Assessment of Maternal and Neonatal SARS-CoV-2 Viral Load, Transplacental Antibody Transfer, and Placental Pathology in Pregnancies During the COVID-19 Pandemic

Key Points Question What key biological characteristics of maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and placental function and pathology have implications for vertical transmission and neonatal protection? Findings In this prospective cohort study including 127 pregnancies, there was no maternal viremia, placental infection, or vertical transmission of SARS-CoV-2. Compromised transplacental transfer of anti–SARS-CoV-2 antibodies with robust transfer of influenza-specific immunity and nonoverlapping placental expression of SARS-CoV-2 receptors angiotensin-converting enzyme 2 and transmembrane serine protease 2 were noted. Meaning These findings suggest that, although low rates of maternal viremia and patterns of placental SARS-CoV-2 receptor distribution may underlie the rarity of vertical transmission, reduced transplacental transfer of anti–SARS-CoV-2 antibodies may leave neonates at risk for infection.

The ELISA was developed with TMB and stopped with sulfuric acid. The signal was read at 450 nm and background corrected from a reference wavelength of 570.

Placental Histopathology
Formalin-fixed placentas were examined by an experienced placental pathologist (DJR) and histopathologic diagnoses were rendered in categories (eTable 1), following the Amsterdam guidelines. 8 SARS-CoV-2 positive cases were tested for placental infection by SARS-CoV-2 using RNA in-situ hybridization (RNAish). One full-thickness section of placental parenchyma from each case was cut at 5 microns for RNAish.
RNAish was performed using the RNAscope 2.5 HD Reagent Kit (RED) (ACD Bio, # 322350) and following the manufacturer's instructions for standard conditions as recommended for human lung and placenta. The slide processing method has been previously described. 9,10 Both lung and a known SARS-CoV-2 positive placenta were used as positive controls (eFigure 1). Placenta sections were then hybridized with a predesigned probe spanning COVID-19 sense strand mRNA to detect the coronavirus (V-ncov2019-S, #848561) for 2h at 40°C, processed for standard signal amplification steps, and a red chromogen development was performed using the RNAscope 2.5 HD

Definitions of COVID-19 severity
COVID severity was defined according to NIH and Society for Maternal-Fetal Medicine criteria 13,14 : • Symptomatic or pre-symptomatic disease or presumptive infection is defined as a positive COVID-19 test result with no symptoms.
• Mild disease is defined as flu-like symptoms, such as fever, cough, myalgias, and anosmia without dyspnea, shortness of breath, or abnormal chest imaging.
• Moderate disease is defined by evidence of lower respiratory tract disease with clinical assessment (dyspnea, pneumonia on imaging, abnormal blood gas results, refractory fever of 39.0 °C /102.2°F or greater not alleviated with acetaminophen) while maintaining an oxygen saturation of greater than 93% on room air at sea level.
• Severe disease is defined by a respiratory rate greater than 30 breaths per minute, hypoxia with oxygen saturation less than or equal to 93%, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen of less than 300, or greater than 50% lung involvement on imaging.
• Critical disease is defined as multi-organ failure or dysfunction, shock, or respiratory failure requiring mechanical ventilation or high-flow nasal cannula.

