A 33-year-old black woman at 34 weeks and 4 days of gestation was admitted on October 15, 2020, with a diagnosis of fetal death 14 days after a positive RT-PCR for SARS-CoV-2. The patient (gravida 2, para 1) was 1.80 m tall, weighed 87.7 kg (body mass index (BMI) 27.1 kg/m2), and had a history of previous bariatric surgery (Roux-en-Y gastric bypass in 2012). She started her prenatal care in the 9th week of pregnancy. She regularly used omeprazole to treat gastritis, multivitamins, ferrous sulfate, folic acid, and vitamin D. She denied smoking, alcoholism, or the use of illicit drugs. The cardiovascular exam showed a regular rhythm, 79 bpm, without cardiac murmur and blood pressure level 120 x 70 mmHg. The respiratory clinical examination and laboratory tests were normal, including serology for HIV, toxoplasmosis, and syphilis. The vaccination history showed a complete schedule for tetanus, hepatitis B, and influenza. Morphological ultrasound at 21 weeks and 1 day of gestation, performed on July 13, 2020, was normal, as well as Doppler ultrasonography performed at 28 weeks and 1 day.
Two weeks before admission (October 1, 2020), she complained of flu-like symptoms at a routine prenatal visit, which started on September 25, 2020, when she had myalgia, runny nose, headache, and fatigue. She denied fever and contact with a suspected or confirmed case of COVID-19. The physical examination showed a stable general condition, respiratory rate 25/min, without dyspnea, oxygen saturation of 99% on room air, heart rate 112 bpm, and without fever or other significant findings. Ultrasonography estimated fetal weight at 2,156 g (64.6% percentile), cephalic presentation, 146 bpm fetal heart rate, normal amniotic fluid volume, and elevated posterior grade II placenta. Doppler evaluation was normal, with umbilical artery pulsatility index (PI) 0.60, middle cerebral artery PI 1.66, and cerebroplacental ratio 2.77. The rapid serological tests for SARS-CoV-2 (IgM and IgG, colloidal gold immunochromatography test, Beijing LEPU-Medical Technology, Beijing, China) were negative, but the qualitative RT-PCR on naso- and oropharyngeal swabs [Bio Manguinhos Molecular Kit SARS-CoV-2 [E/RP], Charite Berlin Protocol ] was positive (result released on October 3, 2020). The patient was instructed to remain in isolation at home for 14 days and return to the emergency room in case of signs or symptoms of worsening.
On October 15, 2020, she returned to prenatal care without symptoms of COVID-19 for the previous 8 days. She reported that on October 4, she had a temperature of 37.2 C and maintained flu-like symptoms until October 8, although afebrile. On physical examination, fetal heartbeats were not detected, and the diagnosis of fetal death was confirmed by ultrasound examination, which estimated gestational age at 34 weeks and 4 days. On that occasion, a new rapid serological test (same manufacturer) was positive for IgM and IgG anti-SARS-CoV-2. The induction of delivery was scheduled for the following day. The patient was then submitted to an MRI for better evaluation of fetal and placental morphology and collected new laboratory tests, including serology for SARS-CoV-2 and TORCH (toxoplasmosis, rubella, cytomegalovirus (CMV), herpes simples, parvovirus, Zika (ZIKV), and chikungunya viruses), in addition to blood biochemistry and dosage of inflammatory biomarkers (Table 1). On October 16, she underwent an elective cesarean delivery because she declined to induce labor. The stillbirth weighed 2,145 g, and the amniotic bag was ruptured during the operation, giving rise to a brownish-looking liquid.
Four months later, a complete screening for thrombophilia was performed. The results showed a single alteration of a heterozygous pattern for Leiden Factor V. A timeline of the case evolution is presented in Supplementary Figure 1.
The patient felt the loss of her daughter very much but felt that studying her case could help other families. She recently received the first dose of the anti-COVID-19 vaccine (AstraZeneca), and a few weeks later, she learned that she was pregnant again. Now, she is 7 weeks pregnant (June 2021) and very hopeful for her new opportunity.
An anatomical and advanced MRI protocol with multiple sequences was performed in a 3.0 Tesla MRI scanner (Magnetom Prisma, Siemens Healthcare, Erlangen, Germany), using two 18-channel abdominal coils for detailed analyses of the placenta and fetal organs in the three orthogonal planes. The complete protocol used is described in the Supplementary Material. The main findings are presented in more detail in Figure 1A and Supplementary Figure 2 and consisted mainly of a substantial placental impairment (appearance of a collapsed placenta with no viability). The placenta had a posterior uterine wall insertion, ill-defined cotyledons, fewer lobulations than usual, and large and dilated vessels with massive thrombosis. The fetus presented accumulation of fluid in the pericardium, and pleural and peritoneal space; alterations in the heart, liver, and spleen images; and signs of diffuse cerebral edema.
