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. Author manuscript; available in PMC: 2026 Sep 14.
Published in final edited form as: Placenta. 2025 Sep 14;171:34–44. doi: 10.1016/j.placenta.2025.09.009

SARS-CoV-2 infection disrupts syncytial and endothelial integrity and alters PLGF levels in the placenta

Brittany R Jones 1, Guilherme M Nobrega 1,2, Deepak Kumar 1, Emily Diveley 4, Arthur Antolini-Tavares 2,3, Renato T Souza 2, José Guilherme Cecatti 2, Jeannie C Kelly 4, Maria Laura Costa 2, Indira U Mysorekar 1,5,6
PMCID: PMC13525721  NIHMSID: NIHMS2199028  PMID: 40966962

Abstract

Introduction:

SARS-CoV-2 infection during pregnancy has been associated with an increased risk for several pregnancy-related disorders, particularly preeclampsia (PE). However, there are limited studies determining the impact of SARS-CoV-2 on placental physiology and function.

Methods:

Placental samples were acquired from two large prospective cohorts: STOP-COVID19 and REBRACO studies. Placental villous tissues (VTs) were collected from pregnant women who tested positive for SARS-CoV-2 without PE during pregnancy. Immunohistochemistry and immunofluorescence were used to assess pathological features known to be altered in PE, including 1) syncytial knot formation; 2) alterations in renin-angiotensin system components; 3) and endothelial integrity. Maternal serum was collected to examine AT1 autoantibodies levels using an immunoassay.

Results:

SARS-CoV-2 viral proteins spike, nucleocapsid, and ORF3a were observed in the syncytiotrophoblast layer and stroma of placental VT. SARS-CoV-2-infected placentas exhibited increased numbers of syncytial knots, which were positive for Flt-1 and SARS-CoV-2 viral proteins. In addition, the presence of placental infarctions and excessive fibrin deposits was also observed in infected placentas. Infection was associated with decreased placental expression of PlGF and an increase in the placental Flt-1/PlGF expression ratio, mostly driven by PlGF. No significant changes in maternal serum AT1AA levels were observed. Finally, SARS-CoV-2-infected placentas exhibited a significant decrease in vimentin expression.

Discussion:

SARS-CoV-2 infection negatively impacts placental integrity in the form of increased syncytial knots, dysregulated RAS components, and endothelial damage. Since all these features are similarly disrupted in PE, this could be a mechanism through which SARS-CoV-2 infection during pregnancy increases the risk of a PE-like syndrome.

Keywords: Syncytial knots, placenta; Renin Angiotensin system; Preeclampsia; COVID-19

1. Introduction

In 2019, the outbreak of SARS-CoV-2 sparked a global pandemic (COVID-19), resulting in over 7 million deaths worldwide [1]. Despite ongoing research, understanding of the disease’s progression and its short- and long-term effects, especially during pregnancy, remains limited [2]. Pregnant women face increased risks of severe illness, composite morbidity, and complications such as preterm birth, stillbirth, and preeclampsia (PE) [3–8]. While vertical transmission is rare, infection during pregnancy has been linked to placental abnormalities and fetal/neonatal neurodevelopmental changes [1,9–11]. Histopathological placental changes associated with SARS-CoV-2 infection have been linked to adverse pregnancy outcomes [5,12].

A significant increase in hypertensive disorders in pregnancies complicated by SARS-CoV-2 infection has been noted [13–15] with some studies suggesting a link between the virus and increased PE risk in infected pregnancies [13,16,17]. Pregnant women with moderate to severe COVID-19 infection are at an increased risk to develop pregnancy complications such as early-onset PE. [18,19]. Further, a recent study using transcriptomic profiling of SARS-CoV-2 infected trophoblasts cells revealed a profile aligned with placental dysfunction including upregulation of genes associated with PE, hypertension, and oxidative stress[20]. This has led to the identification of a PE-like syndrome in infected pregnancies, characterized by similarities in the pathophysiology and clinical presentation of PE and SARS-CoV-2 infection during pregnancy [13,15,21–24].

Preeclampsia (PE) is a pathophysiological condition marked by increased vascular resistance, contributing to maternal hypertension and affecting 2–8% of pregnancies globally, making it a leading cause of maternal and perinatal morbidity and mortality [13,21]. Pathologically, PE is linked to elevated syncytial knots, aggregates of fused cells on terminal villi, indicative of maternal vascular malperfusion and hypertensive disorders [25–27]. While syncytial knots are present in ~30% of terminal villi in normal pregnancies [28,29], they are nearly ubiquitous in PE placentas and associated with decidual arteriopathy and accelerated villous maturation [28,30,31]. PE also involves endothelial damage, vasoconstriction, and vascular inflammation [21,32,33]. Dysregulation of the placental renin-angiotensin system (RAS), which normally supports angiogenesis, immune defense, and fetal development, is another hallmark of PE [34,35].

