This study retrospectively analyzed the clinical characteristics of 8 cases of COVID-19 pregnant women in third trimester. The limited data showed that the clinical manifestations of SARS-CoV-2 infected pregnant women were basically similar to those of the general infected population, and there were no serious adverse mother-infant outcomes. As far as we know, for the first time, we performed microscopic observations and immunohistochemical tests at the same time to determine whether the number of inflammatory cells and fetal-derived placental macrophages (Hofbauer cells) in the placenta from pregnant women with COVID-19 has increased. No specific inflammatory pathological changes suggesting SARS-CoV-2 invasion of the placenta were present through the microscopic observation and IHC detection. FISH detection of SARS-CoV-2 RNA in placental tissues and RT-PCR detection of neonatal pharyngeal swabs in all cases were negative. This study suggested no definite evidence pointing to maternal-fetal vertical transmission in pregnant women with COVID-19 in late pregnancy, and provided important clues for further understanding of the clinical characteristics, pregnancy outcomes, and evaluation of intrauterine transmission of SARS-CoV-2 infection in late pregnancy.
The main histopathological features of placenta viral infection showed significant fetal origin inflammatory abnormalities, such as chronic villitis, intervillositis, and funisitis, which occurred in some TORCH agents17, such as cytomegalovirus, Treponema pallidum, Toxoplasma, rubella virus infection and other hematogenously transmitted infections through the placenta18. Fortunately, in our study, no specific pathological changes of inflammatory reactions and no evidence of worse maternal disease were present. Although individual cases showed corresponding inflammation changes in the placental tissue, such as maternal inflammatory response (mild), chronic intervillositis and chronic plasma cell deciduitis, they were not universal. This phenomenon was consistent with the results of the existing limited studies 19, 20. In addition, we were the first to perform IHC analysis on inflammatory cells, especially Hofbauer cells, in late placenta tissues from COVID-19 pregnant women. As Hofbauer cells can harbor live virus such as ZIKA virus13, HIV virus21 and Cytomegalo virus22, and serve as reservoirs within the placenta, it is one of the important ways to transmit pathogens to fetal-placenta tissues by infecting Hofbauer cells23,24. However, H&E staining and IHC showed no significant infiltration of T cell or evidence of villous stomal macrophages hyperplasia. FISH analysis further enhanced the evidence that no virus directly infected the placenta, which was similar to the results of previous limited studies19, 25.
On the other hand, the stage of gestation at the time of infection may affect whether SARS-CoV-2 virus was vertical transmission. Stage of gestation has been proved as an important factor affecting the mechanisms of maternal-fetal vertical transmission 26. For example, in the early infection of rubella virus, more than 50% of fetuses were infected vertically through the uterus, but as the pregnancy time increases, the risk of vertical transmission was significantly reduced27. The phenomenon was also present in the ZIKA virus. Since higher ZIKA virus titers were detected in amniotic epithelial cells from mid-gestation, suggesting a greater susceptibility of virus infection in the placenta from the second trimester or earlier compared to late-gestation placentas10. As with previous studies19, 28, 29, our study mainly included pregnancy women with infection in the third trimester and found no no evidence of vertical transmission, further suggesting that the placenta may play a greater and powerful barrier role to prevent SARS–CoV2 infection in the third trimester, and the specific resistance mechanism still needs to be further studied.
Although the defense mechanism of placenta to restrict microorganisms from entering the fetus is largely unclear, existing evidence suggested that syncytiotrophoblasts can effectively resist numerous pathogens, and CTB also has an innate defense mechanism against intracellular pathogens10. Impressively, the syncytiotrophoblast layer has strong resistance to various viruses such as HCMV, HSV1, and ZIKA30–32 in the late pregnancy. For example, trophoblasts are sensitive to ZIKA virus at the earliest stage of trophoblast development, but become more and more resistant when the syncytium forms in late pregnancy33. So whether trophoblasts play a part in the mechanism of placental resistance of the SARS–CoV2 virus in late pregnancy will be the direction of our further research.
Notably, another most striking observation in the placentas (all 8 cases) was the prominent and diffuse increase of syncytial knots, which was one of the features of MVM. As first described by Tenney and Parker 34, syncytial knots were the aggregations of syncytiotrophoblast nuclei, and their increase may involve nearly all terminal villi in preeclampsia, whereas they were only appeared in 10%-15% normal terminal villi 35. Moreover, exposure of the placenta to conditions such as hypoxia, hyperoxemia, or oxidative stress may cause an increase in syncytial knots 36. And our results were consistent with the existing evidence on the pathology of placentas with coronavirus infection, which exhibited a few abnormalities about MVM19, 37, such as increased syncytial knots, different degrees of fibrin deposition in intervillous and subchorion, which could also be observed in this study. Given that all cases collected in this study were asymptomatic or with mild syndrome, so the results suggested that mild symptoms of SARS-CoV-2 infection might induce the decline in oxygenation within the intervillous space and cause a degree of placental injury, although there was no clear evidence of SARS-CoV-2 infection of the placenta in the third trimester. This is of great significance to the safety of mothers and fetuses in late pregnancy.
Consistent with a recent case report11, FISH was performed to detect SARS-CoV-2 RNA in the placenta, and no evidence of SARS-CoV-2 invasion in the late gestation placenta was present. None have demonstrated the presence of the SARS-CoV-2 virus by RT-PCR from existing limited studies in the placenta tissue 19. Although the recent case report suggested the presence of SARS-CoV-2 in 3/11 swabs of the placenta or membrane by RT-PCR 38, swab samples rather than tissue samples of the placenta or membranes might increase the possibility of virus droplet contamination in the hospital environment or virus exposure during delivery, so they could not be used as direct evidence of vertical transmission. Compared with RT-PCR, FISH analysis directly used tissue samples for detection, which displayed the precise cell location of fusion genes and relevant information on the anatomical distribution of the placenta23, and helped to provide clues for exploring the mechanism of placental virus infection or defense. Above all, it can be seen that FISH is practicable and can provide more information to diagnosis SARS-CoV-2 invasion of the placenta.
This study still has some limitations. First of all, the cases collected in this study were all mild patients, and it was still unknown whether patients with severe infections in pregnancy will develop intrauterine infection, which is the direction for further research in later research. Secondly, a recent report suggested that positive SARS-CoV-2 infection in the second trimester pregnancy women can lead to miscarriage, and the evidence of SARS-CoV-2 infection in the placenta had aslo been found 11. So further cases including different gestation stage women of COVID-19, especially in the first and second trimester, need to be collected to study the effect to maternal and fetus safety.
In summary, we found no evidence of vertical transmission in the third trimester placenta of COVID-19 pregnancy women by observing histological changes and nucleic acid test, we also analyzed whether the number of the inflammatory cells and macrophages cells increased by immunohistochemistry. Although the sample size of this study was limited, considering the important adverse effects of this ongoing global public health emergency, our results were very useful for understanding the clinical characteristics of COVID-19 infection in late-stage pregnant women and whether it has the potential for vertical transmission. It was important and provided a certain basis for the best clinical management of late pregnant women.