A SARS-CoV-2 Delta Variant Case Manifesting as Extensive Placental Infection and Fetal Transmission

Abstract

Introduction

Studies indicate a very low rate of SARS-CoV-2 detection in the placenta or occasionally a low rate of vertical transmission in COVID-19 pregnancy. SARS-CoV-2 Delta variant has become a dominant strain over the world and possesses higher infectivity due to mutations in its spike receptor-binding motif.

Case Presentation

To determine whether SARS-CoV-2 Delta variant has increased potential for placenta infection and vertical transmission, we analyzed SARS-CoV-2 infection in the placenta, umbilical cord, and fetal membrane from a case where an unvaccinated mother and her neonate were COVID-19 positive. A 35-year-old primigravida with COVID-19 underwent an emergent cesarean delivery due to placental abruption in the setting of premature rupture of membranes. The neonate tested positive for SARS-CoV-2 within the first 24 h, and then again on days of life 2, 6, 13, and 21. The placenta exhibited intervillositis, increased fibrin deposition, and syncytiotrophoblast necrosis. Sequencing of viral RNA from fixed placental tissue revealed SAR-CoV-2 B.1.167.2 (Delta) variant. Both spike protein and viral RNA were abundantly present in syncytiotrophoblasts, cytotrophoblasts, umbilical cord vascular endothelium, and fetal membranes.

Conclusion

We report with strong probability the first SARS-CoV-2 Delta variant transplacental transmission. Placental cells exhibited extensive apoptosis, senescence, and ferroptosis after SARS-CoV-2 Delta infection.

Established Facts

• SARS-CoV-2 was detected in the placenta.

• Maternal-fetal transmission at a very low rate.

• SARS-CoV-2 Delta was a dominant strain and possesses high infectivity.

Novel Insights

• SARS-CoV-2 Delta variant case manifesting as extensive placental infection: intervillositis, fibrin deposition, syncytiotrophoblast necrosis.

• Spike protein and viral RNA of SARS-CoV-2 Delta variant detected in syncytiotrophoblasts, cytotrophoblasts.

• Spike protein and viral RNA of Delta variant found in fetal umbilical cord vascular endothelium and fetal membrane.

• SARS-CoV-2 Delta variant case manifesting as extensive placental infection and fetal transmission.

Introduction

SARS-CoV-2 primarily infects cells in the lung through the binding of its spike (S) protein to the cell surface receptor Angiotensin converting enzyme 2 (ACE2). Cells in the placenta express ACE2, albeit at relatively low levels [1], suggesting the potential for placental infection of SARS-CoV-2 in COVID-19 pregnant patients. Studies have demonstrated a very low rate, 3–5%, of SARS-CoV-2 detection in the placenta in COVID-19 pregnancy [2, 3], and the major cell type infected by SARS-CoV-2 in the placenta is the syncytiotrophoblast (STB) [4, 5, 6]. Early in the COVID-19 pandemic, vertical transmission was not demonstrated in COVID-19 pregnancy [7]. As the pandemic advanced, occasional reports showed a low rate (1.6–3.2%) of vertical transmission in COVID-19 pregnancy [6, 8]. SARS-CoV-2 IgM is present in same neonatal blood samples [9], implicating fetal presence of the virus. However, based on recently defined criteria of SARS-CoV-2 vertical transmission by the World Health Organization [10], these reported cases may not be true congenital infections.

The SARS-CoV-2 Delta variant (B.1.617.2) emerged in India in late 2020 and has been predominant versus the wild-type Wuhan-1 and other earlier variants including B.1.617.1 (Kappa) and B.1.1.7 (Alpha) [11]. Due to mutations in its S receptor-binding motif, the SARS-CoV-2 Delta variant confers increased infectivity and a modest loss of sensitivity to neutralizing antibodies and vaccines and breakthrough infections [11]. It also possesses higher efficiency of replication and S-mediated cell entry compared to other variants [11]. Because the SARS-CoV-2 Delta variant S protein is in a constant cleaved state [11], it can infect cells with low expression of ACE2 and other cell entry factors. All these characteristics of SARS-CoV-2 Delta variant point to a higher potential for placental infection and increased potential for vertical transmission.

