key: cord-0905294-9183n7hc authors: Wu, Hao; Liao, Shujie; Wang, Yiming; Guo, Ming; Lin, Xingguang; Wu, Jianli; Wang, Renjie; Lv, Dan; Wu, Di; He, Mengzhou; Hu, Bai; Long, Rui; Peng, Jing; Yang, Hui; Yin, Heng; Wang, Xin; Huang, Zhixiang; Lan, Ke; Zhou, Yanbin; Zhang, Wei; Xiao, Zhenyu; Zhao, Yun; Deng, Dongrui; Wang, Hongmei title: Molecular evidence suggesting the persistence of residual SARS‐CoV‐2 and immune responses in the placentas of pregnant patients recovered from COVID‐19 date: 2021-07-22 journal: Cell Prolif DOI: 10.1111/cpr.13091 sha: ab271da3dc85c4744e607926ee9749d5e69e9519 doc_id: 905294 cord_uid: 9183n7hc OBJECTIVES: Recent studies have shown the presence of SARS‐CoV‐2 in the tissues of clinically recovered patients and persistent immune symptoms in discharged patients for up to several months. Pregnant patients were shown to be a high‐risk group for COVID‐19. Based on these findings, we assessed SARS‐CoV‐2 nucleic acid and protein retention in the placentas of pregnant women who had fully recovered from COVID‐19 and cytokine fluctuations in maternal and foetal tissues. MATERIALS AND METHODS: Remnant SARS‐CoV‐2 in the term placenta was detected using nucleic acid amplification and immunohistochemical staining of the SARS‐CoV‐2 protein. The infiltration of CD14+ macrophages into the placental villi was detected by immunostaining. The cytokines in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens at delivery were profiled using the Luminex assay. RESULTS: Residual SARS‐CoV‐2 nucleic acid and protein were detected in the term placentas of recovered pregnant women. The infiltration of CD14+ macrophages into the placental villi of the recovered pregnant women was higher than that in the controls. Furthermore, the cytokine levels in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens fluctuated significantly. CONCLUSIONS: Our study showed that SARS‐CoV‐2 nucleic acid (in one patient) and protein (in five patients) were present in the placentas of clinically recovered pregnant patients for more than 3 months after diagnosis. The immune responses induced by the virus may lead to prolonged and persistent symptoms in the maternal plasma, placenta, umbilical cord, cord blood and amniotic fluid. As of April 19, 2021, more than 146 million individuals have been diagnosed with coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Data from WHO). The major manifestations of COVID-19 are observed in the respiratory organs. [1] [2] [3] [4] Patients may also show gastrointestinal and neurological symptoms and myocardial dysfunction. [5] [6] [7] [8] [9] Most patients develop unilateral or bilateral pneumonia, which is diagnosed by radiological examination. 10 In patients with severe COVID-19, the disease can rapidly progress into acute respiratory distress syndrome (ARDS), severe sepsis with shock or multiple organ failure within 1 week due to the onset of a 'cytokine storm'. 11 SARS-CoV-2 is primarily transmitted through the respiratory tract and infects airway epithelial cells, vascular endothelial cells and macrophages. 12, 13 The cellular entry of SARS-CoV-2 is mediated by the spike (S) protein. The binding of the S protein to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) exposes a cleavage site on the S protein. Transmembrane protease serine 2 recognizes this cleavage site and proteolytically cleaves the S protein to initiate fusion and endocytosis. 14, 15 Cells in various human tissues, including the small intestine, testes, kidney, heart, thyroid, adipose and placenta, show high ACE2 expression, whereas the lung cells show moderate expression. 16, 17 Consistently, autopsies in cases of severe COVID-19 have shown that in addition to the lungs, the virus infects various tissues, including the heart, kidneys and liver, among others. 4 These findings provide evidence of the systemic spread of SARS-CoV-2 in the body during infection. Following infection, an immune response is typically induced against the pathogen, and patients with severe COVID-19 may suffer from lymphocytopenia and macrophage activation syndrome. 18, 19 Moreover, there are reports of increased secretion of a series of cytokines and chemokines in the plasma, including interleukin (IL)-2, IL-7, IL-10, granulocyte colony-stimulating factor, interferon (IFN)-gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1 alpha and tumour necrosis factor-alpha (TNFα). The plasma cytokine profile of patients was shown to be associated with COVID-19 severity. [20] [21] [22] Pregnant women are a high-risk population for severe and exhibit high mortality owing to their unique immune status. 21, 23 Maternal inflammatory responses at the maternal-foetal interface, mediated through macrophages and T cells, are induced after SARS-CoV-2 infection, and these responses could persist for as long as 3 months after COVID-19 recovery. 4, [24] [25] [26] During pregnancy, the placenta acts as a transient endocrine organ that supports foetal growth by extracting nutrients from the maternal blood and serving as a barrier against pathogens or mediators of the maternal immune system. The placental villus is a functional unit of the placenta, composed of a layer of mononucleated cytotrophoblast cells and an outer multinucleated syncytiotrophoblast. 27 Within the placental villi, Hofbauer cells (placental macrophages), placental fibroblasts and foetal endothelial cells are located adjacent to the foetal capillaries. The presence of Hofbauer cells is important for a successful pregnancy, as it regulates placental morphogenesis and the immune system. 