Case reports and rare case series have demonstrated variable placental pathology in the setting of maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In rare small studies demonstrating infection of the placental parenchyma, histologic manifestations have included variable degrees of histiocytic intervillositis, perivillous fibrin deposition, and syncytiotrophoblast necrosis.
To characterize the placental pathologic features of SARS-CoV-2–infected placentas, irrespective of fetal-maternal transmission, and to examine the frequency of C4d activation in such cases.
A retrospective study of 7 placentas from mothers with active SARS-CoV-2 infection and placental infection as demonstrated by RNA in situ hybridization was conducted.
There were 6 placentas from live-born neonates (5 singletons, 1 nonfused diamniotic-dichorionic twin placenta), and 1 was from a stillbirth. A total of 5 of the 8 neonates (including the stillbirth) tested negative for SARS-CoV-2, and all were negative for neonatal infection. The remaining 3 neonates were well at time of discharge. All placentas were positive for SARS-CoV-2 infection by RNA in situ hybridization and demonstrated variable degrees of histiocytic intervillositis, perivillous fibrin deposition, and trophoblast necrosis. Three cases demonstrated features of fetal vascular malperfusion. CD68 highlighted intervillous histiocytes. C4d expression was present along the villous borders in 6 of 7 cases.
SARS-CoV-2 placentitis is defined by the triad of histiocytic intervillositis, perivillous fibrin deposition, and trophoblast necrosis. The features may occur in cases without confirmed transplacental transmission. The damage caused by SARS-CoV-2 placentitis is likely mediated by complement activation.
With the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel single positive-stranded RNA virus responsible for the respiratory illness coronavirus disease 2019 (COVID-19),1 there has been growing concern over the effects of the virus on fetuses and neonates. Case reports and small cohort studies have established a possible relationship between SARS-CoV-2 and stillbirth2–5 or spontaneous abortion.6 Additionally, increasing evidence supports the (rare) occurrence of intrauterine transplacental transmission.7 Despite the critical role that the placenta plays in mediating these consequences, few publications have described the histopathologic features of placental SARS-CoV-2 infection. Fewer still have attempted to identify if particular features of placental infection portend an increased likelihood of vertical transmission7 or other poor fetal outcomes.
Results of early studies examining the effects of maternal SARS-CoV-2 infection on the placenta suggested an association between maternal SARS-CoV-2 infection and maternal or fetal vascular malperfusion8–15 ; however, later studies failed to support these conclusions.13–17 These early studies were largely retrospective reviews of placentas from all SARS-CoV-2–infected mothers, irrespective of active placental infection (ie, no confirmation of SARS-CoV-2 infection in the placental parenchyma). More recently, case reports with confirmatory testing for active placental infection have highlighted recurring features of chronic histiocytic intervillositis,4,18–25 villous trophoblast necrosis,4,18,23–26 and, to varying extents, intervillous fibrin deposition.4,24,25,27–29 In rare instances, no significant pathology was noted in cases with in situ hybridization (ISH) positivity.30
In this retrospective cohort study, we evaluate the features common to probable intraplacental SARS-CoV-2 infection, as confirmed by positive RNA ISH, to define so-called SARS-CoV-2 placentitis. We also attempt to provide a mechanistic understanding of the pathobiology of SARS-CoV-2 placental infection by staining a subset of cases with C4d, a proxy for complement activation. We posit that the deposition of C4d along the microvillous border of the syncytiotrophoblasts, a finding previously noted in cases of chronic histiocytic intervillositis, may cause the subsequent fibrin deposition and histiocytic influx that ultimately leads to the sequelae of SARS-CoV-2 placentitis, a hypothesis that has yet to be fully explored.31
MATERIALS AND METHODS
This study was approved by the Mass General Brigham Institutional Review Board for the Massachusetts General Hospital, Boston (2020P001116).
All placental specimens with positive RNA ISH for SARS-CoV-2, seen in either routine practice or in consultation at the Massachusetts General Hospital between March 2020 and April 2021, were included. The placentas were re-reviewed by an experienced perinatal pathologist (D.J.R.) to confirm diagnoses. Clinical histories were culled from the medical record.