Multiple studies, including from our group, have shown that SARS-CoV-2 infects multiple placental compartments, particularly the villous syncytiotrophoblast, Hofbauer cells, and fetal membranes across pregnancy stages [17,36]. Severe infection is associated with endothelial injury and platelet stress, disrupting vascular homeostasis and causing thrombosis in organs like the lungs, kidneys, and heart [37,38].

SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as its receptor, a key regulator of the pro- and anti-inflammatory pathways in the renin-angiotensin system (RAS) [39–41]. ACE2 is expressed in placental cells, and infection reduces its expression in trophoblasts, disrupting RAS balance and activating the pro-inflammatory AT1 pathway. This leads to increased levels of anti-angiogenic sFlt-1, decreased pro-angiogenic PlGF, vasoconstriction, endothelial damage, and potential fetal growth restriction [40,42,43]. PlGF supports feto-placental circulation and trophoblast development, and an elevated sFlt-1/PlGF ratio strongly predicts PE risk [44,45]. We previously showed that SARS-CoV-2 spike protein binding to ACE2 increases sFlt-1, and that infected pregnant women had elevated AT1R autoantibodies, both markers of PE and RAS dysregulation [17]. Placental pathology in infected pregnancies resembles that of PE, including chorangiosis, high-grade villitis, trophoblastic destruction, fibrin deposition, and acute chorioamnionitis [13,22,37,46].

These findings suggest a cellular and molecular pathological link between SARS-CoV-2 infection and PE. However, a comprehensive histopathological analysis considering the PE-like syndrome induced by COVID-19 and including PE and SARS-CoV-2 as key determinants, has yet to be conducted. In this study, we aimed to examine the impact of SARS-CoV-2 on placental pathology, syncytial knot formation, and renin-angiotensin system dynamics. We compared placental samples from women exposed to SARS-CoV-2 during pregnancy, without PE, across two separate cohorts, to explore the mechanisms underlying these pathological changes. Our results indicate that SARS-CoV-2 infection disrupts placental integrity, characterized by increased syncytial knots, dysregulated RAS components, and endothelial damage.

2. Methods

2.1. Participants and biological samples

This study is a case series of placental samples collected from two large prospective cohorts: Safety, Testing/Transmission, and Outcomes in Pregnancy with COVID-19 (STOP-COVID19) collected in St. Louis, USA (December 2020 to July 2022) and the Brazilian Network of COVID-19 in Obstetrics (REBRACO) multicentric study group from participants enrolled in the coordinating center in Campinas, São Paulo, Brazil (May 2020 to June 2021) [17,33,47]. Inclusion criteria were term births, singletons, unvaccinated against SARS-CoV-2, and with placental villous tissue (VT) samples collected for analysis. Exclusion criteria included pre-term birth, pre-eclampsia, eclampsia, and stillbirths. Placental tissues were collected from pregnant women who tested positive for SARS-CoV-2 (mild to severe cases) or negative. For both cohorts, VT was collected at delivery. Maternal peripheral blood serum was collected at delivery for the REBRACO cohort.

2.2. Clinical Characteristics

Medical charts and study interviews were reviewed to retrieve information on general characteristics, comorbidities, maternal outcomes, and pregnancy and perinatal outcomes (Supplementary Figure 1). General characteristics included age (≤24, 24–35, ≥36), race/ethnicity [White, Black/African American, Asian, Latinos, Native American, Multiracial (Pardo)]. In the STOP-COVID19 study, race and ethnicity were classified following the criteria of the National Institute of Health (NIH). The NIH describes race and ethnicity as Black or African American, Hispanic or Latino, Asian, American Indian or Alaska Native, Native Hawaiian or Other Pacific Islander, Multiracial, and White. The REBRACO considered the IBGE criteria for racial and ethnicity status. The IBGE classifies the Brazilian population into five categories based on skin color by asking participants to self-identify as White, Black, “Pardo” (brown), Yellow (East Asian), or Indigenous[48]. Comorbidities including diabetes mellitus, chronic hypertension, obesity, asthma, anemia, STORCH infections, or others (thyroid disease, alopecia areata, Sjogren’s syndrome, chronic kidney disease, cardiovascular disease, sickle cell anemia, and epilepsy) were reported. For maternal outcomes, the variables included were confirmed SARS-CoV-2 diagnosis (yes/ no, and gestational trimester at diagnosis), symptomatic SARS-CoV-2 infection, and SARS-CoV-2 epidemiological strain (Wuhan/WT, Delta, Gamma, and Omicron). SARS-CoV-2 infection was confirmed by RT-qPCR using upper respiratory secretion samples (nasopharyngeal swabs) or saliva. For pregnancy and perinatal outcomes, delivery route (C-section, vaginal) and Apgar 5 minutes score < 7 were considered.