The case we presented here shows massive infection of SARS-CoV-2 Delta of placental cells on both maternal and fetal sides in a third trimester COVID-19 patient with premature rupture of membrane and subsequent preterm birth. The newborn was SARS-CoV-2-positive by PCR testing and developed modest respiratory symptoms. The placenta showed robust inflammatory responses with increased senescence and apoptosis. As the SARS-CoV-2 Delta variant's dominance persists, there is increased potential for placental infection and vertical transmission. This emphasizes crucial roles of vaccination, testing, and specialized obstetric care.

Case Report

Methods of Research

Immunocytochemistry Staining

Formalin-fixed, paraffin-embedded (FFPE) placenta tissues were sectioned at 10 μM in the Pathology Core Lab of the University of Maryland Baltimore. The sections were deparaffined and rehydrated followed by antigen retrieval (citrate buffer, pH6) and H₂O₂ treatment (2%, 15 min). Immunostaining was performed as described [12]. Sections were incubated in blocking solution (phosphate-buffered saline containing 4% normal goat serum, 0.2% Triton-100), then with the primary antibodies at 4°C overnight. After 1 h incubation with biotinylated secondary antibody, followed by 1 h with ABC solution (VectorLab, Elite Kit, Cat# PK6100, 1:500), antibody labeling was demonstrated using precipitation in diaminobenzidine (10 mg/50 mL) chromogen in 0.175 M sodium acetate + 0.003% H₂O₂ (brown products). Sections were counterstained with hematoxylin, dehydrated, and coverslipped. Non-COVID-19 placental FFPE was included in all the staining and displayed negative. Following antibodies were used in this study: spike protein (Abcam, #ab272504), nucleocapsid protein (Novus, #NB100-56576), RORrT (Sigma-Millipore, #MABF81), p-STAT1 (Thermofisher, PA5-97355), DPP4 (Cell Signaling, #67138S), p-H2A.X (Ser139) (Cell Signaling, #9718S), H3K9Me3 (Sigma-Millipore, #SAB5700163), ASCL4 (Abcam, #ab155282), Syndecan (Cell Signaling, #3050S), biotin-conjugated Goat anti-mouse IgG (Vector Lab, #BA-9200), biotin-conjugated goat anti-rabbit IgG (Vector Lab, #BA-1000).

Immunofluorescent Dual Labeling

Fluorescent dual labeling was performed by following the procedure as previously described [13]. After deparaffinization, rehydration, and antigen retrieval, sections were incubated with blocking solution, then with mouse anti-syndecan antibody and rabbit anti-S protein antibody by following the procedure at 4°C overnight. After phosphate-buffered saline washes, the sections were incubated with Alexa 488-Goat anti-mouse IgG (Thermofisher, Cat# A11029, 1:1,000) and Alexa 594-Goat anti-rabbit IgG (Thermofisher, Cat# A11037, 1:1,000) for 1 h before Sudan Black B treatment (0.05% in 70% ethanol for 5 min) and counterstaining with 4′,6-diamidino-2-phenylindole (DAPI).

RNAscope in situ Hybridization

We performed in situ RNA hybridization in FFPE slides using RNAScope 2.5 HD Detection (RED) Kit (ACD, CA) according to manufacturer's protocol. Following deparaffinization and antigen retrieval, slides were hybridized with the probe, V-SARS-CoV-2-S (Ref#: 854841, ACD) at 40°C for 2 h. Following buffer rinses, further hybridization includes Amp 1, 30 min; Amp 2, 15 min; Amp, 30 min; Amp 4, 15 min; Amp 5, 30 min; and Amp 6, 15 min at 40°C with interval rinsing. RNA signals were detected by incubating with a mixture of RED-B and RED-A in a ratio of 1:60 for 10 min at room temperature followed by counterstaining in 50% hematoxylin for 2 min.

