Novel Ratio Soluble Fms-like Tyrosine Kinase-1/Angiotensin-II (sFlt-1/ANG-II) in Pregnant Women Is Associated with Critical Illness in COVID-19

Background: In healthy pregnancies, components of the Renin-Angiotensin system (RAS) are present in the placental villi and contribute to invasion, migration, and angiogenesis. At the same time, soluble fms-like tyrosine kinase 1 (sFlt-1) production is induced after binding of ANG-II to its receptor (AT-1R) in response to hypoxia. As RAS plays an essential role in the pathogenesis of COVID-19, we hypothesized that angiogenic marker (sFlt-1) and RAS components (ANG-II and ACE-2) may be related to adverse outcomes in pregnant women with COVID-19; Methods: Prospective cohort study. Primary outcome was severe pneumonia. Secondary outcomes were ICU admission, intubation, sepsis, and death. Spearman’s Rho test was used to analyze the correlation between sFlt-1 and ANG-II levels. The sFlt-1/ANG-II ratio was determined and the association with each adverse outcome was explored by logistic regression analysis and the prediction was assessed using receiver-operating-curve (ROC); Results: Among 80 pregnant women with COVID-19, the sFlt-1/ANG-II ratio was associated with an increased probability of severe pneumonia (odds ratio [OR]: 1.31; p = 0.003), ICU admission (OR: 1.05; p = 0.007); intubation (OR: 1.09; p = 0.008); sepsis (OR: 1.04; p = 0.008); and death (OR: 1.04; p = 0.018); Conclusion: sFlt-1/ANG-II ratio is a good predictor of adverse events such as pneumonia, ICU admission, intubation, sepsis, and death in pregnant women with COVID-19.

In healthy pregnancies, RAS components are present in the placental villi and contribute to placental invasion, migration, and angiogenesis [7]. Furthermore, RAS components help promote placental circulation and blood flow, facilitating fetal oxygenation [8]. Expression of ACE-2 and TMPRSS2 decreases in late gestation [9]. This downregulation of RAS is associated with adverse maternal-perinatal outcomes, particularly pre-eclampsia and fetal growth restriction [10]. To date, there are no original studies in pregnant women with COVID-19 reporting blood concentrations of ACE-2 and ANG-II, two molecules that are potentially involved in the pathogenesis of severe disease in COVID-19 [11,12].
On the other hand, soluble fms-like tyrosine kinase 1 (sFlt-1) is a protein related to placental hypoxia, endothelial damage, sepsis, and acute lung injury (15) [13]. sFlt-1 production is induced when ANG-II binds to its receptor (AT1) as a response to hypoxia [14]. It is known that sFlt-1 causes endothelial dysfunction, sensitizing the endothelial cells to the effect of ANG-II in the whole endothelium and placenta [15]. In critically ill non-pregnant COVID-19 patients, there is an upregulation of sFlt-1, suggesting that this protein might play a role in the COVID-19-associated systemic endothelial dysfunction [16,17].
We hypothesized that the primary endothelial dysfunction component characterized by an imbalance between sFlt-1 as an angiogenic marker and RAS components (ANG-II and ACE-2) may be related to adverse outcomes in pregnant women with COVID-19.
Therefore, this study aims to investigate the association between serum concentrations of sFlt-1 and RAS components to adverse outcomes in pregnant women with COVID-19.

Study Design and Participants
We conducted a prospective cohort study at the National Institute of Perinatology "Isidro Espinosa de los Reyes" and the General Hospital of Mexico "Dr. Eduardo Liceaga", both third reference hospitals in Mexico City. Inclusion criteria were all pregnant women who arrived at the emergency department with respiratory symptoms and a positive RTqPCR for SARS-CoV-2 between December 2020 and July 2021. The study protocol was prospectively approved by the Ethics and Research Committee of the National Institute of Perinatology (2020-1-32). All enrolled women provided written informed consent.

