The placentas of women who contracted COVID-19 during pregnancy showed evidence of significant injury

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The placentas from 16 women who tested positive for COVID-19 while pregnant showed evidence of injury, according to pathological exams completed directly following birth, reports a new Northwestern Medicine study.

The type of injury seen in the placentas shows abnormal blood flow between the mothers and their babies in utero, pointing to a new complication of COVID-19.

The findings, though early, could help inform how pregnant women should be clinically monitored during the pandemic.

The study was published May 22 in the journal American Journal of Clinical Pathology. It is the largest study to examine the health of placentas in women who tested positive for COVID-19.

“Most of these babies were delivered full-term after otherwise normal pregnancies, so you wouldn’t expect to find anything wrong with the placentas, but this virus appears to be inducing some injury in the placenta,” said senior author Dr. Jeffrey Goldstein, assistant professor of pathology at Northwestern University Feinberg School of Medicine and a Northwestern Medicine pathologist.

“It doesn’t appear to be inducing negative outcomes in live-born infants, based on our limited data, but it does validate the idea that women with COVID should be monitored more closely.”

This increased monitoring might come in the form of non-stress tests, which examine how well the placenta is delivering oxygen, or growth ultrasounds, which measure if the baby is growing at a healthy rate, said co-author Dr. Emily Miller, assistant professor of obstetrics and gynecology at Feinberg and a Northwestern Medicine obstetrician.

“Not to paint a scary picture, but these findings worry me,” Miller said.

“I don’t want to draw sweeping conclusions from a small study, but this preliminary glimpse into how COVID-19 might cause changes in the placenta carries some pretty significant implications for the health of a pregnancy. We must discuss whether we should change how we monitor pregnant women right now.”

Previous research has found that children who were in utero during the 1918-19 flu pandemic, which is often compared to the current COVID-19 pandemic, have lifelong lower incomes and higher rates of cardiovascular disease.

Flu doesn’t cross the placenta, Goldstein said, so whatever is causing life-long problems in those people is most likely due to immune activity and injury to the placenta.

“Our study, and other studies like it, are trying to get on the ground floor for this exposure so we can think about what research questions we should be asking in these kids and what can or should we do now to mitigate these same types of outcomes,” Goldstein said.

Fifteen patients delivered live infants in the third trimester, however one patient had a miscarriage in the second trimester.

“That patient was asymptomatic, so we don’t know whether the virus caused the miscarriage or it was unrelated,” Goldstein said, “We are aware of four other cases of miscarriage with COVID.

The other reported patients had symptoms and three of four had severe inflammation in the placenta. I’d like to see more before drawing any conclusions.”

The placenta is the first organ to form in fetal development. It acts as the fetus’ lungs, gut, kidneys and liver, taking oxygen and nutrients from the mother’s blood stream and exchanging waste.

The placenta also is responsible for many of the hormonal changes within the mother’s body. Examining a woman’s placenta allows a pathologist to follow a retroactive roadmap of a woman’s pregnancy to learn what happened to the baby in utero or what could happen to both the mother and the infant after birth.

“The placenta acts like a ventilator for the fetus, and if it gets damaged, there can be dire outcomes,” Miller said. “In this very limited study, these findings provide some signs that the ventilator might not work as well for as long as we’d like it to if the mother tests positive for SARS-CoV2.”

The placentas in these patients had two common abnormalities: insufficient blood flow from the mother to the fetus with abnormal blood vessels called maternal vascular malperfusion (MVM) and blood clots in the placenta, called intervillous thrombi.

In normal cases of MVM, the mother’s blood pressure is higher than normal. This condition is typically seen in women with preeclampsia or hypertension. Interestingly, only one of the 15 patients in this study had preeclampsia or hypertension.

“There is an emerging consensus that there are problems with coagulation and blood vessel injury in COVID-19 patients,” Goldstein said. “Our finding support that there might be something clot-forming about coronavirus, and it’s happening in the placenta.”

The 16 women in the study delivered their babies at Northwestern Medicine Prentice Women’s Hospital. All tested positive for COVID-19.

Four patients came in with flu-like symptoms three to five weeks before delivery and tested positive for the virus. The remaining patients all tested positive when they came in to deliver.