Statistical Analyses
Differences between SARS-CoV-2 positive cases and controls with respect to demographic variables, viral load, antibody response, and placental pathology were evaluated using appropriate tests (parametric or non-parametric, based on normality of data distribution, with two-sided p-values. These tests included Pearson's chi-squared test, Fisher's exact test, Student's t-test, Mann-Whitney U test, Wilcoxon matched pairs signed rank testing, and Spearman rank-based correlations. To further elucidate associations between COVID-19 disease severity and factors of interest, SARS-CoV-2 positive patients were dichotomized into asymptomatic or mild cases (notated as "mild") versus moderate, severe, or critical cases (notated as "severe"), following criteria set forth by the NIH and SMFM, 13,14 or were analyzed in an ordinal fashion based on their disease severity. Within cases, associations between COVID-19 disease severity and viral load and antibody levels were examined in a continuous fashion with nonparametric testing (Mann Whitney U test); viral load was further categorized as detectable versus undetectable and compared with Fisher's exact or Chi square tests.
Correlation analyses between maximum maternal viral load, antibody response, and COVID-19 severity were performed using Spearman rank-based testing. For paired maternal and neonatal cord blood antibody analyses, data were analyzed using Wilcoxon matched pairs signed rank test. Differences in antibody transfer ratios © 2020 Edlow AG et al. JAMA Network Open.
between RBD, N and flu HA were determined using one-way ANOVA with Tukey's posthoc testing to determine the source of differences, p<0.05 Based on the effect size observed for reduced transplacental antibody transfer of RBD and N, we had > 95% statistical power to detect the observed significant differences with an alpha set at <0.05. The impact of maternal medical comorbidities on the relationship between COVID-19 severity and viral load, antibody transfer, or placental pathology was examined using stratified analyses. Statistical significance was defined as a p-value of <0.05; Bonferroni p-value corrections were utilized for placental pathology analyses (correcting for two comparisons, p<0.025). Analyses were performed using GraphPad negative). Not all participants for whom placenta was available had a completed set of matched maternal and umbilical cord blood, accounting for the difference between the number of placentas analyzed versus ELISAs performed. Viral load quantification was performed throughout the study period, ultimately including a larger subset of patients as matched samples were not required for these analyses (N=107).       Titers are represented as the OD450 value subtracted from the reference OD570 value and are shown as the average of two replicates. For asymptomatic patients (light red circles), the interval from "symptom onset" to study blood draw was defined as interval from positive SARS-CoV-2 test to maternal blood draw (unable to determine duration of illness). The mean time from symptom onset to blood draw for antibody quantification among symptomatic individuals was 28.6 ± 18.1 days. Many asymptomatic participants have detectable antibody titer, suggesting duration of illness even greater than the estimated interval. Spearman correlations between duration of symptoms and antibody titers amongst symptomatic patients: Maternal IgG RBD: rho=0.42, p=0.03; Maternal IgM RBD: rho=0.21, p=0.31; Maternal IgG N: rho=0.43, p=0.05; Maternal IgM N: rho=0.17, p=0.46; Cord IgG RBD: rho=0.42, p=0.03; Cord IgG N: rho=0.50, p=0.02. Of the 12 SARS-CoV-2 positive women with no detectable anti-RBD or anti-N antibody, eight were symptomatic. 3/12 SARS-CoV-2 positive women with undetectable anti-RBD or anti-N antibodies were preterm (<37 weeks). Four of 12 had >14 days elapse from time of symptom onset to blood draw, the remaining 8 were either asymptomatic or had <7 days from symptom onset to blood draw. eFigure 5. Cord Antibody Titers by Days from Maternal Symptom Onset eFigure 5 and 6: Maternal (A) and cord blood (B) antibody titers by days from maternal symptom onset. IgG against RBD, N and HA depicted on left, IgM on right. Titers are represented as the OD450 value subtracted from the reference OD570 value and are shown as the average of two replicates. For asymptomatic patients (light red circles), the interval from "symptom onset" to study blood draw was defined as interval from positive SARS-CoV-2 test to maternal blood draw (unable to determine duration of illness). The mean time from symptom onset to blood draw for antibody quantification among symptomatic individuals was 28.6 ± 18.1 days. Many asymptomatic participants have detectable antibody titer, suggesting duration of illness even greater than the estimated interval. Spearman correlations between duration of symptoms and antibody titers amongst symptomatic patients: Maternal IgG RBD: rho=0.42, p=0.03; Maternal IgM RBD: rho=0.21, p=0.31; Maternal IgG N: rho=0.43, p=0.05; Maternal IgM N: rho=0.17, p=0.46; Cord IgG RBD: rho=0.42, p=0.03; Cord IgG N: rho=0.50, p=0.02. Of the 12 SARS-CoV-2 positive women with no detectable anti-RBD or anti-N antibody, eight were symptomatic. 3/12 SARS-CoV-2 positive women with undetectable anti-RBD or anti-N antibodies were preterm (<37 weeks). Four of 12 had >14 days elapse from time of symptom onset to blood draw, the remaining 8 were either asymptomatic or had <7 days from symptom onset to blood draw.