28 Although placental SARS-CoV-2 infection may damage the syncytiotrophoblast and disrupt the placental barrier, 29 the presence of SARS-CoV-2 in the placenta post-recovery and the chances of its vertical transmission are debatable, and the impact of SARS-CoV-2 on the placenta and foetus should be investigated further. In this study, we used the placentas, maternal plasma, neonatal umbilical cords, cord blood and amniotic fluid donated at delivery by pregnant patients who had completely recovered from COVID-19 and confirmed the presence of residual SARS-CoV-2 nucleic acid and protein in the placentas of patients with COVID-19 long after initial diagnosis and complete recovery. The longest interval between diagnosis and sampling was approximately 3 months, and we believe that the immune response induced by SARS-CoV-2 may persist even longer. The biological specimens used in this study were collected from 11 pregnant women who had recovered from COVID- 19 Results: Residual SARS-CoV-2 nucleic acid and protein were detected in the term placentas of recovered pregnant women. The infiltration of CD14+ macrophages into the placental villi of the recovered pregnant women was higher than that in the controls. Furthermore, the cytokine levels in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens fluctuated significantly. Our study showed that SARS-CoV-2 nucleic acid (in one patient) and protein (in five patients) were present in the placentas of clinically recovered pregnant patients for more than 3 months after diagnosis. The immune responses induced by the virus may lead to prolonged and persistent symptoms in the maternal plasma, placenta, umbilical cord, cord blood and amniotic fluid. by a designated diagnostic laboratory in accordance with the novel coronavirus pneumonia diagnosis and treatment program (5th edition). Among the women, four (#30, #32, #47 and #49) had an asymptomatic infections. At the time of delivery, the patients met the requirements for clinical discharge or release from quarantine, with approval provided by clinicians. However, owing to the limited information about the pathogen and the lack of sensitive testing methods at the onset of suspected symptoms, the time point of initial infection could not be determined in some patients. All the patients were treated in isolation. The diagnosis was based on SARS-CoV-2 nucleic acid detection from throat swabs, serum antibody testing and chest CT imaging before delivery (Table S2 ). Four patients (#14, #20, #46 and #49) underwent The study was approved by the Research Ethics Committee (refer- RNA extracted from maternal throat swabs, placental villi (10 mg) and neonatal throat swabs were used to test for SARS-CoV-2 S protein using qRT-PCR. The RNA titre was measured using a fluorescent probe targeting the S protein RNA. The sequences of the probes were as follows: CoV-F (5'-TCCTGGTGATTCTTCTTCAGGT-3'), CoV-R ELISA was used to detect SARS-CoV-2-specific IgG and IgM in maternal serum, neonatal serum, umbilical cord, cord blood and amniotic fluid specimens, as instructed. 31 In total, maternal plasma (collected at delivery) from 11 patients, neonatal serum from seven patients (#26, #27, #32, #35, #46, #47 and #49), umbilical cords from five patients (#26, #27, #46, #47 and #49) and amniotic fluid from three patients (#26, #42 and #47) were tested for SARS-CoV-2-specific antibodies. Immunohistochemical staining was performed as described by Fu et al 32 To characterize the cytokine profiles of maternal plasma, placenta, neonatal umbilical cord, cord blood and amniotic fluid specimens, The mean cytokine level in uninfected controls was considered the baseline and the cytokine levels in the specimens were expressed relative to the baseline level and termed "relative secretion levels". For negative controls, we used maternal plasma, placenta, umbilical cord, cord blood and amniotic fluid donated by three, four, two, two and three uninfected pregnant women at delivery respectively. Results are expressed in terms of means ± standard error of the mean. Statistical analysis was performed using a paired-sample t-test with Statistical Package for Social Science (SPSS; SPSS Inc., Chicago, IL, USA). Significance was defined as follows: *, P <.05; **, P <.01; ***, P <.001. The clinical details of the pregnant patients with COVID-19 and their fetuses have been summarized in Tables 1 and 2. The pregnant women had been discharged from the hospital before delivery, and routine prenatal examinations were performed after the completion of the quarantine period (Tables S1 and S2 ). After recovery, the women delivered 11 infants (one set of monochorionic diamniotic twins). Five patients underwent caesarean deliveries, five patients had natural childbirth and one patient experienced foetal malformation-induced labour. The twins were delivered preterm at 36 +4 W, whereas the other infants were full-term newborns (Table S3 ). Subsequent assessments were based on the biological samples obtained. To explore the existence of SARS-CoV-2 in the placenta of clinically recovered patients, we first performed qRT-PCR on isolated total RNAs of the placentas from six patients (#26, #30, #42, #46, #47 and #49). As shown in Figure 1A , the placenta from patient #46 After SARS-CoV-2 infection, the immune response in the patient is activated, and the infiltration of macrophages and lymphocytes into the infected locus is a common mechanism for virus clearance. 18, 35 To clarify the nature of the immune response that might be acti- The infiltration of excess CD14+ macrophages in the placenta may indicate innate immune activation. As shown in Figure 3A and 3B, To determine whether placental SARS-CoV-2 infection affected the pregnancy outcome and led to the intrauterine infection of the foetus, we further investigated the foetal and accessory abnormalities at delivery. Of all newborns, the monochorionic diamniotic twins weighed 2500 g and 2100 g, whereas the other full-term infants had body weights ranging from 2810 g to 3900 g. All infants received 1-minute and 5-minute Apgar scores of 8 -10 (Table S3) . 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