INTRODUCTION
In December 2019, a cluster of four cases of pneumonia of unknown etiology in Wuhan, China, were reported to the World Health Organization (WHO).[1] Since then, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly across the world. As pregnant women are at greater risk of complications and severe disease from infection with other coronaviruses, including severe acute respiratory syndrome (SARS) and Middle Eastern Respiratory Syndrome (MERS), they were identified as a vulnerable group and were advised to take additional precautions as the COVID-19 pandemic unfolded.[2,3]
To reduce transmission risks for both pregnant women and health care workers, the International Federation of Gynecology and Obstetrics (FIGO) recommended the suspension of the much routine antenatal care and replacement with video or telephone consultations whenever possible.[4,5]
It is unclear whether and how SARS-CoV-2 can be transmitted from the mother to the fetus. Some cases of perinatal transmission have been described; however, it is unclear if these occurred via the transplacental or the transcervical route or through environmental exposure.
The impact of maternal SARS-CoV-2 infection on placental histopathology is not well known. Histopathologic examination of placental tissue can contribute significant information regarding the health of both mother and fetus. Viral infections in pregnancy are associated with specific placental findings such as lymphoplasmacytic villitis with associated enlargement of villi and intravillous hemosiderin deposition in case of maternal cytomegalovirus infection and intervillositis in Zika virus and Dengue virus.[6]
It is important to clarify whether and how SARS-CoV-2 reaches the fetus to prevent neonatal infection, optimize pregnancy management, and eventually better understand SARS-CoV-2 biology. We postulate a comprehensive study to determine any significant histopathological change occurring in the placenta of women with SARS- COV-2 infection in pregnancy.
Aim
The aim of the study was to determine if significant placental histopathological changes occurred after the diagnosis of SARS-CoV-2 infection in pregnancy.
MATERIALS AND METHODS
This was a cross-sectional study reviewed and approved by the ethics committee (Research Review Board; Ethical number: IEC/GDMC/2021/107) of Government Doon Medical College and Hospital, Dehradun, India, carried out at the Department of Pathology, Government Doon Medical College and Hospital. In this study, placenta samples from 50 pregnant women were taken that were confirmed with COVID-19 infection by reverse transcriptase-polymerase chain reaction (RT-PCR) from August 2020 to October 2020. Fifty non-infected women without evidence of inflammatory infiltrates were selected from previous records and included as controls, matched by age and body mass index. The samples were collected during routine cesarean section/normal delivery after full informed consent prior to delivery by the Department of Obstetrics and Gynecology.
Precautions taken during the transfer of specimen from the department of obstetrics and gynecology to department of pathology as per the WHO guidelines
Placenta specimens were sent in sealed double leak-proof specimen containers. The specimens were completely immersed in 10% buffered formalin.[7] All specimens as well as histopathology requisition forms were labeled properly with patient details and clearly marked in red regarding the positive status of COVID-19. Requisition forms were ensured to be free from contamination with blood, bodily fluids, or any infective material (mostly were filled by pathology residents through telephonic communication with the gynecology residents) All specimens were processed after 48 h of formalin fixation according to the World Health Organization (WHO) guidelines. All personnel handling the clinical specimen followed standard Centers for Disease Control and Prevention (CDC) precautions and guidelines including hand hygiene and the use of personal protective equipment (PPE) such as laboratory coats or gowns, gloves, eye protection, or a disposable mask and face shield, to help protect the skin and mucous membrane of the eyes, nose, and mouth. Before starting the grossing, all doctors and technicians followed the standard precautions. Historical controls of 50 term placentas from healthy women were taken that underwent singleton delivery between February 2019 and June 2019.
Inclusion criteria
All COVID-19 positive mothers giving birth by elective lower segment cesarean or normal vaginal delivery at Government Doon Medical College, Dehradun, Uttarakhand, India, who had signed informed consent.
Those who did not fulfill any of the exclusion criteria listed below.
Exclusion criteria
(a) Age less than 18 years old or more than 45 years old, (b) any other active contagious viral infection or transfusion-related infection such as Hepatitis A, Hepatitis B, HIV, and syphilis, (c) previous or current serious blood disorder or cancer or blood transfusion within 12 months, (d) less than 36 weeks gestation at the time of delivery or with multiple pregnancies, (e) use of intravenous drugs/shared needles/prior treatment with clotting factors such as factor VIII or factor IX, (f) active genital herpes, chickenpox, meningitis, shigella, or salmonella infections, food poisoning, viral infection.
Stepwise gross examination of the placenta
Before starting the gross examination, an additional 24 h of formalin fixation of specimens was done in a dedicated fume cabinet.[8] Grossing was done in a dedicated, certified, class II biological safety hood. The placenta was first oriented with the maternal surface down and membrane up encircling the fetal surface. The gestational sac was opened and a 2 to 3 cm strip was cut from the ruptured margin to the placental margin. This strip was rolled along a probe with amnion inward and sectioned transversely. Insertion of membranes was noted and then trimmed from the placental disc. The length of the umbilical cord length was measured and it was removed at its insertion. Several transverse sections were made along the length. The number of blood vessels was noted. The placental disc was then weighed and measured along three dimensions in cm.
