key: cord-0766788-wue6ise9
authors: Edlow, Andrea G.; Li, Jonathan Z.; Collier, Ai-ris Y.; Atyeo, Caroline; James, Kaitlyn E.; Boatin, Adeline A.; Gray, Kathryn J.; Bordt, Evan A.; Shook, Lydia L.; Yonker, Lael M.; Fasano, Alessio; Diouf, Khady; Croul, Natalie; Devane, Samantha; Yockey, Laura J.; Lima, Rosiane; Shui, Jessica; Matute, Juan D.; Lerou, Paul H.; Akinwunmi, Babatunde O.; Schmidt, Aaron; Feldman, Jared; Hauser, Blake M.; Caradonna, Timothy M.; De la Flor, Denis; D’Avino, Paolo; Regan, James; Corry, Heather; Coxen, Kendyll; Fajnzylber, Jesse; Pepin, David; Seaman, Michael S.; Barouch, Dan H.; Walker, Bruce D.; Yu, Xu G.; Kaimal, Anjali J.; Roberts, Drucilla J.; Alter, Galit
title: Assessment of Maternal and Neonatal SARS-CoV-2 Viral Load, Transplacental Antibody Transfer, and Placental Pathology in Pregnancies During the COVID-19 Pandemic
date: 2020-12-22
journal: JAMA Netw Open
DOI: 10.1001/jamanetworkopen.2020.30455
sha: b22b4b09b30d665c48cb008090e4aaf8e1f5d1a4
doc_id: 766788
cord_uid: wue6ise9

IMPORTANCE: Biological data are lacking with respect to risk of vertical transmission and mechanisms of fetoplacental protection in maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. OBJECTIVE: To quantify SARS-CoV-2 viral load in maternal and neonatal biofluids, transplacental passage of anti–SARS-CoV-2 antibody, and incidence of fetoplacental infection. DESIGN, SETTING, AND PARTICIPANTS: This cohort study was conducted among pregnant women presenting for care at 3 tertiary care centers in Boston, Massachusetts. Women with reverse transcription–polymerase chain reaction (RT-PCR) results positive for SARS-CoV-2 were recruited from April 2 to June 13, 2020, and follow-up occurred through July 10, 2020. Contemporaneous participants without SARS-CoV-2 infection were enrolled as a convenience sample from pregnant women with RT-PCR results negative for SARS-CoV-2. EXPOSURES: SARS-CoV-2 infection in pregnancy, defined by nasopharyngeal swab RT-PCR. MAIN OUTCOMES AND MEASURES: The main outcomes were SARS-CoV-2 viral load in maternal plasma or respiratory fluids and umbilical cord plasma, quantification of anti–SARS-CoV-2 antibodies in maternal and cord plasma, and presence of SARS-CoV-2 RNA in the placenta. RESULTS: Among 127 pregnant women enrolled, 64 with RT-PCR results positive for SARS-CoV-2 (mean [SD] age, 31.6 [5.6] years) and 63 with RT-PCR results negative for SARS-CoV-2 (mean [SD] age, 33.9 [5.4] years) provided samples for analysis. Of women with SARS-CoV-2 infection, 23 (36%) were asymptomatic, 22 (34%) had mild disease, 7 (11%) had moderate disease, 10 (16%) had severe disease, and 2 (3%) had critical disease. In viral load analyses among 107 women, there was no detectable viremia in maternal or cord blood and no evidence of vertical transmission. Among 77 neonates tested in whom SARS-CoV-2 antibodies were quantified in cord blood, 1 had detectable immunoglobuilin M to nucleocapsid. Among 88 placentas tested, SARS-CoV-2 RNA was not detected in any. In antibody analyses among 37 women with SARS-CoV-2 infection, anti–receptor binding domain immunoglobin G was detected in 24 women (65%) and anti-nucleocapsid was detected in 26 women (70%). Mother-to-neonate transfer of anti–SARS-CoV-2 antibodies was significantly lower than transfer of anti-influenza hemagglutinin A antibodies (mean [SD] cord-to-maternal ratio: anti–receptor binding domain immunoglobin G, 0.72 [0.57]; anti-nucleocapsid, 0.74 [0.44]; anti-influenza, 1.44 [0.80]; P < .001). Nonoverlapping placental expression of SARS-CoV-2 receptors angiotensin-converting enzyme 2 and transmembrane serine protease 2 was noted. CONCLUSIONS AND RELEVANCE: In this cohort study, there was no evidence of placental infection or definitive vertical transmission of SARS-CoV-2. Transplacental transfer of anti-SARS-CoV-2 antibodies was inefficient. Lack of viremia and reduced coexpression and colocalization of placental angiotensin-converting enzyme 2 and transmembrane serine protease 2 may serve as protective mechanisms against vertical transmission.

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.

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 

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. 

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. 

Research electronic data capture (REDCap)--a metadata-driven methodology and workflow process for providing translational research informatics support

Rapid establishment of a COVID-19 perinatal biorepository: early lessons from the first 100 women enrolled

Research Use Only 2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Primers and Probes

Identification of importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens

Nasal-associated lymphoid tissues (NALTs) support the recall but not priming of influenza virus-specific cytotoxic T cells

Identification and validation of reference genes for expression studies in human keratinocyte cell lines treated with and without interferon-gamma -a method for qRT-PCR reference gene determination

New real-time reverse transcriptaseinitiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma

Sampling and Definitions of Placental Lesions: Amsterdam Placental Workshop Group Consensus Statement

Single-cell sequencing of neonatal uterus reveals an Misr2+ endometrial progenitor indispensable for fertility

RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues

Neonatal Morbidity of Small-and Large-for-Gestational-Age Neonates Born at Term in Uncomplicated Pregnancies

Preterm neonatal morbidity and mortality by gestational age: a contemporary cohort

Coronavirus (COVID-19) Infection in Pregnancy: Information for Health Care Professionals

percentile 20 (45%) 17 (57%) 9 (64%) 0.39 a A SARS-CoV-2 positive mother was more likely to have maternal vascular malperfusion lesions as disease severity increased (OR 2.09 (95% CI 1.11-3.97) using a univariate logistic regression model to test the hypothesis of an ordered relationship between increasing SARS-CoV-2 severity and maternal vascular malperfusion on placental pathology, p=0.02). Remainder of p-values generated from Pearson's chi square test, with Bonferroni correction (adjusted p-value threshold p<0.025).