SARS-CoV-2 RNA ISH was performed using RNA-scope 2.5 LS Probe-V-nCoV2019-S Cat No. 848568 and RNA-scope 2.5 LS Reagent Kit-RED Cat No. 322150 (Advanced Cell Diagnostics), on an automated BondRx platform (Leica Biosystems). A representative 5-μm–thick section of formalin-fixed, paraffin-embedded full-thickness parenchymal sections were used for each case. Ideal blocks were chosen based on the presence of chronic histiocytic intervillositis, perivillous fibrin deposition, or villous necrosis. All steps from baking for 1 hour at 60°C to counterstain with hematoxylin were done on a BondRx machine. RNA unmarking was done using Bond Epitope Retrieval Solution 2 for 15 minutes at 95°C followed by protease treatment for 15 minutes and probe hybridization for 2 hours. Signal was amplified by a series of signal amplification steps followed by color development in red using Bond Polymer Refine Red Detection (Leica Biosystems) in the forms of red dots.
In a subset of cases, representative placental parenchymal sections were cut from formalin-fixed, paraffin-embedded tissue blocks and immunohistochemically stained for C4d (mouse monoclonal antibody, clone SP91, Leica Biosystems RTU PA 0792, ER1 citrate buffer) and/or CD68 (mouse monoclonal antibody, KP1, Biocare RTU IP-033-AA, ER1 citrate buffer).
A total of 7 placentas with positive RNA in situ hybridization were identified from the routine cases at the Massachusetts General Hospital and the private consultations of one of the authors (D.J.R.). The cases included have not been previously reported.
Clinical variables are presented in detail in Table 1. Maternal age at delivery ranged from 24 to 33 years (mean, 29.3 years). The timing of maternal COVID-19 testing was known in 5 cases and ranged from 2 to 42 days prior to delivery (mean, 15.6 days; median, 10 days). Maternal symptoms of COVID-19 were generally mild (eg, fever, anosmia, rhinorrhea, myalgias); 2 patients were asymptomatic. All births but one (case 1) were singletons. Gestational age ranged from 28 to 39 2/7 weeks (mean, 35 2/7 weeks; median, 37 1/7 weeks). The method of delivery was known in 5 cases (2 vaginal deliveries, 3 cesarean deliveries); indications included spontaneous labor (1 case), gestational hypertension (1 case), fetal intolerance to labor (2 cases), and abruption (1 case). One case (case 7) was a stillbirth. One neonate demonstrated complications of prematurity (case 3); all remaining neonates were well. Neonatal SARS-CoV-2 testing was performed in 3 cases (case 1, case 2, and case 3), with negative results in all. The stillborn infant also underwent SARS-CoV-2 testing via nasopharyngeal swab, which was negative.
Gross, Histopathologic, and Immunohistochemical Features
The gross pathologic findings are summarized in Table 2. In most cases, gross examination was remarkable for a variegated placental parenchyma, with some demonstrating marked fibrin deposition (Figure 1), mimicking that which may be seen with massive perivillous fibrin deposition.
All placentas, with the exception of Twin B's from case 1, demonstrated histiocytic intervillositis defined by patchy to diffuse involvement of the intervillous spaces by a monomorphic monocyte macrophage population (Figure 2). At least focal neutrophilic infiltrate was also present in the intervillous spaces, typically admixed with the histiocyte population (Figure 3). Adjacent to foci of inflammation, trophoblast necrosis was seen with associated degeneration of villi (Figure 4). Perivillous fibrin deposition was seen in all cases but Twin B's from case 1, and was analogous to the amount seen in cases of massive perivillous fibrin deposition (Figure 5). Features of fetal vascular malperfusion were seen in 3 cases, with 1 case notable for intravascular thrombi (case 4; Figure 6). Chronic villitis was present in 2 cases and acute chorioamnionitis in 1. Two cases demonstrated possible features of maternal vascular malperfusion in the form of placental infarction (case 5) and villous agglutination (case 3); however, the infarctions and villous agglutination were favored to be secondary to the virally induced massive perivillous fibrin deposition and histiocytic intervillositis rather than of de novo maternal vascular disease.