Reverse-Transcription PCR and Sequencing

Total RNA extraction from formalin-fixed placental tissue was performed with PureLink FFPE RNA Isolation Kit (Thermofisher, Cat# K156002). Formalin-fixed placenta tissue blocks were homogenized in 300 μL melting buffer, 20 μL proteinase K added, and incubated at 60°C for 30 min with occasional mixing until tissue debris was not visible in the solution. Total RNA was isolated and purified with the spin column.

Reverse transcription was conducted with Superscript III first-strand synthesis system for RT-PCR (Thermofisher, Cat# 18080-051). One microgram of total RNA was reverse transcribed to complementary DNA (cDNA) with random hexamer and yielded 20 μL of cDNA.

Mutation of threonine amino acid residue to lysine at 478, leucine to arginine at 452, and proline to arginine at 681 in the S protein are critical for SARS-CoV-2 Delta variant transmissibility. To identify the Delta variant, we designed two PCR primer pairs flanking the K417 and asparagine at 501 (Fig. 1) according to the SARS-CoV-2 viral genome sequence (Genbank Accession# MN_908947.3). Forward primer F1, 5′-GGTGATGAAGTCAGACAAATCGC; forward primer F2, 5′-ACAGGCTGCGTTATAGCTTG; common reverse primer R, 5′-CACAAACAGTTGCTGGTGCAT. PCR reactions contain 50 μL of GoTaq Green Master mix (Promega, Cat# M7122), 5 μL of 10-μ forward primer (final = 0.5 μ), 5 μL of 10-uM reverse primer (final = 0.5 μ), 10 μL cDNA, and 30 μL of water. For the negative control, the cDNA was replaced with the same volume of water. For PCR amplification, we first tested annealing temperatures at 55°C, 58°C, and 60°C (Fig. 1) and found Ta = 60°C is optimal. PCR products generated at Ta = 60°C were used for DNA sequencing. PCR thermocycler parameters: denaturation at 94°C for 5 min, followed by 40 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, final extension at 72°C for 5 min. Expected PCR products were 369 bp with primer pair F1/R and 289 bp with F2/R. PCR products were visualized by agarose gel electrophoresis (Fig. 1). For sequencing, PCR products were purified from agarose gel with QIAquick gel extraction kit (Qiagen, Cat# 28704) and submitted for sequencing at the Center for Innovative Biomedical Resources, University of Maryland Baltimore.

Fig. 1.

Sequencing confirms SARS-CoV-2 Delta variant in case #30738. Two PCR primer pairs were designed to amplify viral genomic fragments flanking K417 and N501 of S protein. Expected PCR products are 289 bp and 369 bp. PCR products were generated from fixed placental tissue RNA at optimal annealing (Ta) temperature of 60°C. The protein sequence translated from PCR-amplified DNA fragment was aligned with wild-type Wuhan-1 SARS-CoV-2 (Genbank accession #MN_908947.3). Comparison of amino acid mutations in S protein of different SARS-CoV-2 variants demonstrated L452R and T478K mutations but lacking of E484 and N501 mutation in #30738 placenta, indicating the SAR-CoV-2 strain in case #30738 placenta is Delta [14]. K, lysine; N, asparagine; L, leucine; R, arginine; T, threonine; E, glutamic acid.

TUNEL Staining

TUNEL staining was performed with ApopTag Red in situ apoptosis detection kit (EMD Millipore, Cat# S7165). Sections were equilibrated and incubated with TdT enzyme solution for 1 h at 37°C, treated with working strength stop/wash buffer, followed by incubation with anti-digoxigenin conjugate for 30 min at room temperature. To quench the autofluorescence background, sections were treated with Sudan Black B as described above, then 4′,6-diamidino-2-phenylindole counterstained.

