key: cord-0763159-l5kxome5
authors: Shook, Lydia L; Brigida, Sara; Regan, James; Flynn, James P; Mohammadi, Abbas; Etemad, Behzad; Siegel, Molly R; Clapp, Mark A; Li, Jonathan Z; Roberts, Drucilla J; Edlow, Andrea G
title: SARS-CoV-2 placentitis associated with B.1.617.2 (Delta) variant and fetal distress or demise
date: 2022-01-13
journal: J Infect Dis
DOI: 10.1093/infdis/jiac008
sha: 584dfb81d02d84787ed337a8731938aa9d3b98a0
doc_id: 763159
cord_uid: l5kxome5

There is limited information on the specific impact of maternal infection with the SARS-CoV-2 B.1.617.2 (Delta) variant on pregnancy outcomes. We present two cases of intrauterine fetal demise and one case of severe fetal distress in the setting of maternal infection with Delta-variant SARS-CoV-2. In all cases, fetal demise or distress occurred within 14 days of COVID-19 diagnosis. Evaluation revealed maternal viremia, high nasopharyngeal viral load, evidence of placental infection with Delta-variant SARS-CoV-2, and hallmark features of SARS-CoV-2 placentitis. We suggest that Delta-variant SARS-CoV-2 infection during pregnancy warrants vigilance for placental dysfunction and fetal compromise regardless of disease severity.

A c c e p t e d M a n u s c r i p t Background SARS-CoV-2 infection acquired during pregnancy is associated with an increased risk of adverse pregnancy outcomes including preeclampsia and preterm birth, particularly in cases of severe and critical maternal illness [1] . Although fetal growth restriction and stillbirth have been associated with some cases of maternal SARS-CoV-2 infection [1, 2] , the extent to which SARS-CoV-2 infection itself might drive placental dysfunction leading to these pregnancy outcomes is not known, as a characteristic histopathological signature associated with maternal SARS-CoV-2 infection was not clearly identified in initial reports [3, 4] . Ultimately, an increased risk of stillbirth associated with maternal SARS-CoV-2 infection had not been consistently observed prior to the emergence of new variants of concern [5] .

In April 2021, the Royal College of Physicians of Ireland and the Institute of Obstetricians and Gynaecologists issued a statement warning the public of 6 cases of stillbirth and one case of second trimester pregnancy loss associated with "SARS-CoV-2 placentitis"; results indicated a temporal link of this placental condition with the emergence of the B.1.1.7 ("Alpha") variant [6] .

However, no other groups identified increased risk of adverse fetal outcomes associated with the Alpha variant, which was rapidly eclipsed by the B.1.617.2 ("Delta") variant. Recently published data from the Centers for Disease Control and Prevention (CDC) demonstrate a 4-fold increased risk of stillbirth in deliveries with COVID-19 relative to those without COVID-19 during the period of Delta predominance in the US, a sharp increase when compared with prior epochs in the pandemic, raising concerns that Delta-variant infection may be contributory [7] . This population-level study was not able to shed light on potential pathogenesis of the stillbirths, and scant information exists on the impact of SARS-CoV-2 variants on placental histopathology, although increased severity of maternal disease with Delta-variant COVID-19 in pregnancy has been observed [8, 9] . Here we present two cases of intrauterine fetal demise and one case of fetal distress requiring emergent cesarean delivery in the setting of confirmed maternal infection with the SARS-CoV-2 Delta variant, via sequencing of viral RNA isolated from the placenta, nasopharyngeal swabs, and maternal blood, with placental histopathologic features consistent with SARS-CoV-2 placentitis. At the time of delivery, placental biopsies were collected from the maternal and fetal sides of the placenta. Maternal plasma and nasopharyngeal swabs were also collected on admission to the hospital for delivery. Total RNA was extracted from placental homogenates, plasma samples, and nasopharyngeal swabs, and SARS-CoV-2 viral load was quantified using the US CDC 2019-nCoV_N1 primers and probe set as previously described [3] . The limit of quantification for viral load was 40 SARS-CoV-2 RNA copies/mL and positive values below that were set at 1.0 log 10 RNA copies/mL. SARS-CoV-2 RNA extracted from placenta homogenates, plasma, and nasopharyngeal swabs was used as a template to amplify and sequence the viral genome. Sequencing was done using the QIAseq SARS-CoV-2 Primer Panel (Qiagen) as instructed by the manufacturer's manual. PCR products were purified with AMPure XP beads (Beckman Coulter) and subsequently sequenced with the Illumina MiSeq sequencer, utilizing V2 chemistry. Raw Illumina reads were trimmed, and sequences were assembled by Sequencher 5 (Gene Codes Corp) and QIAGEN CLC Genomics Workbench (Qiagen). SARS-CoV-2 region-specific PCR amplification and sequencing was performed for regions with suboptimal sequencing coverage.

Placentas were sent for full pathologic examination and were examined grossly and sampled per the Amsterdam Consensus recommendations. At least 4 sections of placenta were sampled A c c e p t e d M a n u s c r i p t (umbilical cord, membranes, and at minimum three sections of parenchyma). Samples were fixed in formalin, processed, embedded and stained with hematoxylin and eosin (H&E) for histopathologic examination per routine. One parenchymal section was chosen to study SARS-CoV-2 by RNA in situ hybridization (RNA-ISH) as previously described [10] . An experienced perinatal pathologist (D.J.R.) reviewed the gross and microscopic pathology, interpreted the RNA-ISH and provided diagnoses. Table 1 illustrates clinical case information for the three patients included in this report.

Maternal age ranged from 30 to 39, all SARS-CoV-2 infections were mild in severity per NIH criteria, all SARS-CoV-2 infections occurred in the third trimester (gestational age at infection 30 to 35 weeks), and all patients were unvaccinated. Two cases (Case 1 and Case 3) resulted in intrauterine fetal demise, and one (Case 2) in a live birth after emergent cesarean delivery for Category 3 fetal heart tracing. The number of days from initial COVID-19 symptoms to pregnancy outcome ranged from 5 to 10.

Of the two cases of intrauterine fetal demise (Cases 1 and 3), the antenatal courses were uncomplicated. Both patients had negative cell free DNA screening for common aneuploidies and a normal second trimester detailed anatomic survey ultrasound. Serologies for toxoplasmosis, cytomegalovirus (CMV), parvovirus B19, and syphilis were negative for acute infection. No gross anatomic abnormalities were detected at birth in either fetus. Because neither patient with fetal demise consented to fetal autopsy, fetal examinations including SARS-CoV-2 testing were not able to be performed.

In Case 2, the patient presented for evaluation 5 days after the onset of COVID-19 symptoms after reporting decreased fetal movement to her outpatient provider. A Category 3 fetal heart rate tracing concerning for significant acidemia was identified on fetal heart rate monitoring, which prompted immediate delivery by cesarean section (emergent). Maternal laboratory evaluation from blood draw on admission was notable for D-dimer level of 49,884 ng/mL, undetectable fibrinogen 

All three placentas revealed characteristic gross and microscopic findings of SARS-CoV-2 placentitis [10, 11] : the triad of histiocytic intervillositis, increased perivillous fibrin, and villous trophoblastic necrosis (Figure 1 : A,C,E), and a positive SARS-CoV-2 RNA-ISH signal in the villous trophoblast ( Figure 1 : B, D, F), and firm and mottled appearing parenchyma grossly ( Figure 1G ).

Other pathological findings present in each case are listed in Supplementary Table 1 .

RNA isolated from biopsies obtained from the maternal and fetal sides of the placenta demonstrated detectable SARS-CoV-2 in all three cases (viral load range 3.3 to 6.2 log 10 SARS-CoV-2 copies/ng tissue, Supplementary CoV-2 viral loads were detected in Case 2 and Case 3 (7.4 and 5.5 log 10 copies RNA/mL, respectively, not available for Case 1); sequencing confirmed the Delta variant.