Five patients never developed symptoms, others were symptomatic at delivery.

Between 30 and 40 patients deliver at Prentice daily. The team began testing placentas of COVID-19-positive mothers in early April. Fourteen of the live-born infants in the study were born full term and with normal weights and Apgar scores. One live-born infant was premature.

“They were healthy, full-term, beautifully normal babies, but our findings indicate a lot of the blood flow was blocked off and many of the placentas were smaller than they should have been,” Miller said.

“Placentas get built with an enormous amount of redundancy. Even with only half of it working, babies are often completely fine. Still, while most babies will be fine, there’s a risk that some pregnancies could be compromised.”

In February, before the pandemic was known to have reached Chicago, Goldstein assembled his research team.

“If you get the flu and you’re pregnant, we know nothing about what that looks like in your placenta, so I began thinking how we’d study this flu-like epidemic if it came through Chicago,” Goldstein said. “We started setting things up and then lo and behold, the epidemic came here, so we were ready.”

Other Northwestern co-authors include Elisheva D. Shanes, Leena B. Mithal and Hooman A. Azad.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus causing the deadly pandemic of coronavirus disease 2019 (Covid-19). The risks and specific effects of SARS-CoV-2 in pregnant women remain unknown.

No adverse outcomes were reported among a small cohort of pregnant women who presented with Covid-19 in the late third trimester of pregnancy in Wuhan, China1. However, little is known about maternal and neonatal outcomes as a result of infection in the first and second trimesters of pregnancy.

Hypertensive disorders of pregnancy (HDP) complicate 2-8% of pregnancies and rarely occur in the second trimester2. HDP in women with Covid-19 have been noted within limited case reports from China and New York City3,4.

At the same time, a subset of non-pregnant patients with Covid-19 have demonstrated significant abnormalities of liver enzymes as well as coagulopathy5,6. These laboratory abnormalities significantly overlap with findings seen in severe preeclampsia, a subset of HDP, defined by associated findings of hemolysis, elevated liver enzymes, and low platelets as well as proteinuria and elevated blood pressures.

This leads to a diagnostic dilemma when faced with pregnant patients with Covid-19, hypertension, and coagulopathy.

We present a case of a woman with Covid-19 in the second trimester of pregnancy with severe hypertension, coagulopathy and preeclampsia.

This case highlights the association between Covid-19 and HDP, and demonstrates clear SARS-CoV-2 invasion of the placenta, with associated placental inflammation distinct from typical preeclampsia.

Case Report:

A previously healthy 35-year old gravida 3 para 1011 woman presented at 22 weeks gestation with symptoms of Covid-19 infection. Ten days prior to admission, she developed fever and cough, with acute worsening in the four days prior to admission including fever, malaise, nonproductive cough, diffuse myalgias, anorexia, nausea, and diarrhea.

On the morning of presentation, the patient awoke with vaginal bleeding and abdominal pain. Her initial vital signs showed that she was afebrile with a pulse of 110, respiratory rate of 22 per minute and an oxygen saturation of 99% on room air.

Her blood pressure was elevated to 150/100. Physical exam was notable for dark blood in the vaginal vault without cervical dilation. SARS-CoV-2 RNA was detected by RT-PCR in a nasopharyngeal swab obtained from the patient on admission.

Her past medical history was significant for psoriasis without current symptoms. The patient had a prior pregnancy that was complicated by term gestational hypertension which resolved with delivery. Her current antepartum course was notable for normal blood pressure and normal baseline preeclampsia evaluation.

The patient was admitted to labor and birth. Her chest x-ray was significant for a hazy opacity throughout the left lung. Transabdominal ultrasound revealed an active fetus, normal amniotic fluid volume, estimated fetal weight within expected range, and a posterior fundal placenta with a retroplacental clot concerning for placental abruption.

Laboratory studies revealed elevated liver transaminases, profound thrombocytopenia, and increased urine protein consistent with preeclampsia, as well as prolonged partial thromboplastin time and decreased fibrinogen consistent with disseminated intravascular coagulation (Table I).