Results

Maternal and Neonatal Course

The 35-year-old primigravida has a past medical history of morbid obesity. She was unvaccinated. She developed nasal congestion, ageusia, and anosmia at 30^3/7 weeks, seeking medical care 4 days later, and was diagnosed with COVID-19 by PCR. Her initial presentation did not require hospitalization, but by 31^5/7 weeks she presented in preterm labor, complaining of leakage of fluid. She was subsequently diagnosed with premature rupture of membranes, started on latency antibiotics, magnesium sulfate, antenatal corticosteroids, and transported to our hospital. On admission, patient did not require oxygen supplementation, although she did have compensated metabolic acidosis, a bicarbonate drip was initiated. Her contractions persisted, and at 32 weeks she underwent emergent cesarean section for nonreassuring fetal heart rate, suspected placental abruption. A female infant weighing 1,350 g was born with Apgar scores of 5 and 8 at 1 and 5 min, respectively. Umbilical cord arterial blood showed a pH of 7.25 and base excess of −4.2. Histopathologic examination of the placenta revealed acute and chronic intervillositis, increased perivillous fibrin deposition, and focal STB necrosis (Table 1; Fig. 3a).

Table 1.

Clinical diagnosis of case #30738

Clinical exam	Results
Age, years	35
Group	4 (staining percentage 4)
Category	3 (FBV)
Adverse outcome	Yes
BMI	33.6
GA at diagnosis, weeks	30.6
GA at delivery, weeks	32
PMH	No
DM	No
GDM	No
HTN	No
HDP	No
PPROM	Yes
GA at PPROM, weeks	31.5
Iatrogenic PTD	No
Outcome	Live birth
MOD	Cesarean section
Placenta pathology	Inflammation/infection changes
Oxygen	No
IMV	No
APGAR_5	8
pH of umbilical cord artery blood (ApH)	7.25
NICU	No
LOS	14
Neonate COVID-19 test on DOL 2	COVID-19 positive
Repeat test on DOL 6, 13, and 21	COVID-19 positive
Pathology analysis	Results
Placenta	Third trimester, cesarean section
Inflammation	Placental disc with acute and chronic intervillositis, increased perivillous fibrin deposition, and focal syncytiotrophoblast necrosis
Umbilical cord	Trivascular umbilical cord with no diagnostic abnormalities recognized
Fetal membrane	With no diagnostic abnormalities recognized

BMI, body mass index; GA, gestation age; PMH, past medical histology; DM, diabetes mellitus; GDM, gestational diabetes mellitus; HTN, hypertension; HDP, hypersensitive disorders in pregnancy; PPROM, prelabor premature rupture of membranes; MOD, method of delivery; IMV, intermittent mandatory ventilation; APGAR_5, APGAR score at 5 min; NICU, neonatal intensive care unit; LOS, length of stay; DOL, day of life, FBV, fetal blood vessel.

Fig. 3.

SARS-Cov-2 Delta variant infection induces placental inflammation and cell death. a Hematoxylin-eosin staining showed significant perivillous fibrin deposition and intervillositis in the SARS-CoV-2 Delta variant-infected placenta. Increased RORγT + T-regulatory cells and phospho-STAT1 signal were detected in the placental villi. b Apoptotic cells were depicted in syncytiotrophoblast (STB) and intravillous cells (IVC). c Infected placenta displayed increased DPP4 senescent cells in STB, and upregulated DNA damage marker γH2A.X and H3K9Me3 in the nuclei of STB and IVC. d SARS-CoV-2 Delta variant induced ASCL4, a marker for cell ferroptosis. Counterstaining: hematoxylin.

The maternal postpartum course was complicated by acute blood loss anemia with a hemoglobin nadir of 6.5 g/dL from 13.2 g/dL. The patient was asymptomatic and declined blood transfusion. She was discharged home on postoperative day 3.

