key: cord-0860841-09tsgs2f authors: Torres‐Torres, J.; Espino‐y‐Sosa, S.; Poon, L. C.; Solis‐Paredes, J. M.; Estrada‐Gutierrez, G.; Espejel‐Nuñez, A.; Juarez‐Reyes, A.; Etchegaray‐Solana, A.; Alfonso‐Guillen, Y.; Aguilar‐Andrade, L.; Hernández‐Pacheco, J. A.; Villafan‐Bernal, J. R.; Martinez‐Portilla, R. J. title: Increased levels of soluble fms‐like tyrosine kinase‐1 are associated with adverse outcomes in pregnant women with COVID‐19 date: 2021-10-19 journal: Ultrasound Obstet Gynecol DOI: 10.1002/uog.24798 sha: 71bd2bf9c44f58fc92aae10e0e3a5e0ae722bec1 doc_id: 860841 cord_uid: 09tsgs2f BACKGROUND: Besides the lungs, the placenta and the endothelium are organs affected by SARS‐CoV‐2. Soluble fms‐like tyrosine kinase‐1 (sFlt‐1) and placental growth factor (PlGF) are markers of endothelial dysfunction and could potentially serve as predictors of severe COVID‐19. We aimed to investigate the association between serum concentrations of sFlt‐1 and PlGF with the severity of COVID‐19 in pregnancy. METHODS: We performed a prospective cohort carried out in a tertiary hospital in Mexico City. We included all symptomatic pregnant women with a positive RT‐qPCR test for SARS‐CoV‐2 infection. The primary outcome was severe pneumonia due to COVID‐19; secondary outcomes were intensive care unit (ICU) admission, viral sepsis, and death. sFlt‐1 levels were expressed as multiples of the median (MoM). The association between sFlt‐1 and each adverse outcome was explored by a logistic regression analysis and the prediction was assessed using a receiver‐operating‐curve (ROC). RESULTS: Among 113 pregnant women with COVID‐19, higher sFlt‐1 MoM levels were associated with an increased probability of severe pneumonia in women with COVID‐19 (odds ratio [OR]: 1.817; 95% CI: 1.365‐2.418), ICU admission (OR: 2.195; 95% CI: 1.582‐3.047), viral sepsis (OR: 2.318; 95% CI: 1.407‐3.820), and maternal death (OR: 5.504; 95% CI: 1.079‐28.076). At a 5% false‐positive‐rate, sFlt‐1 showed a 38.7%, 55.6%, 66.7%, and 80% detection rate for severe COVID‐19 pneumonia, ICU admission, viral sepsis, and death, respectively. CONCLUSIONS: sFlt‐1 MoMs are higher among pregnant women with severe COVID‐19 and has the capability to predict serious adverse events such as severe pneumonia, ICU admission, viral sepsis, and death This article is protected by copyright. All rights reserved. Pregnancy is considered a major risk factor for adverse outcomes in women with SARS-CoV-2 infection. Findings from recent studies among women of reproductive age infected with SARS-CoV-2 have shown that pregnancy increases the odds of death (odds ratio [OR] 1.65; 1.30-2.09), pneumonia (OR: 1.99; 1.81-2.19) , and admission to intensive care unit (ICU) (OR: 2.25; 1.86-2.71) 1 2 . In Mexico, the year 2020 ended with a maternal mortality ratio (MMR) of 46.6, which represents an increase of 33.2% compared to 2019 (MMR 31.1) 3 , with COVID-19 as the leading cause of maternal death overtaking obstetric hemorrhage and preeclampsia 4 . Other than the lungs being the most affected organs as a result of SARS-CoV-2 infection, there is evidence suggesting that the most severe forms of COVID-19 may also affect the vascular endothelium 5 , causing significant systemic effects such as hypertension, renal impairment, thrombocytopenia, and liver injury [6] [7] [8] [9] . Maternal SARS-CoV-2 infection generates inflammatory changes in the placenta due to direct viral invasion into the parenchyma and blood vessels; it causes chorionic villi inflammation, lymphoplasmacytic villitis, and several degrees of chorangiosis, mainly associated with low-grade placental hypoxia 10 . As a result of this inflammatory response, the placenta produces soluble fms-like tyrosine kinase 1 (sFlt-1) proteins by stimulating the toll-like receptors (TLR) along with a critical tisular inflammatory response 10, 11 . The sFlt-1 protein is a splice variant of receptor 1 for the vascular endothelial growth factor A (VEGF-A) and placental growth factor (PlGF). When binding to its circulating ligand, sFlt-1 inhibits the VEGF-A pathway causing endothelial and cell homeostasis impairment 12 . There are reports demonstrating an upregulation of sFlt-1 in critically ill non-pregnant COVID-19 patients 13 , suggesting that sFlt-1 could play an important role in the COVID-19associated systemic endothelial dysfunction, which also involves the human placenta 14, 15 . As endothelial dysfunction becomes more evident among severe forms of COVID-19, angiogenic biomarkers may potentially serve as valuable tools for predicting severe COVID-19 with profound endothelial dysfunction. Therefore, we hypothesize that these angiogenic markers are predictive of adverse events related to SARS-CoV-2 infection in pregnant women, such as severe pneumonia, ICU admission, viral sepsis, or death. This study aimed to investigate the association between serum concentrations of sFlt-1 and PlGF and the severity of COVID-19 among symptomatic pregnant women. This article is protected by copyright. All rights reserved. We conducted a prospective cohort study at General Hospital of Mexico "Dr. Eduardo Liceaga", a tertiary care