Blood smear revealed normochromic normocytic red cells with unremarkable morphology, atypical lymphocytes, suggestive of viral infection, and severe thrombocytopenia (Supplemental Figure S1). Rotational Thromboelastometry (ROTEM) demonstrated severe deficiencies in clot formation.

The patient was resuscitated with 4 units of cryoprecipitate, 4 pools of packed platelets, 2 grams of tranexamic acid (TXA), 5 grams of fibrinogen concentrate, and 2 units of fresh frozen plasma.

This resulted in the improvement of her coagulopathy, but the thrombocytopenia and elevated blood pressure persisted.

The combination of hypertension, proteinuria, elevated transaminases, and low platelets supported the diagnosis of severe preeclampsia, for which delivery is the definitive treatment. Multidisciplinary consultations were performed via telemedicine from maternal-fetal medicine, neonatology, and infectious disease.

The patient opted for termination of the pre-viable pregnancy to reduce the risk of serious maternal morbidity or death, which was performed via dilation and evacuation under general endotracheal anesthesia.

Intraoperative findings included a retroplacental clot and were otherwise unremarkable. On post-operative day 1, she was extubated and weaned to room air; however lymphopenia developed.

Hydroxychloroquine was initiated as investigational treatment for Covid-19. Her coagulation markers improved and she was discharged to self-isolation on post-operative day three (Figure 1).

Home blood pressure monitoring was initiated with close provider telemedicine support. An emergency room visit on post-operative day four was required to titrate antihypertensives. She consented to pathology examination and to release of tissue for research-related testing per our IRB-regulated protocol.

RESULTS:

Microbiologic Investigation:

Using the US CDC qRT-PCR assay, the placenta (3 x 107 virus copies/mg) and umbilical cord (2 x 103 virus copies/mg) were positive for SARS-CoV-2 RNA (Figure 2A). Fetal heart and lung tissues were also tested and met the human RNA control (RNase P) standards and were negative for virus RNA (Figure 2).

Maternal samples were also collected post-operatively; and while the oral and nasal swabs were negative, the saliva and urine were still positive for SARS- CoV-2 (Figure 2A). Virus from the placenta was whole genome sequenced (Yale-050) and was phylogenetically similar to other SARS-CoV-2 detecting locally (Connecticut, USA) and abroad (Europe and Australia) (Figure 2B). In addition, the SARS-CoV-2 genome from the placenta did not contain any unique amino acid substitutions compared to other sequenced SARS-CoV-2.

Serologic testing:

Levels of anti-SARS-CoV-2 IgG and IgM antibodies in the case study patient were among the highest observed in 56 Covid-19+ patients admitted to Yale New Haven Hospital. Further quantification revealed end-point dilution titers of 1:1,600 for IgM and 1:25,600 for IgG (Supplemental Figure S2).

Pathology:

On gross examination the placenta showed a marginal adherent blood clot associated with a focal placental infarct supportive of the clinical diagnosis of abruption and was otherwise unremarkable.

On histological examination the placenta was remarkable for the presence of diffuse perivillous fibrin and an inflammatory infiltrate composed of macrophages as well as T- lymphocytes, as demonstrated by immunohistochemistry for CD68 (Figure 3 A-C) and CD3 (Figure 3F).

No dark pigment was noted in the intervillous space. Maternal vessels did not show features of decidual vasculopathy. The fetal organs were grossly and microscopically unremarkable (Supplemental Figure S3). SARS-CoV-2 localized predominantly to the syncytiotrophoblast cells of the placenta, as demonstrated by immunohistochemistry for the SARS-CoV-2 spike protein (Figure 3G-H), and by SARS-CoV-2 RNA in situ hybridization (Figure 3I).

Electron Microscopy:

Electron microscopic analysis of immersion-fixed placental tissue showed relatively well-preserved ultrastructure of the placenta (Figure 4). Analysis of placental region adjacent to

the umbilical cord identified virus particles within the cytosol of placental cells. The size of these virus particles were 75-100nm in diameter, which is consistent with the size and appearance of SARS-CoV-212.