Upon delivery, the neonate required noninvasive positive pressure ventilation due to acute respiratory failure and was admitted to the neonatal intensive care unit. Nasopharyngeal SARS-CoV-2 PCR testing was positive within the first 24 h (Table 1). A confirmatory test in another 24 h was also positive. Repeat tests on day of life 6, 13, and 21 were all positive. The neonate progressed to room air and is developing normally.

Identification as a SARS-CoV-2 Delta Case

To determine Delta variant identity, we designed two primer pairs (F1/R, F2/R) flanking the K417 and N501 of S protein and sequenced the PCR-amplified fragments from placental tissue RNA (Fig. 1). Comparison of translated protein sequence with the wild-type Wuhan-1 (Genbank accession #MN_908947.3) showed the presence of the L452R and T478K mutations and the absence of E484 and N501 mutation (Fig. 1) [14]. These findings confirmed this case as the SARS-CoV-2 Delta variant [14].

Determination of Placental and Fetal Infections

RNAscope in situ hybridization demonstrated abundant presence of SARS-CoV-2 RNA in the STB, cytotrophoblast, and fetal vascular endothelium (Fig. 2a). Immunohistology analysis indicated intensive labeling of S protein and nucleocapsid protein in the STB and intravillous cells (IVC) (Fig. 2b). Intravillous cell-cell fusion was also observed (Fig. 2b) [15, 16]. Dual-fluorescent labeling showed colocalization in the STB, as well as in the IVC and fetal blood vessel endothelium (Fig. 2c).

Fig. 2.

SARS-CoV-2 viral RNA, S protein, and nucleocapsid protein are detected in the placenta, umbilical cord, and fetal membranes. RNAscope in situ hybridization demonstrated SARS-CoV-2 viral RNA presence in STB, cytotrophoblast, and fetal vascular endothelium (a). Extensive S protein and nucleocapsid protein were depicted in the STB and IVC in the placenta. Intravillous cell-cell fusion was also observed (b). Dual fluorescence labeling of S protein with syncytiotrophoblast marker syndecan confirmed viral presence in STB, IVC, and fetal vascular endothelium (c). For (a–c), arrows pointed to the labeled STB and arrowheads to IVC and fetal vascular endothelium. Electron microscopic analysis identified viral particles in STB ranging from 50 to 100 nm (d). S protein (e) and SARS-CoV-2 viral RNA (f) existed in the vascular endothelium of umbilical cord vessels (arrows pointed). S protein (g) and SARS-CoV-2 genome (h) were also found in the fetal membranes including cells in the chorion and chorionic vessels. RNAscope or diaminobenzidine-stained sections were counterstained with hematoxylin, and fluorescent-labeled sections with 4′,6-diamidino-2-phenylindole.

Electron microscopic analysis identified SARS-CoV-2 viral particles in the STB (Fig. 2d). The viral particles were 50–100 nm in diameter (Fig. 2d), consistent with the size and appearance of SARS-CoV-2 in the placenta [2].

Further analysis showed S protein (Fig. 2e, g) and SARS-CoV-2 viral RNA (Fig. 2f, h) in the cord vascular endothelium, and cells in the chorion and chorionic vessels. Non-COVID-19 control placental FFPE was included in all the staining and displayed negative (data not shown).

Extensive Cell Death in the Placenta

Pathology analysis showed the presence of massive perivillous fibrin deposition and chronic intervillositis in the placenta (Fig. 3a). Further analysis indicated increased RORγT + T-regulatory cells and phospho-STAT1 signaling in the placenta (Fig. 3a), suggesting JAK-STAT signal pathway is involved in SARS-CoV-2-induced placental inflammation. TUNEL staining revealed extensive cell apoptosis in the STB (Fig. 3b). DPP4, a senescence marker, was detected in syncytiotrophoblasts and IVC (Fig. 3c). The DNA damage marker, phospho-histone H2A.X (γH2A.X), and the heterochromatin marker H3K9Me3 were detected in the infected placenta (Fig. 3c). The ferroptosis marker ASCL4 (Acyl-CoA synthetase long-chain family member 4) was also present in the placenta (Fig. 3d).