Outcome
The primary outcome was pregnant women with severe pneumonia. Secondary outcomes were ICU admission, intubation, viral sepsis, and maternal death as a direct result of SARS-CoV-2 infection. Severe pneumonia was defined according to the American Thoracic Society criteria, which include either one major criterion (septic shock with need for vasopressors, or respiratory failure requiring mechanical ventilation) or three or more minor criteria (respiratory rate ≥ 30 breaths/min; PaO 2 /FIO 2 ratio ≤ 250; multilobar infiltrates; confusion/disorientation; uremia [blood urea nitrogen level ≥ 20 mg/dL]; leukopenia [white blood cell count < 4000 cells/µL]; thrombocytopenia [platelet count < 100,000/µL]; hypothermia [core temperature < 36 • C]; hypotension requiring aggressive fluid resuscitation) [19,20]. ICU admission was decided according to the Quick Sequential Organ Failure Assessment (qSOFA) score, where a score of ≥2 points would require ICU admission [21]. Viral sepsis was defined according to the Sepsis-3 International Consensus [22] associated with SARS-CoV-2 infection [23].

Statistical Analysis
Descriptive and inferential statistics were used. Quantitative variables were reported as the median and interquartile range (IQR), while qualitative data were reported as numbers and percentages. Differences between variables among COVID-19-severity were compared using the Mann-Whitney U test or X 2 test. We performed a correlation between all biochemical parameters to explore possible candidates for multiple logistic regression. All significant candidates were explored in an adjusted logistic regression to establish independent predictors for adverse outcomes. To explore a possible endothelial dysfunction, we performed sFlt-1/PlGF and sFlt-1/ANG-II ratios. Forward and backward stepwise logistic regression analyses were performed to assess the association between independent variables and primary and secondary outcomes including all possible candidates in the correlation analysis. The adjusted model's performance after logistic regression was evaluated by receiver-operating-curve (ROC) analysis to estimate the area under the curve. p-values < 0.05 were considered statistically significant. (StataCorp. 2020. Stata Statistical Software: Release 17. College Station, TX, USA: StataCorp LLC.).

Description of the Cohort and Characteristics of the Study Population
A total of 80 pregnant women with SARS-CoV-2 infection were included for the analysis. Twenty-five (31.25%) had severe COVID-19 disease and 55 were classified as non-severe. There were two (2.5%) maternal deaths. Baseline characteristics were similar between groups (Table 1).

Correlation between sFlt-1 and ANG-II
Spearman s Rho test was used to identify the relationship of sFlt-1 and ANG-II, and a significant correlation was found among severe pneumonia (r = −0.453; p < 0.001) (Figure 1).

Hypothetical Molecular Mechanisms Contributing to the Pathogenesis of Severe COVID-19 in Pregnant Women
In pregnant women with severe pneumonia by COVID-19, plasma levels of ANG-II are reduced, and plasma levels of sFlt-1 are increased, compared to those with non-severe disease. This leads to an imbalance in the sFlt-1/ANG-II ratio. Although the molecular mechanisms involved in the production of ANG-II and sFlt-1 were not explored in our work, current evidence allows us to propose a hypothetical pathway on how SARS-CoV-2 infection in the placenta affects the RAS signaling pathway contributing to the pathogenesis of severe COVID-19 in pregnant women (Figure 3). (2) Accumulation of ANG-II on the cell surface enhances its binding to the AT-1 receptor (AT-1R), promoting downstream signaling, followed by rapid endocytosis of the ANG-II/AT-1R complex [24]. (3) By avoiding the endosome/lysosome degradation, the excess of ANG-II is accumulated in endothelial cells [25]. (4) ANG-II binds to mitochondrial AT-1R [26], inducing cellular senescence with positive regulation of reactive oxygen species (ROS) [27]. (5) Over-activation of AT-1R on the cell membrane leads to increased PKC and calcineurin activity [25]. (6) Transcription factors NF-kB and NFAT are activated and translocated to the nucleus, leading to an increase in gene expression and release of Flt-1 [25]. (7) Flt-1 alternative splicing generates sFlt-1 isoform [25]. (8) The excess of sFlt-1 protein is released into the circulation causing endothelial dysfunction.