2.3. H&E Staining and Immunohistochemistry (IHC)

Formalin-fixed paraffin embedded placental VT samples were sectioned at 5 μm thickness and were sequentially rehydrated using ethanol-water gradients. Sections were stained with Harris hematoxylin for 3 minutes and briefly rinsed in deionized water before destaining in 1% acid alcohol and soaked in 1% sodium bicarbonate for 5 minutes. Sections were counterstained with eosin, dehydrated using ethanol water-gradients and mounted with Permount mounting media. Slides were visualized using a 3D HISTECH slide viewer (3DHISTECH, Ltd).

2.4. Quantification of Syncytial Knots

Syncytial knots were quantified in H&E-stained sections using a method based on a previous study [31]. The primary investigator (BJ) was blinded to COVID-19 diagnosis and clinical characteristics of participants. Syncytial knots were defined as a multilayered aggregate of at least 5 syncytiotrophoblastic nuclei protruding from the villous surface that were not in direct contact with any adjacent villi. Syncytial knots were only included if they were, I) suspended between the intervillous space, or II) located on the edge of the syncytium. The number of syncytial knots within four individual 4 mm2 quadrants per slide were counted and averaged (Supplementary Figure 2). Any syncytial aggregates connecting two villi were excluded.

2.5. Immunofluorescence analysis

The primary investigator (BJ) was blinded to COVID-19 diagnosis and clinical characteristics of participants during IF staining. Antigen retrieval was performed on VT sections as described for IHC, except for staining for SARS-CoV-2 viral proteins. Sections were washed with PBS and blocked with 5% horse serum with 0.3% Triton X for 1 hour at room temperature. After blocking, tissue sections were incubated overnight at 4 °C with primary antibodies specific for SARS-CoV-2 Nucleocapsid (1:300, Invitrogen), Spike (1:500, Invitrogen), ORF3a (1:200, R&D Systems), Cytokeratin 5 (1:500, Abcam) FLT1 (1:200, R&D Systems), PlGF (1:100, biorbyt), and Vimentin (1:100, NOVUS). Following washing (PBS with 0.1% Triton X), sections were stained with Alexa Fluor-conjugated secondary antibodies at room temperature for one hour: anti-chicken (1:200, Invitrogen), anti-mouse (1:500, Invitrogen), anti-rabbit (1:200 and 1:500, Invitrogen), and anti-goat (1:300, ThermoFisher). Following additional washing, autofluorescence was quenched using the Vector TrueView Autofluorescence Quenching Kit (Vector Laboratories, CA). Sections were counterstained with Hoescht dye before imaging with a Nikon AX-R Point Scanning Confocal microscope (Nikon, NY) at 20X, 40X, and 60X. For quantification, the mean fluorescence intensity of four random fields at 20X was averaged for each slide.

2.6. Angiotensin II Type 1-Receptor Autoantibody (AT1-AA) ELISA

Levels of AT1AA were measured in maternal peripheral serum from uninfected and SARS-CoV-2-infected pregnant women using a commercial ELISA kit (MyBiosource). All samples were run in duplicate as per manufacturer’s instruction and read using a microplate reader at 450 nm. The author was blinded to COVID-19 diagnosis and clinical characteristics of participants.

2.7. Data Analysis

Cases of both cohorts were merged and classified into two groups: no diagnosis of SARS-CoV-2 infection during pregnancy, SARS-CoV-2(−); and positive SARS-CoV-2 diagnosis at any point during pregnancy, SARS-CoV-2(+). The number of samples for SARS-CoV-2(−) and SARS-CoV-2(+) groups were determined based on the minimum detectable effect, given a Type 1 error rate of 0.05 and a desired statistical power of 80%. Normality tests were performed on sample data prior to analysis. Comparisons between groups for categorical variables were performed using Odds Ratio with 95% confidence interval (CI), and uncorrected Chi-square test or Fisher’s exact (expected results less than 5) for clinical characteristics using EPInfo (CDC, v7.2.5 2021). Linear regression analysis was performed to assess PlGF and sFlt-1/Flt-1 levels in placental tissue and maternal serum. AT1 levels were similarly evaluated using linear regression with adjustment for gestational age. Statistical analysis for continuous variables was performed using nonparametric Mann-Whitney U test for IHC, IF, ELISA experiments and post-hoc analyses using Graphpad 10.3.0. P < 0.05 was considered statistically significant.