Discussion

The criteria for vertical transmission of SARS-CoV-2 have been recently established [10]. In our case, a positive nasopharyngeal test within 24 h of birth and persistence of positivity in four subsequent tests affirm neonatal infection. Although we were unable to test IgM in neonatal blood, we revealed the robust presence of SARS-CoV-2 S protein and RNA in fetal membranes and umbilical cord. Additionally, since the neonate was delivered via caesarean section, contamination with maternal blood is less likely. Based on these observations, this SARS-CoV-2 Delta case fits the defined criteria for congenital infection [10]. Membrane rupture and prematurity may not be the causes of vertical transmission because SARS-CoV-2 is only present in the blood vessels of the fetal membrane and within the umbilical cord. This suggests maternal-fetal transmission of SARS-CoV-2 across the placenta to the fetal circulation.

Studies have shown that placental SARS-CoV-2 positivity does not correlate with neonate positivity [3, 17, 18]. Even in the infrequent case of placental infection, the presence of SARS-CoV-2 is mostly confined to STB [5]. We found significant placental SARS-CoV-2 infection within cytotrophoblast and fetal endothelium, consistent with the high infectivity of the SARS-CoV-2 Delta variant as described in vitro studies [11]. The placental damage we observed may be due to heavy viral load, facilitated by subclinical inflammation, and altered amniochorion characteristics commonly seen in case of raptured membranes independent of COVID-19 status. Placental damage in COVID-19 patients is associated with neonatal neurological symptoms [5]. The neonate in our case exhibited respiratory distress. Although development to date is as expected, it is too early to determine. Moreover, it may be difficult to separate such effects from the consequences of prematurity.

In summary, we report with strong probability the first SARS-CoV-2 Delta variant transplacental transmission. With the widespread dominance of the SARS-CoV-2 Delta variant and its potential for vertical transmission, the number of affected neonates is likely to rise. Such information could provide valuable insight in counseling of pregnant women and efforts to increase vaccination acceptance.

Statement of Ethics

The study was reviewed and approved by the Institutional Review Board at the University of Maryland, Baltimore, protocol HP-00099298. The article was prepared following CAseREports guidelines (CARE) [19]. The patient consented to publication for the use of their information for research and education purposes.

Conflict of Interest Statement

The authors have declared that no conflict of interest exists.

Funding Sources

The authors were supported by NIH Grants R01 HD099843, R01 HD102206, and R01 HD102206.

Author Contributions

W.-B.S. designed the study; conducted RNA extraction from formalin-fixed tissue and RT-PCR; conducted DNA sequencing and analysis; identified SARS-CoV-2 Delta variant in the maternal-fetal transmission case; performed immunohistology and dual immunofluorescence staining; and wrote the manuscript. S.T. supervised the placental sample collection; collected clinical diagnostic data including COVID-19 test for mother and neonate. B.W. performed RNAscope in situ hybridization on the placenta, umbilical cord, and fetal membranes. L.C. collected placental sample and assisted S.T. to conduct/collect clinical diagnostic data including COVID-19 test for mother and neonate. C.H. supervised maternal and fetal COVID-19 PCR testing and performed prenatal and postnatal care for the mother and neonate. J.L. provided thoughtful discussion regarding SARS-CoV-2 sequencing. E.A.R. supervised the project and provided grant support. M.B.F. assisted identification of Delta variant and provided thoughtful discussion. P.Y. supervised the project, provided grant support, designed the study, analyzed data, and finalized the manuscript.

Data Availability Statement

The data used to support the findings of this study are included within the article.