Main Findings
A high ratio of sFlt-1/ANG-II was associated with a 1.31-fold increase in severe pneumonia and higher odds of ICU admission, viral sepsis, and maternal death. So, this ratio can be considered as a high-performance prognostic marker in pregnant women with COVID-19.

Comparison with Existing Literature
Studies in non-pregnant individuals have reported higher serum levels of sFlt-1 in patients with pneumonia due to COVID-19 compared to non-COVID-19-pneumonia [28]. Negro and colleagues have reported higher levels of sFlt-1 among deceased compared to COVID-19 survivors [29]. This report has demonstrated higher sFlt-1 levels in pregnant women with COVID-19 severe pneumonia. Other studies have reported lower plasma ACE-2 levels in non-survivors than in critically ill patients that have survived COVID-19 [30], however, our study has failed to demonstrate a difference in this parameter between those with critical illness and non-critical illness. We have found lower plasma levels of ANG-II in pregnant women with severe pneumonia by COVID-19. Lower ANG-II levels have been found in previous studies among people with acute respiratory distress syndrome not related to SARS-CoV-2. Studies in non-pregnant participants with severe COVID-19 have shown lower serum levels of ANG-II among deceased patients when compared to survivors [30]. A possible explanation of the ANG-II downregulation could be a defect in the endothelial-bound ACE activity due to endothelial injury [31].
We have shown that the sFlt-1/ANG-II ratio could be a potential predictor of adverse events such as severe pneumonia, ICU admission, intubation, viral sepsis, and death among pregnant women who tested positive for SARS-CoV-2 infection. This ratio should be tested in a larger cohort to prove its utility before its clinical use.
In relation to preeclampsia, no significant differences in the incidence among patients with severe and non-severe COVID-19 have been observed; this finding is contradictory to previous studies in which higher rates of preeclampsia have been demonstrated in cases of severe COVID-19 [32][33][34].

Strengths and Limitations
The strength of our study is the number of adverse events that allowed us to make statistical inferences for outcomes such as severe pneumonia. Furthermore, the baseline clinical characteristics between groups were similar, which decreases the probability of selection bias.
The limitations are, despite being a cohort, the analysis was carried out cross-sectionally, which does not allow us to infer the causal relationship between the sFlt-1/ANG-II ratio and adverse outcomes, and the ORs could be overestimated. Although the sFlt-1/ANG-II ratio has a positive predictive value for predicting the severity of COVID-19 in the short term, the results need clinical validation in a new cohort.

Clinical Interpretation
In this study, a negative correlation has been found between the plasma concentrations of ANG-II and sFlt-1. A high sFlt-1/ANG-II ratio is associated with several adverse outcomes related to COVID-19, such as severe pneumonia, ICU admission, intubation, viral sepsis, and death. The sFlt-1/ANG-II ratio may allow the development of predictive models for the identification of high-risk pregnant women in need of intensive surveillance and aggressive supportive treatment upon admission to the hospital, thus preventing clinical deterioration.

Conclusions
sFlt-1/ANG-II ratio is a promising predictor for adverse outcomes such as pneumonia, ICU admission, intubation, viral sepsis, and death in pregnant women with COVID-19. However, further research in a larger prospective cohort is needed to validate the association and accuracy of the sFlt-1/ANG-II ratio for the prediction of adverse events among pregnant women with COVID-19.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/v13101906/s1, Table S1: Pregnancy outcome of the study population, Table S2: Association between sFlt-1/ANG-II ratio and each adverse outcome of COVID-19. Funding: sFlt-1 and PlGF assays for this study were donated by Roche Diagnostics ® and did not have any influence on the design of this study, analysis, results, or interpretation of the results.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Instituto Nacional de Perinatologia (Register number 2020-1-32) on 16 December 2020.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.