3. Results

3.1. Clinical Characteristics

Details of the demographics for the STOP-COVID119 (n = 38) and REBRACO (n = 38) cohorts are shown in Supplementary Table 1. We note no significant differences in clinical characteristics including pregnancy and perinatal outcomes, most of the comorbidities, and age (p-value=0.2547) between the STOP-COVID19 and REBRACO cohorts. We saw significant differences in maternal outcomes as SARS-CoV-2 infection presented more in the 1st trimester in the STOP-COVID19 (p-value<0.0001) cohort and primarily in the 3rd trimester in the REBRACO cohort (Supplementary Table 1). There was no difference in 2nd trimester infections between the two cohorts, however, the STOP-COVID19 study consisted primarily of SARS-CoV-2 strains Delta and Omicron (p-value<0.0001) whereas the REBRACO study participants were primarily infected with Wuhan and Gamma strains (p-value<0.0001). Sub-analyses of comorbidities between 1st/2nd trimester and 3rd trimester groups revealed no significant differences. However, we found that women infected earlier in pregnancy were more likely to be symptomatic (p-value=0.0264, Supplementary Table 2). We also observed 1st/2nd trimester infections caused by Delta, Wuhan, and Gamma variants compared with 3rd trimester infections which consisted primarily of infections with the Wuhan and Gamma variants (p-value<0.0001, Supplementary Table 2). Race could not be compared because the USA and Brazil classify race/ethnicity differently (Supplementary Table 3). Given the lack of difference in most comorbidities, age, and pregnancy outcomes, we combined both cohorts when evaluating demographic differences between SARS-CoV-2(−) and SARS-CoV-2(+) groups (Table 1). When the two cohorts were combined, maternal age, comorbidities, and pregnancy outcomes were not significantly different between SARS-CoV-2(−) and SARS-CoV-2(+) cases. The exceptions to this were obesity, which was higher in the SARS-CoV-2(+) group (p-value =0.0075), and anemia (p-value =0.0123), which was higher in the SARS-CoV-2(−) group (Table 1).

Table 1:

Sociodemographic data, comorbidities, maternal, and neonatal outcomes for SARS-CoV-2 negative (n=29) and SARS-CoV-2 positive (n=47) cases.

Variable SARS-CoV-2 (-) SARS-CoV-2 (+) Odds Ratio (95% CI) p-value
N 29 47
General Characteristics
Age
 ≤ 24 4 13.8% 12 25.5% - 0.3325**
 24 – 35 21 72.4% 26 55.3% -
 ≥ 36 4 13.8% 9 19.1% -
Comorbidities (n = 27) (n = 42)
Diabetes Mellitus 4 14.8% 10 23.8% 1.7969 (0.5009 ~ 6.4456) 0.5412**
Chronic Arterial Hypertension 4 14.8% 9 21.4% 1.5583 (0.4311 ~ 6.4495) 0.5469**
Obesity 6 22.2% 23 54.8% 4.2368 (1.4170 ~ 12.6265) 0.0075*
Asthma 5 18.5% 2 4.8% 0.2200 (0.0394 ~ 1.2291) 0.1018**
Anaemia 6 22.2% 1 2.4% 0.0854 (0.0096 ~ 0.7562) 0.0123*
STORCH 1 3.7% 5 11.9% 3.5135 (0.3874 ~ 31.8647) 0.3922**
Others 2 7.4% 4 9.5% 1.3158 (0.2239 ~ 7.7315) 1.0000**
Maternal Outcomes (n = 29) (n = 47)
Hemorrhage 0 - 3 5.7% - 0.2826**
Pregnancy and Perinatal Outcomes
Delivery Route
 C-section 8 24.2% 21 44.7% 2.1202 (0.7824 ~ 5.7455) 0.1362*
 Vaginal 21 63.6% 26 55.3%
Apgar 5 minutes < 7 1 3.0% 0 - - 0.3816**
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Others: thyroid disease, alopecia areata, Sjogren’s syndrome, chronic kidney disease, cardiovascular disease, sickle cell anemia, and epilepsy.

*

Chi-Square

**

Fisher’s

3.2. Placentas from SARS-CoV-2 infection during pregnancy are positive for viral proteins

Within the infected cohort, we observed localized foci of SARS-CoV-2 viral proteins spike and nucleocapsid in the STB layer and stroma in the chorionic villi (Figure 1A, Inset A and B), consistent with studies that have shown SARS-CoV-2 infects multiple compartments of the placenta[17,36,49]. SARS-CoV-2 accessory protein, ORF3a was observed in the STB layer of placental sections from infected pregnancies, indicative of active viral replication (Figure 1B, Inset C and D). Additionally, colocalization of spike and ORF3a was observed within placental villi of the SARS-CoV-2 (+) group (Figure 1C, Insets E and F).

Figure 1: Foci of SARS-CoV-2 infection in the placenta.

