, omphalocele, duodenal atresia, other), genitourinary
(hydronephrosis/ureteropelvic junction obstruction, autosomal recessive
polycystic kidney disease, multicystic/dysplastic kidney, posterior
urethral valves, renal agenesis, other), skeletal (skeletal dysplasia,
club feet, other), umbilical cord, craniofacial (cleft palate, other),
hydrops fetalis, other and any anomalies. To further describe
malformations, a ‘write in’ option was used, where sections were
individually reviewed to ensure appropriate categorization. For example,
‘craniofacial anomalies’ category was added after data inspection
revealed that these were not consistently categorized. In addition, a
single diagnosis for ‘hydrops’ category was added, and when multiple
abnormalities were noted and consistent with hydrops (i.e. ascites,
pleural effusions, skin oedema). Other structural malformations were
defined based on ‘write in’ option, where sections were individually
reviewed to ensure that they were appropriately categorized. Multiple
structural malformations (any malformation) were classified according to
each system for which an abnormality was present.
Placental and stored/frozen fetal liver, fetal muscle and cord blood
tissue biospecimens were collected from stillbirth cases. Sizes of
placental biopsies varied, but they were as large as 1
cm3.16 In macerated fetuses, fresh
samples were obtained from the placenta. DNA from placental biopsies was
stored at −20°C for 2 to 5 years before microarray analysis, which was
performed at a single laboratory (Columbia University Medical Center).
DNA from stored frozen muscle and liver specimens was extracted
immediately before microarray analysis and were used when placental DNA
was unavailable for analysis. Fetal muscle, cord blood or fetal liver
were used for chromosomal microarray assessment when placental DNA was
unavailable (n=106 [19.9%]).
DNA Samples were analyzed using the Affymetrix Genome Wide Human single
nucleotide polymorphism (SNP) Array 6.0 and the Chromosome Analysis
Suite, version 1.0.1, and the NetAffx annotation database, version 28
for microarray analysis (Affymetrix). Data were aligned to the Human
Genome release 18 (hg18). CNVs with ≥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.4, 17 Due to improving resolution for
determination of pathogenicity of CNVs, the number of novel structural
variants is constantly increasing.18 An efficient
computational analysis may be required to update pathogenicity score of
manually defined variants of unknown clinical significance (VOUS) CNVs.
Therefore, we implemented the latest ACMG guidelines19in high-throughput CNV analysis to classify and update pathogenicity of
CNVs previously categorized as VOUS by using ClassifyCNV
tool.20 Using the ACMG guideline, we classified CNVs
into two groups: abnormal CNVs, defined as pathogenic CNVs (including
aneuploidy) or VOUS, and normal CNVs, defined as no CNVs >
500 kb or benign CNVs.21-23 As such, the abnormal CNVs
and normal CNVs groups were compared in statistical analysis and only
pathogenic CNVs (excluding aneuploidy, sex-chromosome and VOUS CNVs)
were discussed.
We used the Wald Chi-squared test and a two-by-two table to compare the
proportions of abnormal CNVs and normal CNVs between stillborn fetuses
with and without fetal structural malformations. Other categorical
measures were similarly compared between abnormal CNVs and normal CNVs.
To compare continuous measures, ANOVA statistics 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.20