The placenta weighted 470 g (80th percentile for gestational age) and had a paramarginally inserted three-vessel umbilical cord with a reddish color, and the chorionic plate vessels were highly dilated and congested and had magistral distribution; the fetal surface was reddish, and the membranes were hypotransparent (Figure 1B, left and middle). The maternal surface was compact, wrinkled, and yellow-tan with patchy hemorrhages, resembling maternal floor infarction and poorly delimited and frayed lobes. Cut sections demonstrated extensive whitish irregular deposits, areas of intervillous and subchorionic thrombi, and recent infarcts. Histologically, it showed massive diffuse inflammatory alterations in regions with a predominance of acute and chronic intervillositis (Supplementary Figures 3F,G) and intervillous neutrophilic microabscesses associated with chronic and proliferative villitis and acute deciduitis (Figure 1B, right; and Supplementary Figure 3F). There were also signs of severe maternal vascular malperfusion represented by extensive and diffuse intervillous/perivillous fibrin deposition with areas of intervillous laminated thrombi and a large subchorionic recent thrombus at the umbilical insertion (Supplementary Figure 3C). Areas of recent infarct characterized by collapsed and necrotic villi with partial or total loss of trophoblast layer (Supplementary Figure 3H) and hypovascular or avascular stroma were observed. The decidua showed focal laminar necrosis and some vessels with fibrinoid necrosis and thrombi. Signs of acute fetal vascular malperfusion, including ectasia and recent thrombi, were noted in large vessels from the chorionic plate and stem villous vessels (Supplementary Figure 3C). There were also stem villous vessels with subintimal fibrin deposition and occlusive thrombus with recanalization (Supplementary Figure 3D) and few villi with stromal-vascular karyorrhexis (Supplementary Figure 3E). Occasional groups of preserved chorionic villi showed delayed villous maturation.
The organs were fixed in 10% buffered formalin, and fragments of all sampled tissues were processed for paraffin embedding and were stained with hematoxylin and eosin. The skin was detached from the surface of the body due to postmortem autolysis compatible with the estimated time of fetal death (>48 h) (Supplementary Figure 4). All organs of the thoracic and abdominal cavities were congested and soft due to postmortem autolysis (Supplementary Figure 5). At removal, the brain weighed 280 g. The gyri were wide, congested, and extremely soft due to autolysis (Supplementary Figure 6). Histologically, the brain and spinal cord showed no signs of inflammation or necrosis. The meninges and choroid plexus were immunostained for T lymphocytes, which was negative. The olfactory bulb had occasional T lymphocytes. The pituitary gland presented small foci of calcification. The vagus nerve and dorsal root ganglia were normal, as were the muscles (psoas, diaphragm, and abdominal). Histology of the lungs showed mild thickening of the pleura and alveolar septa, with mild infiltration of lymphocytes (Figure 1C, left) without signs of fibrosis as seen with Gomori's staining. Immunohistochemistry for leukocyte common antigen (LCA) showed small aggregates of lymphocytes in pleura and pulmonary septa. They were CD3 T lymphocytes (Figure 1C, middle) and can be compared with a negative control lung of a patient without COVID-19 (Figure 1C, right). In the kidneys, occasional interstitial lymphocytes immunostained with LCA were seen. Most of the other organs showed signs of maceration, particularly the liver, adrenal gland, spleen, pancreas, and intestines.
Freshly collected samples from the fetal and maternal faces of the placenta, umbilical cord, heart, lungs, liver, kidney, intestines, cerebellum, olfactory bulb, and brain cortex were properly stored in RNA stabilization solution and later processed for viral RNA extraction and identification by RT-PCR. The complete experimental protocol, positive and negative controls, and standards for result interpretation are described in the Supplementary Material. Results of the RT-PCR assay were considered positive if either N1 or N2 SARS-CoV-2 nucleocapsid fragments had a cycle threshold of <40. The standard curve and data analysis were prepared as described previously. The mean and standard deviations of absolute quantification (number of copies/reactions) of N1 and N2, and the mean and standard deviations of crossing points (Cp) values of Hs_RPP30 (Homo sapiens Ribonuclease P protein subunit p30) were calculated from data obtained in all analyzed distinct fragments from the same postmortem tissue.
SARS-CoV-2 was detected in high amounts in the placenta. These amounts were also at least an order of magnitude higher than the average for nasopharyngeal swabs (positive control). Additionally, the new coronavirus was detected by qRT-PCR in the umbilical cord and the following fetal tissues: the salivary gland, trachea, olfactory bulb, lungs, liver, and kidney, but not in the heart or brain (Table 2). To confirm the presence of the virus in fetal tissues, sections of the lung, heart, and brain were immunostained against spike protein. As a result, positive immunolabeling was identified in the fetus's lung, brain, and heart and was absent in the immunofluorescence negative lung control of a non-COVID case (Figure 2).