Figure 1:

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(A) Detection of SARS-CoV-2 proteins spike (green), nucleocapsid (red), and colocalization (inset A and B) in villous tissue from SARS-CoV-2 positive term placentas (magnification scale 20X; scale bar represents 50 μm), Nuclei (blue). (B) Presence of SARS-CoV-2 ORF3a (green) in STB layer (Inset C and D); STB layer outlined with CK5 (red) (magnification scale 20X; scale bar represents 50 μm). (C) SARS-CoV-2 proteins spike (green) and ORF3a (red) detected in villous tissue and colocalization of (Inset E and F) of viral proteins in STB layer (magnification scale 20X; scale bar represents 50 μm).

3.3. SARS-CoV-2 infection is associated with increased syncytial knot formation

Syncytial knots were observed in both uninfected and SARS-CoV-2-infected placental sections (Figure 2A) localized on the edge of the villi (Inset A, C, and D) or suspended between villi (Inset B, E, and F). However, the number of syncytial knots was significantly increased in SARS-CoV-2-infected VT (p-value<0.0001, Figure 2B). We also included PE cases in our overall cohort and observed significant increase in syncytial knot formation SARS-CoV-2 infected placental VT tissue (p-value<0.0001, Supplementary Figure 3). Syncytial knots were found to express SARS-CoV-2 spike, nucleocapsid, and ORF3a proteins (Figure 1, Inset A and Inset C). Placental sections were also examined for other pathological markers or damage using histology. In addition to increased syncytial knots, placentas from infected pregnancies exhibited placental infarctions (10.6%) and excessive fibrin deposition (21.3%), suggesting placental damage (Figure 2C and 2D).

Figure 2: Increased formation of syncytial knots in SARS-CoV-2 infected placentas.

Figure 2:

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(A) Presence of SK in SARS-CoV-2 positive placentas. Syncytial knots were located in the maternal circulation (Inset A, C, and D) or suspended between villi (Inset B, E, and F). (B) Semiquantitive analysis of syncytial knots formation in uninfected and SARS-CoV-2 infected placentas. Values are expressed as mean +/− standard error of the mean (SEM); * p <0.05, ** p < 0.01, ***: p < 0.001; ns: non-significant. (C-D) Presence of placental infarction (C), and excessive fibrin deposition (D) in SARS-CoV-2 infected placentas.

3.4. SARS-CoV-2 infection is associated with decreased PlGF

We previously reported a significant decrease in ACE2 expression in SARS-CoV-2-infected placentas, suggesting that placental RAS is dysregulated by infection[17]. In our current study, we observed the presence of Flt-1 and PlGF in the STB layer with puncta staining in the stroma in both the uninfected and infected placental sections (Figure 3). Flt-1 was also detected in syncytial knots (Figure 3A, Inset A and B), including in knots suspended between villi (Figure 3A Inset B). Further, SARS-CoV-2-infected placentas showed a significant decrease in expression of PlGF, mainly in focal areas of the VT (Figure 3A and 3B; p-value<0.0001). While there was no significant difference in Flt-1 expression between the two groups (Figure 3B, p-value=0.0664), the placental Flt-1/PlGF ratio was significantly higher in SARS-CoV2 (+) placentas (Figure 3C; p-value=0.0002), suggesting dysregulation of RAS[50–53]. Upon inclusion of PE cases in the overall cohort; we observed a significant increase in Flt-1/PlGF ratio (p-value<0.0001), a significant decrease in PlGF (p-value<0.0001), and no changes in Flt-1 levels (p-value=0.0664) in SARS-CoV-2 infected placental VT tissue (Supplementary Figure 3). We compared placental averages versus serum levels only in the REBRACO cohort for Flt-1, PlGF, and the Flt-1/PlGF ratio and observed no significant correlation (Supplementary Figure 4). Next, we evaluated the levels of AT1AA in the maternal serum (Figure 3D). We saw no change in the levels of AT1AA (p-value=0.6484). We observed no significant correlation between AT1 levels and gestational age at the time of blood draw (Supplementary Figure 4). In addition, there no were significant changes in AT1 levels between pregnant women taking hypertensive medication and pregnant women not on hypertensive medication (Supplementary Figure 4). Together, our findings demonstrate SARS-CoV-2 infection leads to reduced expression of PlGF, resulting in a higher placental Flt-1/PlGF ratio.

Figure 3: SARS-CoV-2 infection is associated with changes in RAS components in the placenta.