hospital in Mexico City between May 2020 and February 2021. Inclusion criteria were all pregnant women who arrived at the emergency department with clinical suspicion of SARS-CoV-2 infection, which was later confirmed with positive RT-qPCR and fulfil criteria for hospitalization, between July 2020 and January 2021. Criteria for hospitalization were a positive RT-qPCR, obstetric conditions that required immediate care, and a maternal conditions according to the National Protocol for SARS-CoV-2 in pregnancy which includes any of the following characteristics for hospitalization, 1) a CURB-65 score for pneumonia >1 point, 2) persistent fever >39 degrees Celsius, 3) comorbidities such as chronic hypertension, chronic pulmonary obstructive syndrome, pregestational diabetes, taking immunosuppressant drugs, and 4) chest x-ray with signs of pneumonia 16 . Patients with negative RT-PCR for COVID-19 and those asymptomatic were excluded since they did not warrant hospital admission or additional biochemical evaluation according to our protocol. The protocol was approved by the Ethics and Research Internal Review Board (DI/20/112/04/24) of the General Hospital in Mexico City. All patients authorized and provided informed consent. The following data were collected from the medical records: age, gestational age at admission, pregestational body mass index (pBMI) (kg/m 2 ), mean arterial pressure (MAP) at admission, chronic hypertension, pre-gestational diabetes, preeclampsia, pneumonia, sepsis, acute renal failure, organ dysfunction, and mortality. Participants' blood samples were obtained at hospital admission as routine testing for COVID-19, and the following laboratory results were recorded: leukocytes, neutrophils, hemoglobin, hematocrit, platelets, glucose, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides, cholesterol, D-dimer, C-reactive protein (C-RP), and procalcitonin were determined. The blood sample for sFlt-1 measurements was also drawn at admission. At admission, an additional blood sample was processed by centrifugation (1000xg/10min), and plasma was aliquoted, and stored at -70ºC until analysis. PlGF (Elecsys PlGF, Roche ® ) and sFlt-1 (Elecsys sFlt-1, Roche ® ) levels were measured by electrochemiluminescence using an automated analyzer (Cobas-e411, Roche ® ) according to the manufacturer's instructions. Primary outcome was severe pneumonia due to defined Descriptive and inferential statistics were used. Variable distribution was assessed using the Kolmogorov-Smirnoff test. Quantitative variables were reported as the median and interquartile range (IQR), while qualitative data were reported as numbers and percentages. Differences between variables among severe and non-severe COVID-19 were compared using the Mann-Whitney U test or X 2 test. PlGF and sFlt-1 biomarkers were log-transformed and converted to their multiples of the median (MoM) 24, 25 according to the Fetal Medicine Foundation algorithms 26 . sFlt-1 and PlGF MoM were compared between groups using the Mann-Whitney U test. A gestational-age adjusted logistic regression analysis was performed to assess the association between sFlt-1 MoM and the primary and secondary outcomes. When more than 5 available participants in each outcome a gestational-age-adjusted logistic regression was performed, when five or less patients in an outcome an unadjusted logistic regression was performed. For model construction, we used forward and backward stepwise logistic regression analysis in which a p-value ≤0.05 was used for model-inclusion and a pvalue > 0.05 was the criteria for model elimination. We use the variance inflation factor (VIF) to identify the influence of independent variables on other variables. When the VIF values were >4 they required investigation, whereas VIF values higher than 10 were a criterion for correction indicating serious multicollinearity. The performance of the model after logistic regression was evaluated by receiver-operating-curve (ROC) analysis. A ROC analysis was also used to determine the best cut-off value of sFlt-1 MoM for the prediction of adverse A total of 134 pregnant women with suspected SARS-CoV-2 infection were included in the original cohort. Two patients were excluded for being negative for COVID-19 by RT-PCR. Two patients were excluded for being negative for COVID-19 by RT-PCR. Twenty-one were excluded because although they were COVID-positive, they did not warrant hospital admission or additional biochemical evaluation as they were asymptomatic. Among the 113 included women, 32 (27.43%) had severe COVID-19 as defined by the composite definition, including 5 (4.42%) maternal deaths. Clinical and biochemical characteristics as well as cause of death in included in Supplemental Table 1 . Baseline characteristics were similar between women with non-severe and severe COVID- The AUC of sFlt-1 for the prediction of severe COVID-19 was 0.715 (95% CI: 0.582-0.828). The AUCs of sFlt-1 for the prediction of ICU admission, viral sepsis, and maternal death were 0.815, 0.939, and 0.976, respectively (Table 4, Figure 1 ). The detection rate for maternal death at a 5% and 10% false-positive