Sections were made in the parenchyma at 1 to 2 cm intervals with the maternal surface upward.
A total of four sections of the placental disc were submitted, two sections from the maternal surface and two sections from the fetal surface. Sections were also submitted from any area looking abnormal on gross examination. Two sections each of membrane roll and umbilical cord were also submitted. Sections underwent routine processing, embedding, sectioning at 5 μm, and staining with hematoxylin and eosin (H and E). Histologic examination was performed.
Systematic approach for histopathological examination of the placenta
Different sections of the placenta were examined at 10× and 40× magnifications to look for the following: cord examined for the presence of three blood vessels, any thrombus or abnormality. Membranes were examined for the presence of chorioamnionitis, Maternal surface was observed for features of maternal vascular malperfusion (MVM) (villous infarction and necrosis as well as villous agglutination and crowding). The fetal surface was examined for features of fetal vascular malperfusion (chorangioisis). Other pathological findings: villitis, villous edema, intervillous fibrinoid, and intervillous hemorrhage, calcification, and endovasculosis.
RESULTS
Gross examination of control placentas showed three vessels, membranes were unremarkable, and the cut surface was spongy brown. One showed a small 1 cm area of hemorrhage near cord insertion and membranes were unremarkable. Microscopic examination in controls showed normal villous maturation and vasculature. As of now, our study is the largest in relation to sample size. All placentas delivered were full-time third trimester. The mother's age ranged from 19 years to 36 years (median: 27 years).
Gross findings in the placenta
On gross examination of placentas, we found that the membrane was thick and opaque in 4 out of 50 (8%) specimens and was transparent in the remaining 46 (92%) specimens of the placenta. Tan areas/infarct was noted in 20 of 50 (40%) cases in both fetal and maternal surface, whereas 30 of 50 (60%) placentas were grossly unremarkable. The cord was normal in all cases with all three lumens intact. In terms of fetal outcome, there were 43 of 50 (86%) full-term normal deliveries, 3 (6%) lower segment cesarean section, and 4 (8%) intrauterine death.
Microscopic findings in the placenta
On microscopic examination, H&-stained sections showed various changes and abnormal morphological findings in the placenta. Chorioamnionitis referred to as infiltration of the membrane by predominantly acute inflammatory cells was found in 9 out of 50 (18%) cases, villitis was seen in 4 (8%) cases, and significant areas of infarction and necrosis were seen in 11 (22%) cases. Crowding and agglutination of villi were observed in 12 (24%) cases [Figure 1c].
;)
Figure 1: Findings of the placenta with COVID-19. (a and b) Microscopic images showing chorangioisis and Tenny–Parker changes in term placenta (hematoxylin and eosin [H and E]. 40×). (c) Microscopic images showing endovasculosis in term placenta (H and E, 40×)
Features of stem villous vascular obliteration (endovasculosis) were seen in 7 (14%) patients [Figure 1c]. The major finding was chorangiosis, which is defined as >10 capillaries. More than 10% of villi was seen in 28 (56%) cases [Figure 1a and b]. Tenney–Parker change was seen in 13 (26%) patients. Intervillous fibrinoid deposition was observed in 37 (74%) patients [Figure 1a and b] along with significant areas of intervillous hemorrhage (which consisted of clots of layered red cells and fibrin/fibrinoid, the proportion of fibrin/fibrinoid increases as the lesion ages in the intervillous space, whereas normal blood is present between intervillous spaces) were seen in 37 (74%) patients and 7 (14%) patients showed significant calcification.
Features such as avascular fibrotic villi were seen in 14 (28%) cases, distal villous hypoplasia and edema were observed in 3 (6%) cases each [Table 1].
Table 1: Histopathological findings of placentas delivered by patients with COVID-19
DISCUSSION
There is limited research on this topic as the SARS–CoV-2 infection is relatively new and we are still trying to understand many aspects of the pathophysiology of the disease. This study is conducted on 50 placentas of pregnant women affected with COVID-19 in the last trimester. The median age of women in the study was 27 years.
In terms of fetal outcome, the majority of the cases were delivered full-term by normal vaginal delivery, with three cases delivered by cesarean sections and four cases of intrauterine death. The most prominent histological finding in the placenta of pregnant women affected with COVID-19 was chorangiosis, which is a feature of fetal vascular malperfusion. Chorangiosis has been known to be associated with decreased maternal oxygen saturation.[9,10] A recent study also showed most common lesions were of fetal vascular malperfusion.[11]
Another significant finding in our study is the increased incidence of features of MVM such as villous crowding and agglutination along with villous infarction and necrosis. MVM has been associated with oligohydramnios, fetal growth restriction, preterm birth, and stillbirth.[12,13,14,15] Study conducted by Shanes et al.[6] (2020) also highlighted the increased incidence of features of MVM and chorangiosis.
COVID-19 patients in our study also showed an increased incidence of intervillous hemorrhage, intervillous fibrinoid deposition, and Tenney–Parker changes along with areas of necrosis and infarction in a significant number of cases. