Researchers from all over the world are actively exploring SARS-CoV-2 virus and related COVID-19 disease. These studies are of particular importance for the cohort of pregnant women because of the urgency of the challenge; new information about COVID-19 may contribute to the future health of the next generation. To date, there are no reliable reports of increased obstetric disorder prevalence, as well as of possible perinatal SARS-CoV-2 transmission. Possible correlation between COVID-19 in pregnant/puerperant women and the development of fetal distress syndrome, as well as with thrombocytopenia and liver dysfunction in newborns has been shown [1, 2]. According to researchers from Zurich, COVID-19 causes systemic inflammation of lung vasculature (vasculitis) affecting multiple organs and systems; this has no apparent connection with pneumonia. Inflammation involves endothelium, the internal lining of various organs’ blood vessels [3]. Impaired placenta development during early pregnancy associated with systemic endothelial damage in patients with COVID-19 may result in oxidative stress in the placenta, and in a number of complications [4, 5]. Literature analysis shows that systemic inflammatory response syndrome (SIRS) in pregnant women with COVID-19 involves multiple pathogenetic mechanisms resulting in inflammation activation and immune response alteration. Understanding of SIRS pathophysiologal mechanisms involved in mother-placenta-fetus system in patients with COVID-19 promotes improvement of diagnostic methods and deciding pathogenetically based treatment for a number of obstetric complications [5, 6].

The study was aimed to reveal histological and immunohistochemical (IHC) features of placental tissue in pregnant women with COVID-19 at different stages of gestation, and to examine the contribution of these abnormalities to pathogenesis of the disease involving mother-placenta-fetus system.

METHODS

From March to May 2020 in the 1st Infectious Diseases Department of the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V. I. Kulakov the “red zone” was established for examination and treatment of patients with COVID-19, among them of pregnant women. A total of 190 beds for patients with COVID-19 and 60 beds for obstetric patients were prepared. Biological material samples (maternal venous blood, cord blood, amniotic fluid, placental tissue) were deposited in the certified biobank of the Center. The index group included pregnant women with verified COVID-19 diagnosis. The average age of the patients was 30.3 ± 6.25 years. The patients were enrolled once they were admitted. In order to attain the envisaged goals, clinical and laboratory investigation was performed, and pregnancy management was provided in 66 women with COVID-19 (placental tissue was assessed in 42 cases that ended in delivery, and in one fatal case), and 40 pregnant women of comparison group with no clinical manifestations and laboratory signs of viral infection.

Inclusion criteria: group I — COVID-19 confirmed by PCR test; group II — no COVID-19 based on physical examination data and PCR test. Exclusion criteria: multiple pregnancy.

Macroscopic and microscopic examination was carried out of 42 placental tissue samples obtained from puerperant women with COVID-19, and one dead patient, as well as of 40 placental tissue samples obtained from healthy puerperant women of the comparison group. Paraffin-embedded placental tissue blocks taken from women with uncomplicated pregnancy who delivered in 2017–2018 were used as additional comparison group and to exclude the suppressed disease. Histological assessment (hematoxylin and eosin staining) and IHC analysis of serial sections were performed. ICH analysis of 10% formaline (pH 7.4) fixed 4-µm paraffin sections was carried out using Ventana immunostainer (Roche; UK) with closed detection kit. The automated staining protocol included all stages of standard IHC staining. Ultra View Universal DAB Detection Kit (Ventana Medical Systems, Inc.; USA) was used for visualization. Primary monoclonal SARS-CoV-2 nucleoprotein antibody (N-protein (NP), clon 1518 (1:1000); Bialexa, Russia) was used for detection of viral particles. The control of NP antibody to SARS-CoV-2 expression assessment was the most important issue. Antibodies to SARS-CoV-2 were previously checked by immunoblotting techniques with the use of recombinant protein. Morphometry was used to evaluate the degree of the villous tree syncytiotrophoblast membrane damage. Assessment was carried out with NIS-Element AR3 image analysis system (Nikon; Czech Republic) for Nikon ECLIPSE 80i microscope. Statistical data processing was performed using the SPSS Statistics ver. 21 software package for Windows. 

RESULTS

Analysis of COVID-19 patients’ anamnestic and clinical characteristics yielded the following results: the average age of the patients was 30.3 ± 6.25 years, the average BMI value was 27.1 ± 4.6 kg/m², the average duration of the disease was 17.6 (6–34) days, and the length of hospital stay was 14.9 (4–30) days. Negative PCR test confirming the absence of SARSCoV-2 virus in the oropharyngeal mucosa was obtained within 15.6 (6–31) days (on average). The major clinical manifestations observed in patients were as follows: loss of smell (34.9%), hyperthermia (33.3%), and cough (51.5%). Asymptomatic COVID-19 was observed in 15 patients (22.7%). Patients with mild, moderate and severe disease accounted for 25 (38%), 20 (30.2%) and 6 (9.1%) cases respectively.

All pregnant women received low molecular weight heparin and interferon alpha-2b. Antimicrobial therapy included amoxicillin–clavulanic acid combination (46%) and macrolides (28%). Carbapenems were considered as the drug of choice in severe cases. Supplemental oxygen was required in six cases (9%), of those in four cases (6%) it was used in the intensive care unit, and in two cases (3%) it was used during artificial ventilation. Corticosteroids (dexamethasone 12 mg/day for 3–4 days) and immunoglobulin therapy (0.5 g/kg for 3–4 days) were also prescribed. One maternal death from pulmonary embolism and multiple organ dysfunction syndrome progression associated with severe hematological disease occurred on day 33 after admission. Spontaneous abortion occurred at 21–22 weeks of gestation. Labor and prolongation of pregnancy occurred in 42 (63.6%) and 20 (30.3%) cases respectively, and spontaneous abortions occurred in four cases (6.1%) out of 66. Deliveries were performed in 42 women, of those eight cases of preterm labor (19%), and 34 cases of labor at term (81%). Caesarean section was performed in 17 patients (40.5%), and vacuum extraction vaginal delivery due to fetal distress was performed in two patients (4.8%); normal vaginal delivery occurred in 23 patients (54.7%). The average birth weight was 3,283 ± 477 g, and the average birth length was 52 ± 2.75 cm.

The newborns were assigned Apgar score at one minute (7.8 ± 0.6) and five minutes (8.7 ± 0.5) of age. No cases of COVID-19 in newborn infants were registered. The infants were isolated from their mothers immediately after birth, until their mothers tested negative twice for SARS-CoV-2. After getting a negative PCR test result the women resumed breastfeeding. All newborn infants were tested for SARS-CoV-2 immediately after birth, as well as at three and 10 days of age. All newborns tested negative for the virus. This could indicate lack of vertical transmission of the infection. 

Placental tissue samples obtained from 42 patients who delivered in the red zone, one dead patient and 40 healthy women of the comparison group were sent to the Anatomical Pathology Department for further histological assessment and IHC analysis. Histological assessment revealed fewer than expected inflammatory conditions of placenta (umbilical cord, fetal membranes, and villous tree) in patients with COVID-19 (less than 10%). Focal forms of villitis prevailed (>3 affected fields of view out of 10 viewed at 200x magnification), which could be detected only when using IHC staining with antibodies to CD4 (marker of lymphocytes and macrophages) (fig. 1А–F and fig. 2А–D). In-depth analysis of complex placental changes in the index group patients revealed significant differences (р < 0.05), such as predominance of terminal villi hypercapillarization (moderate and severe degree) or pathological immaturity of the villous tree (see fig. 1А–F). In patients with COVID-19, histological features of the villous tree corresponded to moderate and severe branching angiogenesis (angiogenesis with predominant vascular branching) (score 23 and 32, scales by Berniske, Kaufman, 2006). This indicated the compensatory mechanisms involvement and was more often characteristic of intrauterine hypoxia. The placental villous infarction rate was significantly higher in patients with novel coronavirus infection; it was mainly associated with severe forms of the disease (р < 0.05) (see fig. 1А–F). The rate of intravillous hemorrhage and blood clots in the lumen of blood vessels showed the upward trend (see fig. 1А–F). In the placenta of the woman with leukemia (the fatal case), up to 90% of the villous tree was affected, surrounded by infarcted fields, multiple hemorrhages and hematomas (subamniotic, intervillous, retrochorial), and massive fibrinoid deposition making it possible to distinguish only a few villi, some of which were dystrophic (see fig. 1А–F). At the same time, in patients with COVID-19 who gave birth to healthy babies, the analysis of placental villous tree and the degree of villous syncytiotrophoblast damage revealed intact syncytiotrophoblast membranes, and no cells sloughed off the surface of the villi. In our opinion, the key consideration is the fact that in women with COVID-19 the trophoblast damage degree was less than 10%, i. е. it could be considered the normal value [7]; when entering the mother’s bloodstream, the destroyed trophoblast compartments did not trigger maternal systemic inflammatory response observed in preeclampsia. During evaluation of IHC study results with the use of primary antibody to SARS-CoV-2 N-protein, lack of staining in most placental villi in women with mild forms of the disease drew the attention. In women with moderate and severe infection, the following features were detected: focal staining of individual syncytiotrophoblast villi membrane and cytoplasm, syncytial knots, small areas of the basement membrane, endothelium of individual villous vessels, and macrophages (fig. 3А–F). No viral particles were detected in fetal membranes and umbilical cord. On the contrary, in patient with leukemia (the fatal case), individual villi positively stained with antibody to virus could be distinguished among the infracted fields and hemorrhages (fig. 3; dark areas). Positively stained areas matched the villous syncytiotrophoblast contour; some villi with evident dystrophic changes were embedded in fibrinoid deposition (see fig. 3А–F). Thus, the function of fibrinoid produced primarily of coagulated plasma proteins in the sites of villous tree damage is to separate maternal and fetal blood circulation in case of placental damage; when the viral particles enter the intervillous space, it seeks to localize and inactivate viral particles in order to prevent them from entering the fetal bloodstream. There were no signs of vasculitis and perivasculitis in blood vessels of patients with COVID-19 who gave birth to healthy babies, which was in contradiction to data on lung injury and damage to other organs resulting from granulomatous inflammation reported by a number of authors [8, 9]. In the index group, IHC staining with primary antibody to TNFα revealed moderate staining of cytotrophoblast and syncytiotrophoblast membrane and cytoplasm, macrophage and lymphocyte cytoplasm, epithelium, as well as syncytial knots, functioning as the zones of trophiblast proliferation, extravillous cytotrophoblast and decidual cells. Prominent staining was observed in the perivillous fibrinoid (fig. 4А–D). This demonstrated elevated plasma levels of proinflammatory markers compared to placenta. Assessment of IL8 revealed more prominent staining in the listed above structures, primarily in syncytiotrophoblast (fig. 5А–F). As is known, syncytiotrophoblast forms the placental barrier, which functions as the main barrier for the virus. Downregulation of proinflammatory cytokine IL4 expression was observed in placenta of the index group patients with СOVID-19 (fig. 6А–D). 

DISCUSSION

Thus, elevated proinflammatory cytokine synthesis and reduced levels of anti-inflammatory cytokines are observed in patients with novel coronavirus infection. The most prominent staining was detected in fibrinoid deposits. It should be noted that not only immune cells, but also placental structures are able to produce proinflammatory and anti-inflammatory cytokines, even when binding the innate immune receptors. Of particular note is the fact that even in case of 5–10 times increase in plasma levels of proinflammatory markers (cytokine storm), the placental levels of those were comparable with values characteristic of uncomplicated pregnancy. At the same time, upregulation of proinflammatory factors (TNFα, IL8) expression and downregulation of anti-inflammatory factors (IL4) were observed in placental structures of patients with moderate and severe СOVID-19.

An important reason for impairment of umbilical blood flow is blood clot and sludge formation in the umbilical cord and chorionic blood vessels, also referred to as fetal thrombotic vasculopathy [10, 11]. Hypercapillarization of terminal villi is a compensatory mechanism for hypoxia, which is attributable to elevated level of angiogenic factors promoting growth of new blood vessels, such as VEGF. In case of prominent terminal villi hypercapillarization (32–33 points in accordance with the scale) [12] rearrangement occurs during angiogenesis resulting in slowing down in microcirculation and fetoplacental blood flow. Therefore, platelet aggregation in the capillary lumen increases, and microthrombi are formed. Upregulation of these processes results in focal villous stromal fibrosis; changes initially focused on hypoxia negative impact compensation facilitate gradual transition to decompensation. In our study, the frequency of moderate and severe terminal membrane hypercapillarization in the index group was significantly higher than in comparison group. The study results showed higher proportion of infarctions, as well as the presence of chorionic vessels thrombosis, and intervillous and subamniotic hematomas in the index group compared to controls. In addition to mechanical factors, blood clot formation is caused by violation of blood rheological properties (slowdown in blood flow, decreased diameter of blood vessels), and coagulation system disorders. These factors are characteristic of novel coronavirus infection. Morphological features of placenta suggested that acute fetal hypoxia during labor occurred in cases of chronic fetal hypoxia in the mother-placenta-fetus system or any other disturbing factor influence during pregnancy [13, 14]. As a result, the villous tree rearrangement occurred, and the subclinical placental insufficiency developed. The influence of additional triggers during labor (uterine contractile activity, umbilical cord compression, etc.) resulted in decompensation with clinical manifestations of acute fetal hypoxia.

Evaluation of IHC analysis results revealed no significant increase in the proinflammatory marker levels against the background of decreased inti-inflammatory marker levels in patients with moderate and severe infection. 

CONCLUSION

In the recent decade, advances in immunology and broader understanding of innate and acquired immunity mechanism resulted in growing interest in studying the individual differences of immune response associated with infectious inflammatory diseases. Studying the immune response differences in individuals with predominant Th1 or Th2 response, which largely determines the clinical, morphological and immunological features of the inflammatory response, is the most productive. Thus, pregnancy may be considered as a factor contributing to more favorable course of COVID-19. The reported higher proportion of hypoxia during labor may be associated with coagulation system disorders and result from higher proportion of chorionic blood vessel thromboses, as well as from intervillous, subamniotic, retrochorial hematomas, and villous tree infarctions. Placenta functions as an “airbag” for the fetus, and the intact placental barrier protects the fetus against COVID-19. However, under certain conditions, in case of “broken” placental barrier the novel coronavirus infection may be dangerous for the fetus: the infection can induce the cytokine-storm-type changes. Placenta minimizes the burden on the fetus. The vast majority of babies are born healthy, and the placental levels of proinflammatory cytokines are comparable with those in comparison group. Intact placenta and placental barrier protect the unborn baby against infections, and the impact on the child’s development is minimal. Taking into account higher proportion of hypoxia occurring during labor in infants born from mothers with COVID-19, the obstetrical tactics for mothers with COVID-19 should be decided individually based on the risk factors; continuous cardiotocography should be used during labor. It may be appropriate to conduct IHC analysis of placenta in puerperant women with suspected novel coronavirus infection COVID-19 using primary antibodies to Sars-CoV-2 in order to fine-tune the tactics of neonatal management and to predict possible neonatal complications.