rate were 80% and 100%, respectively. The best cut-off value of sFlt Table 3 ). The principal findings of this study are that higher levels of sFlt- which is known as preeclampsia-like syndrome. Even though we couldn't find no significant differences in the incidence of preeclampsia among severe and non-severe COVID-19 pneumonia in our sample, this study shows that abnormal angiogenic status could be identify actual PE in pregnant women with COVID-19, regardless of the degree of severity 23 . This article is protected by copyright. All rights reserved. The main strength of our study was that COVID-19 cases were consecutively recruited in a study with a specialized database built for research purposes, minimizing potential biases. In addition, biochemical analysis for sFlt-1 and PlGF were blinded to clinicians to also avoid selective outcome bias. Another strength was the inclusion of five maternal deaths, which are very uncommon events in other consecutive cohort studies [34] [35] [36] . Reason for this is that our hospital is a third level reference center for COVID-19 in pregnancy and we gather most severe cases in our country. Baseline clinical characteristics between pregnant women with severe and non-severe COVID-19 were similar, which diminishes the probability of selection bias. Further, the complete data for each patient allowed us to have sufficient precision to rule out other causes of sFlt-1 elevation, such as preeclampsia, fetal growth restriction, placental abruptio, and HELLP syndrome. We also must acknowledge some limitations. First, the number of rare outcomes such as death and sepsis were scarce (five and six patients, respectively), and the results should be considered with caution as confidence intervals for the association between sFlt-1 and these rare severe adverse outcomes are large. Another limitation can be that we only included women in the second half of pregnancy which does not allow us to understand how useful sFlt-1 can be in the first half of pregnancy but helps us recognise the need to further investigate risk factors in this population. The small sample did not allow us to statistically adjust for several covariates for outcomes, such as maternal death, and since death is a scarce outcome, external validation of this model can quite challenging. However, maternal death is rare globally, which make us think that including 5 maternal deaths is a methodological strength but a statistical limitation. Finally, although sFlt-1 MoM has good performance to provide short-term prediction of severe pneumonia in COVID-19, ICU admission, viral sepsis, and maternal death, the results need clinical validation in independent cohorts before its implementation as a prognostic marker in real life settings. Although fetal growth restriction was more prevalent among severe COVID-19 than in nonsevere COVID-19, fetal growth restriction was not associated with severe COVID-19 in the final multivariate logistic model and the only independent predictor was sFlt-1 MoM. Our data has suggested that sFlt-1 could serve as a biomarker of endothelial dysfunction This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Accepted Article Pregnant women with SARS-CoV-2 infection are at higher risk of death and pneumonia: propensity score matched analysis of a nationwide prospective cohort (COV19Mx) Update: Characteristics of Symptomatic Women of Reproductive Age with Laboratory-Confirmed SARS-CoV-2 Infection by Pregnancy Status -United States Molecular Insights into the Thrombotic and Microvascular Injury in Placental Endothelium of Women with Mild or Severe COVID-19 COVID-19: consider cytokine storm syndromes and immunosuppression Protocolo de la Federación Mexicana de Colegios de Obstetricia y Ginecología para sospecha de SARSCoV-2 en mujeres embarazadas Head-to-head comparison of qSOFA and SIRS criteria in predicting the mortality of infected patients in the emergency department: a meta-analysis The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) SARS-CoV-2 and viral sepsis: observations and hypotheses Unfractionated heparin displaces sFlt-1 Extra-placental expression of vascular endothelial growth factor receptor-1, (Flt-1) and soluble Flt-1 (sFlt-1), by peripheral blood mononuclear cells (PBMCs) in normotensive and preeclamptic pregnant women by platelet-monocyte aggregates contributes to the pathogenesis of preeclampsia Enhanced Serum Levels of sFlt1: Impact on Rates of Maternal and Perinatal Mortality and Vertical Transmission in Pregnancies Complicated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Co-V-2) Infection: A Systematic Review Effect of coronavirus disease 2019 (COVID-19) on maternal, perinatal and neonatal outcome: systematic review Maternal and Neonatal Morbidity and Mortality Among Pregnant Women With and Without /mL) 1789 MoM: Multiples of the median; UtAPI: uterine artery pulsatility index; SpO2: Oxygen saturation; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LDH: Lactate dehydrogenase; C-RP: C-reactive protein; GUT: General Urine test; PlGF: Placental growth factor -U test for continuous variables expressed as median and interquartile