Figure 3:

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(A) Immunofluorescence images of uninfected and SARS-CoV-2 infected villous tissue showing expression of PlGF (red), Flt-1 (green) (magnification scale 20X; scale bar represents 100 μm). Presence of Flt-1 in SK of SARS-CoV-2-infected placentas (Inset A and B). (B-C) Quantification of mean fluorescence intensity of PlGF and Flt-1 in uninfected and infected placentas (B) and Flt-1/PlGF ratio (C). (D) Predelivery of angiotensin II type 1-receptor autoantibody (AT1-AA) levels in sera of uninfected and SARS-CoV-2-infected pregnant women. Bars represent means and error bars represent standard error of the mean (SEM); *: * p <0.05, ** p < 0.01, ***: p < 0.001; ns: nonsignificant difference.

3.5. SARS-CoV-2 infection is associated with endothelial cell dysfunction in the placenta

Severe SARS-CoV-2 infection can lead to endothelial injury, causing excessive platelet stress, inflammation, and disruption of the homeostatic balance in the vasculature, as seen in the lungs of COVID-19 patients [38]. We examined SARS-CoV-2-infected placental VT sections and noted morphological changes, including chorangiosis (36.2%), suggesting vascular changes in the chorionic villi (Figure 4A). Furthermore, expression of vimentin, a key intermediate filament protein expressed in vascular endothelial cells and other mesenchymal-derived cells, where it plays a critical role in maintaining endothelial barrier function and vascular integrity, was significantly decreased in SARS-CoV-2-infected samples (Figure 4B and C, p-value=0.0003) compared to the uninfected samples and we observed similar results after including PE cases (Supplementary Figure 3, p-value=0.0003).. Together, these findings suggest SARS-CoV-2 infection may impact endothelial cell integrity in the placenta even at term.

Figure 4: SARS-CoV-2 infection impacts endothelial integrity of the placenta.

Figure 4:

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(A) Foci of chorionic villi with more than ten capillaries in more than ten villi, under objective lens of 10x magnification, characterizing chorangiosis (scale bar represents 100 μm). (B) Expression of vimentin (red) in villous tissue of uninfected (Inset A and B) and infected placentas (Inset C and D) (magnification scale 20X; scale bar represents 100μm). (C) Quantification of mean fluorescence intensity of vimentin in uninfected and SAR-CoV-2 infected placentas, bars represent means and error bars represent standard error of the mean (SEM); *: * p <0.05, ** p < 0.01, ***: p < 0.001; ns: nonsignificant difference.

4. Discussion

In this study, our findings reveal several key pathological alterations in the placenta associated with SARS-CoV-2 infection during pregnancy. First, we detected SARS-CoV-2 viral proteins—spike, nucleocapsid, and ORF3a—within the syncytiotrophoblast layer and stroma of the placental villous tissue, indicating active viral replication in the placenta. Additionally, SARS-CoV-2 infection was associated with an increase in syncytial knot formation, a hallmark of placental dysfunction. Notably, placental growth factor (PlGF) expression was significantly reduced in SARS-CoV-2 infected placentas, leading to a higher placental Flt-1/PlGF ratio, primarily driven by lower PlGF levels. This suggests a potential disruption of the local renin-angiotensin system. Furthermore, we observed a decrease in vimentin expression, which may indicate impaired endothelial integrity. Infected placentas also displayed signs of placental infarctions and excessive fibrin deposits. These findings demonstrate that SARS-CoV-2 infection negatively impacts placental integrity through increased syncytial knots, dysregulated RAS components, and endothelial damage—features commonly associated with preeclampsia. Therefore, these disruptions may underlie the development of a PE-like syndrome in pregnancies complicated by SARS-CoV-2 infection.

Earlier studies have shown evidence of SARS-CoV-2 virus in STBs, the stromal and other compartments of the placenta, supporting our findings [17]. Further, a study found active in vivo SARS-CoV-2 replication in the placenta following maternal infection, increasing the risk of stillbirths and placentitis [54], suggesting placental involvement in the pathophysiology of these outcomes [54]. Detection of viral proteins spike, nucleocapsid, and ORF3a at delivery, even when infection occurred earlier in pregnancy, indicates persistent viral presence. Persistent spike protein in plasma and immune cells may underlie prolonged post-COVID-19 symptoms through ongoing viral presence and systemic inflammation [55]. Spike protein has also been found in the skull, meninges, and brain tissue of acute and post-mortem COVID-19 cases [55]. Additionally, ORF8 has been detected in fetal tissues, suggesting possible transplacental transfer and potential implications for maternal and fetal health during infection [56].