As previously stated, SARS-CoV-2 RNA ISH was performed on all cases and demonstrated nearly diffuse positivity in the syncytiotrophoblast. A total of 5 of 6 examined cases demonstrated at least focal expression of C4d in the syncytiotrophoblast surface, with 2 cases demonstrating diffuse expression (cases 2 and 6; Figure 7). In a subset of cases (n = 4) CD68 was performed to highlight intervillous histiocytes and the absence of monocyte infiltration in the villous stroma (Figure 2, B and D).
As the COVID-19 pandemic continues, an increasing number of pregnant women worldwide have been and will become infected with SARS-CoV-2. Thus, although placental infection by SARS-CoV-2 is rare, cases are still expected to be seen well into the future. Additionally, with the evolution of new, more virulent variants of SARS-CoV-2,32 it is possible that the devastating consequences of SARS-CoV-2 placentitis, including miscarriage and stillbirth, may become more common. It is interesting on this note that our cases all occurred during the second and third waves of the pandemic in the United States. Recognition of this entity is thus critical.
Others have noted the presence of some of these histopathologic features in SARS-CoV-2 placentitis.33,34 Herein we “officially” define the histopathologic triad of SARS-CoV-2 placentitis, including histiocytic (and neutrophilic) intervillositis, perivillous fibrin deposition, and trophoblast necrosis. We propose that when present such features warrant confirmation of SARS-CoV-2 infection through RNA in ISH or anti-SARS-CoV-2 immunohistochemistry, as available. CD68 is not necessary for diagnosis but may be used to highlight the presence of intervillous histiocytes.
Much of the existing literature on neonatal SARS-CoV-2 placentitis has highlighted vertical transmission as the main outcome of concern.7,35 Of neonates who test positive for SARS-CoV-2 infection, somewhere between 5.7% and 10.6% of infections are congenitally acquired,36 and in some cases of confirmed transplacental infection, neonates have developed severe disease, even requiring intubation.7 However, our study highlights that SARS-CoV-2 placentitis is not pathognomonic of congenital infection. Thus, other consequences, particularly those secondary to histiocytic intervillositis and perivillous fibrin deposition, are of tantamount concern. Both chronic histiocytic intervillositis and massive perivillous fibrin deposition are associated with poor pregnancy outcomes, including stillbirth, miscarriage, and intrauterine growth restriction.37,38 In our study, we witnessed 1 stillbirth where the likely cause of intrauterine death at 33 weeks was SARS-CoV-2 placentitis. Stillbirth caused by SARS-CoV-2 placentitis has been confirmed in at least 3 other cases7 and suspected in several more.3 Additionally, recent reports out of Ireland suggest that the COVID-19 variant B.1.1.7 may be associated with an increased risk of stillbirth due to SARS-CoV-2 placentitis.39,40 Given the marked effects in most of the placentas observed in our study, stillbirth and/or miscarriage appears to be a plausible and serious sequelae of the massive placental damage imposed by SARS-CoV-2. Our findings also raise concern for neurodevelopmental disorders in children affected by SARS-CoV-2 placentitis, secondary to perivillous fibrin deposition analogous in amount to that seen in massive perivillous fibrin deposition.41 Further studies, including those with long-term follow-up, will be needed to assess the long-term effects of SARS-CoV-2 placentitis.
Schwartz and Morotti42 previously noted that placentas with features of SARS-CoV-2 placentitis largely occurred in infected maternal-neonatal dyads; they thus concluded that chronic histiocytic intervillositis and trophoblast necrosis were risk factors for maternal-fetal viral transmission. In our study, all tested neonates were negative for neonatal SARS-CoV-2 infection. Thus, infection of the placenta, even with marked trophoblast necrosis, does not necessarily equate to transplacental transmission. This raises the question of why some neonates do not acquire infection despite placental infection, and further, why most placentas from SARS-CoV-2–infected mothers fail to demonstrate infection at all. The latter question may be partially resolved. A recent study that examined transcriptomic changes specific to immune and nonimmune cell types in placentas from SARS-CoV-2–infected mothers demonstrated 2 key features: (1) a robust increase in natural killer cell expression of genes encoding cytotoxic proteins and (2) endothelial cell upregulation of genes critical in an antiviral response.43 Thus, there is evidence of an intraplacental antiviral response to maternal infection. It is therefore possible that this response, due to maternal factors or perhaps coronavirus variant, is more robust and/or effective in some. Similar mechanisms may be at play in determining which neonates will acquire infection transplacentally. However, it is not entirely understood whether other factors, including viral load or timing of infection, play a role in preventing infection of the neonate.