Figure 1: Case timeline. Clinical Course of 35 year old G3P1011 at 22 weeks gestation with Covid-19 associated with preeclampsia and placental abruption. Top panel: Timeline from onset of symptoms to immediate post-partum period including resuscitation products, hypertension management, and surgical preparation for dilation and evacuation. demonstrates Timeline from onset of symptoms to immediate post-partum period including resuscitation products, hypertension management, and surgical preparation for dilation and evacuation. Bottom panel: Patient’s platelet count (green line), and serum fibrinogen (purple line) throughout hospitalization. Major clinical events highlighted with arrows



Figure 2: Examination of SARS-CoV-2 RNA in Maternal and Fetal Tissue. (A) SARS-CoV-2 qRT-PCR results of fetal and maternal samples using the CDC assay which consists of the N1 and N2 primers/probes targeting the coronavirus nucleocapsid and the RP primers/probe targeting human RNase P as an internal control. Cycle threshold (Ct) values from both N1 and N2 must be below 38 for the result to be positive, as internally validated. Virus titers shown as the average calculation from N1 and N2 Ct values. For the tissues, 80- 160 mg were used for extractions; and for the swabs in viral transport media and other liquid samples, 0.25-0.4 ml were used. (B) The SARS-CoV-2 genome sequenced from the infected placenta was combined with 289 other genomes available from GISAID from around the world. The phylogenetic tree was constructed using IQ- Tree within the Nextstrain Augur pipeline, and the results were visualized using Auspice. Genetic divergence is shown as substitutions per site from the root. An enlarged view of the 18 genomes in the clade that contains the SARS-CoV-2 genome sequenced from the placenta (USA/Connecticut-Yale-050). The clade is defined by 3 nucleotide substitutions, A28881A, G28882A, and G28883C, providing the equivalent of ~95% branch support. The consensus SARS-CoV-2 genome from the placenta (Yale-050) can be found using NCBI BioProject PRJNA614976 and the phylogenetic data can be visualized at: https://nextstrain.org/community/grubaughlab/CT-SARS-CoV-2/paper2. The acknowledgements for the sequences obtained from GISAID can be found at: https://github.com/grubaughlab/CT-SARS-CoV-
Figure 3: Histopathology and Electron Microscopy of placenta. A-C. Section of placenta stained with Hematoxylin & Eosin showing histiocytic intervillositis (40X, 100X, 400X). C. Star indicates intervillous space infiltrated by immune cells. Arrows indicates perivillous fibrin. D, E. Immunohistochemical stain for CD68 showing the majority of intervillous inflammatory infiltrate positive (brown stain) for this macrophage marker (40X, 400X) F. Staining for CD3, a marker of T lymphocytes (100X). G,H. Immunohistochemical staining for SARS-CoV-2 spike protein, demonstrating viral localization predominantly in syncytiotrophoblast cells (50X, 100X). I. In situ analysis for the presence of SARS-CoV-2 RNA shows strong positive staining within the placenta.
Figure 4: Electron microscopy images of coronavirus particles in different placental cell types. A. Schematic illustration of chorionic villi. Cytotrophoblast, syncytiotrophoblast and fibroblast are indicated with pink, purple and yellow, respectively. Right box indicates the location of cell on EM images of  panels C-F. Left box indicates the origin of EM images of panels G-I. B. Toluidineblue staining of the section of which EM images on panels C-F are derived. Arrow originates from the that is depicted on panels G-I. Bar scale represents 25LJm.
C.  Cytotrophoblast (on the left with nucleus) and syncytiotrophoblast (on the right). Bar scale represents 500nm.
D.  Enlarged image of boxed area of panel C. Cytoplasm of a syncytiotrophoblast cells with virus particles (red arrowheads). Bare scale represents 500 nm
E.  Cytoplasm of a cytotrophoblast cell with virus particles indicated by red arrowheads. Bar scale represents 500 nm
F.  A representative high power magnification image of a virus particle. Bar scale represents 100 nm. G. Low power image of the chorionic villi stroma area. Bar scale represents 2LJm.
H. Enlarged image of boxed area indicated on panel G. Cytoplasm of a fibroblast cell viral particles (red arrowheads). Bar scale represents 500 nm I. High-power image of boxed area of panel H. Red arrowhead indicates a virus particle. Bar scale represents 100 nm.
 

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Source:
Northwestern University

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