Syncytial knots are present in the placenta of normal pregnancies; however, an increase presence is associated with placental maturity and are greatly accelerated in PE [28]. In SARS-CoV-2-infected pregnant women, syncytial knots were significantly higher compared to the uninfected, suggesting premature aging of the placenta. Extensive syncytial knotting is associated with accelerated villous maturation, a histopathological marker for placental sufficiency [30,57]. This accelerated maturation is a manifestation of maternal vascular malperfusion, characterized by small or short hypermature villi for the given gestational period. Studies have shown changes in placental maturity via accelerated maturation and increased risk of maternal vascular malperfusion in SARS-CoV-2-infected placentas [58–60]. Furthermore, syncytial knots have been associated with the transfer of sFlt-1/Flt-1 into maternal circulation, raising maternal vascular tension and increasing the risk for PE [61]. Our finding that SARS-CoV-2 infection is associated with increased syncytial knot formation and presence of viral proteins in addition to Flt-1 suggest that increased syncytial knots may contribute to the development of a PE-like syndrome in SARS-CoV-2-exposed pregnancies. The increased presence of syncytial knots in the SARS-CoV-2 (+) group indicates that viral infection alone induces placental dysfunction and villous maturation abnormalities. This effect was further accentuated when we included PE cases, suggesting that SARS-CoV-2 and preeclampsia exert synergistic effects on placental integrity. We also observed placental injury with the presence of placental infarctions, excessive fibrin deposits, and an increase in syncytial nuclear aggregates [12,62–64], consistent with studies on SARS-CoV-2-infected placental pathology and those seen in the preeclamptic placenta [1,13,37,65]. Further, placental injury caused by SARS-CoV-2 infection is also associated with stillbirth with massive perivillous fibrin deposition, trophoblast necrosis, and chronic histiocytic intervillositis [66], which recapitulate anatomopathological findings in clinical studies and noted in placentas from women with PE [22,62,63,66].

SARS-CoV-2 is recognized as a microvascular and endothelial disease, capable of causing endothelial injury [38,67,68]. It can disrupt the vasculature system in organs like the kidney, heart, and brain, suggesting similar effects in the placenta [38,58]. Unvaccinated pregnant women with COVID-19 face higher risks of placental injury from hypoperfusion and inflammation [16,65]. Studies show the virus infects endothelial cells in lung tissues via ACE2, increasing permeability [38,67,69], and is linked to maternal and fetal vascular malperfusion through a hypercoagulable state [58]. Indeed, we observed a significant decrease in the expression of vimentin in SARS-CoV-2-infected VT sections compared to uninfected placentas. The decrease in vimentin expression suggests potential disruption of stromal and vascular integrity; however, we acknowledge that vimentin is not specific to endothelial cells. Nonetheless, the observed increase in syncytial knots—indicative of hypoxic stress and placental maturation abnormalities—the reduction in PlGF expression with a corresponding rise in the Flt-1/PlGF ratio, both of which suggest impaired angiogenic signaling, point toward a pattern of placental dysfunction involving both structural and molecular changes. The vascular dysfunction observed in SARS-CoV-2-infected placentas mirrors endothelial damage seen in PE, where impaired endothelial function is a key feature of the disease. Additionally, SARS-CoV-2 infection has been shown to involve direct interactions between spike proteins and endothelial cells, leading to endothelial dysfunction and microvascular damage in both pulmonary and extrapulmonary systems [70]. Our findings suggest that a similar mechanism may occur in the placenta contributing to a PE-like pathology in SARS-CoV-2-exposed pregnancies.

RAS regulates blood pressure, hydroelectrolyte balance, and vascular resistance in organs including the kidneys, liver, lungs, and placenta [27,71]. RAS imbalance is linked to worse COVID-19 outcomes, including acute respiratory distress and cytokine storm [13,35]. PlGF plays key role in normal pregnancy with studies showing that low levels of PlGF mid-pregnancy increase the risks for adverse pregnancy outcomes [72]. In our study, placental Flt-1/PlGF ratio was higher in SARS-CoV-2 infected placenta samples tissue, mainly driven by reduced PlGF levels. We included PE cases and observed significant reduction in PlGF and a high Flt-1/PlGF ratio, suggesting SARS-CoV-2 infection may independently drive angiogenic imbalance like preeclampsia, potentially through shared mechanisms of placental and endothelial dysfunction. Alterations in the placental Flt-1/PlGF ratio may reflect, at least in part, local disruption of the RAS, where downregulation of ACE2 and the Ang-(1–7) axis shifts the balance toward vasoconstriction and endothelial dysfunction, creating conditions that resemble PE[50–53]. Nevertheless, while changes in PlGF expression and the Flt-1/PlGF ratio may indicate disrupted angiogenic signaling, further studies are needed to fully assess local RAS activity. We also observed no differences in the levels of AT1-AA in maternal sera between SARS-CoV-2-infected and uninfected pregnant women. This could be due to factors such as antihypertensive medications taken by participants, severity of infection, and the timing of sample collection. Further, we compared placental and serum levels of Flt-1, PlGF, and their ratio and observed no significant correlation. Interestingly, it has been reported that serum sFlt-1/PlGF levels from infected participants remain unchanged [73], suggesting that SARS-CoV-2 infection may have localized, focal effects in placenta not reflected in serum/plasma biomarkers. Indeed, we observed foci of infection in placenta, with confocal microscopy revealing localized oxidative stress and DNA damage colocalized with SARS-CoV-2 spike protein [53]. These findings indicate that SARS-CoV-2 infection can induce focal placental damage, leaving lasting scars even if infection occurred earlier in pregnancy, consistent with recent studies [74]. Additionally, our mechanistic work shows that infection of trophoblasts or 3D organoids triggers secretory autophagy and releases extracellular vesicles carrying ORF3a, potentially affecting nearby uninfected tissue [75]. This suggests that focal infection can have systemic effects. Persistent SARS-CoV-2 infection may serve as a viral reservoir, potentially contributing to long COVID [76]. Pregnant women infected with SARS-CoV-2 are reported to experience long COVID symptoms, including cardiovascular issues [77–79]. Thus, even limited maternal exposure leading to placental damage may have broader and longer-term impacts than histopathology, serum markers, or symptomology alone.