Our study demonstrated evidence of C4d deposition along the trophoblastic surface of villi, providing evidence that the mechanism of trophoblast destruction in SARS-CoV-2 placentitis is, at least in part, due to activation of the complement system. It is likely that the complement fixation along the border of villi damages the microvillous absorptive apical syncytiotrophoblastic membrane. Bendon et al,44 who found similar C4d deposition in the setting of chronic histiocytic intervillositis of unknown etiology, have postulated that it is this damage that is to blame for the major sequelae of chronic histiocytic intervillositis (eg, intrauterine growth restriction, fetal death). Additionally, complement system activation likely triggers local cytokine upregulation and subsequent monocyte adhesion.44 Thus, SARS-CoV-2 placentitis likely begins when the virus infects cytotrophoblast43 and syncytiotrophoblast2,17,19,45,46 eliciting complement activation, which through cytokine upregulation then draws monocytes to the site of injury. The inflammatory cytokines simultaneously are postulated to cause a local procoagulant environment, leading to the subsequent deposition of fibrin.43 Trophoblast necrosis may represent the terminal event needed to subsequently allow access to the villous stroma, and thus the fetal vasculature, resulting in transplacental transmission of SARS-CoV-2.7 It is worth noting that C4d expression was not seen in case 1, yet the triad of pathology was present, albeit in only 1 of the 2 twins. It is possible this was secondary to the administration of betamethasone prior to delivery, which blunted complement activation47 ; the timing of the steroid administration may explain that lack of significant placental findings in Twin B despite positive SARS-CoV-2 ISH. Therefore, it is also theoretically possible that corticosteroids, as has been attempted in non–COVID-19-related cases of massive perivillous fibrin deposition,37 could be administered prenatally to dampen the placental response to SARS-CoV-2 infection and thereby decrease the likelihood of poor fetal outcomes. It is also possible that another mechanism is at play here that is yet to be discovered.
Our study is somewhat limited by the absence of neonatal SARS-CoV-2 testing in some cases. There is also the theoretic possibility that some of the neonatal testing represents false negatives; however, it is statistically unlikely that this explains all 5 negative test results. Therefore, we find it improbable that there are many missed cases of transplacental infection represented in our cohort. Additionally, as SARS-CoV-2 ISH is generally only pursued in our practice after increased perivillous fibrin, chronic villitis, histiocytic intervillositis, or trophoblast necrosis has been identified (ie, not all placentas from COVID-19–positive mothers undergo SARS-CoV-2 ISH), there is likely some selection bias in our cohort. Thus, our study's methodology does not allow us to guarantee that all infected placentas display these characteristic features. However, we do believe that the triad of histiocytic intervillositis, perivillous fibrin deposition, and trophoblast necrosis is distinctive and strongly associated with SARS-CoV-2 infection, and that perhaps the features may be in some way related to the acuity of the infection or the intraplacental inflammatory response.
In conclusion, SARS-CoV-2 placentitis is an uncommon complication of maternal COVID-19 and manifests with a distinct triad of histiocytic intervillositis, perivillous fibrin deposition, and trophoblast necrosis. These features warrant confirmation of SARS-CoV-2 infection through RNA in ISH or anti–SARS-CoV-2 immunohistochemistry, as available. The massive placental injury observed in many cases has the potential to cause fetal demise and transplacental infection. Because the triad includes marked perivillous fibrin deposition and histiocytic intervillositis, additional follow-up studies to examine the neurodevelopmental consequences in children affected by SARS-CoV-2 placentitis should be performed to exclude the possibility of long-term sequelae of SARS-CoV-2 placentitis.
The authors would like to acknowledge David Gray, MD, and Megha Joshi, MD, who both contributed cases in the form of consultation.
The authors have no relevant financial interest in the products or companies described in this article.