In this brief report, we present three cases, two of fetal demise (Case 1 and 3) and one case of fetal distress necessitating emergent cesarean delivery (Case 2), all demonstrating maternal and placental infection with the Delta variant and SARS-CoV-2 placentitis [6, 10, 11] . Because neither patient whose pregnancies resulted in fetal demise consented to fetal autopsy, and chromosomal microarray results were not available for one patient (Case 1) at the time of this report, complete information on an anatomic or genetic cause of death is limited. However, in the setting of low-risk cell free DNA screening for common aneuploidies and a normal anatomical survey ultrasound, the likelihood of a genetic etiology for stillbirth is low. No other potential cause of fetal demise was identified on standard laboratory testing. Our results suggest that the two stillbirths were secondary to SARS-CoV-2 placentitis, leading to severe placental damage and insufficiency.

In Case 2, the cause of maternal DIC was not identified. Although placental abruption can cause fetal distress and DIC, the lack of vaginal bleeding and/or contractions on presentation is inconsistent with this diagnosis, and no evidence of abruption was identified at delivery or on placental pathology. Although elevated D-dimer and abnormalities of coagulation have been welldescribed in cases of severe and fatal cases of COVID-19 [12] , DIC has not yet been identified in the setting of otherwise mild COVID-19 disease. SARS-CoV-2 placentitis certainly may have played a role in the development of fetal distress in Case 2, and the fetus may have been vulnerable to placental insults due to small placental size despite normal fetal growth.

With low COVID-19 vaccination rates in pregnant people, recent evidence of risk of greater COVID-19 morbidity in pregnant people infected with variants of concern [8, 9] , and evidencethat the risk of stillbirth is increased with Delta-variant COVID-19 [7] , the impact of SARS-CoV-2 variants of concern on pregnancy warrants continued scrutiny. Prior to this report, a definite link between the Delta variant, SARS-CoV-2 placentitis and stillbirth had not been established. Delta variant infections A c c e p t e d M a n u s c r i p t have been linked to higher viral loads and increased risk of hospitalizations compared to prior variants [13] . It is possible that maternal viremiaa feature not commonly observed in pregnant women in the first wave of the pandemic [3] , but which was observed in both cases with available blood samples at delivery -can overcome placental immune defenses at the level of the syncytiotrophoblast, which comes in direct contact with maternal blood. Evidence suggests that the Delta spike P681R mutation may increase viral infectivity, tissue tropism, and virulence due to enhanced S protein cleavability by furin [14] , a transmembrane serine protease that is widely expressed by the syncytiotrophoblast. In the cases presented here, in which placental weights were smaller than expected for gestational age, it is also possible that preexisting placental compromise was worsened by SARS-CoV-2-driven placentitis. We propose SARS-CoV-2 placentitis as a potential mechanistic link between Deltavariant SARS-CoV-2 infection during pregnancy and adverse pregnancy outcomes.

Similarities in the three cases presented here include third trimester infection, mild COVID-19 disease severity, timing of pregnancy outcome within 14 days of COVID-19 diagnosis, female neonatal sex, small for gestational age placental weight (only in the stillbirth cases), and unvaccinated status. It will be important to elucidate in larger cohorts whether these factors or others predispose to SARS-CoV-2 placentitis and/or stillbirth. Although features of maternal vascular malperfusion, intervillositis, and trophoblastic necrosis have been described in cases of poor pregnancy outcomes and severe or critical maternal COVID-19 disease [15] , the same findings have also been reported in cases of mild COVID-19 disease not resulting in fetal demise or compromise [11] , and thus maternal disease severity may not correlate well with the risk of adverse fetal outcomes. We suggest that infection with Delta variant SARS-CoV-2 during pregnancy, regardless of disease severity, warrants vigilance for signs of fetal compromise in the weeks following SARS-CoV-2 infection, and that antenatal surveillance for placental compromise in the third trimester is warranted. Vaccination against COVID-19 will continue to play a critical role in protecting pregnant people from risks associated with Delta-variant COVID-19. M a n u s c r i p t A c c e p t e d M a n u s c r i p t M a n u s c r i p t 

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Acknowledgements: Drs. A. Huynh, M. Misialek, V. Torous, and J. Watkins for assistance with the gross and histopathologic diagnoses, the pathologist assistants and histopathologists at the MGH for the gross and histological preparations of the cases, and J. Mendez-Pena for the RNA-ISH studies.