Our study is not without limitations. The sample size was too small to compare hypertensive pregnant women with and without SARS-CoV-2 infection. Due to missing clinical data and lack of significant differences, cohorts were combined, limiting in-depth analysis. Participants included high-risk women with chronic hypertension, some on antihypertensives. Severe infection and PE cases were excluded. Additionally, the inability to capture viral strain, infection timing, and severity, along with the absence of active infection at delivery and missing maternal serum data in the STOP-COVID19 cohort, further constrained interpretation. Despite these limitations, we observed notable placental damage, highlighting the need for larger studies with detailed infection timelines to understand long-term impacts on placental health.

In summary, our findings suggest that SARS-CoV-2 infection disrupts placental integrity, with foci of infection in placental villi exacerbating syncytial knot formation, leading to endothelial dysfunction and reduced placental growth factor, potentially contributing to PE-like pathology in infected pregnancies. These results warrant further investigation into the specific locations of viral infection and the long-term implications of maternal infection.

Supplementary Material

1

Highlights.

  • SARS-CoV-2 viral proteins are detected in the placenta indicative of replication

  • SARS-CoV-2 infection is associated with increased formation of syncytial knots in placenta

  • SARS-CoV-2 infected placenta samples exhibit reduced placenta growth factor expression

  • SARS-CoV-2 infected placentas have decreased vimentin indicative of impaired endothelial integrity

Acknowledgments and Funding

This work was supported in part by NIH/NICHD grant R01HD091218 to IUM. BRJ was supported by an NIH Institutional National Research Service Award grant [5T32GM136554-03] and by a grant to Baylor College of Medicine from the Howard Hughes Medical Institute through the Gilliam Fellows Program [GT17071]. GMN was supported by Brazilian Coordination of Superior Level Staff Improvement (CAPES) [grant number 88887.712761/2022-00], by CAPES Institutional Internationalization Program (CAPES/PrInt) [grant number 88887.891986/2023-00] and Santander Bank Short-Term International Fellowship [University of Campinas 2023 grant]. MLC was supported by São Paulo Research Foundation (FAPESP) [grant number 2021/09937-1] and by Brazilian National Council for Scientific and Technological Development (CNPq) [grant number 408407/2021-2 and 308378/2022-9]. IUM and MLC were also supported by the Washington University at Saint Louis, USA, McDonnell Academy seed grant for research on infectious diseases and the impact of COVID-19. We thank Robert M. Lawrence for helpful editorial input. We thank all the REBRACO Study Group collaborators, as the core part of the current study.

Abbreviations

sFlt-1

soluble fms-like tyrosine kinase-1

Flt1

fms-like tyrosine kinase-1

PlGF

placental growth factor

RAS

renin-angiotensin system

AT1-AA

angiotensin II type-1 receptor autoantibodies

VT

placental villous tissue

PE

Pre-eclampsia

STB

syncytiotrophblast

Footnotes

Declaration of interest

IUM serves on the scientific advisory board of Seed Health. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Informed consent statement

All participants in both cohorts signed an informed consent form authorizing the collection, storage, and use of clinical samples and data.

Institutional review board statement

Both study cohorts followed all recommended rules for the use of human biological samples, with approval by the respective responsible Research Ethics Committee board. STOP-COVID19 cohort: IRB# 202012075 REBRACO cohort: IRB #31591720.5.0000.5404 issued by the Research Ethics Committee of the University of Campinas, Campinas, São Paulo state, Brazil.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. The data is not publicly available due to privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2199028-supplement-1.pdf (3.6MB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. The data is not publicly available due to privacy or ethical restrictions.

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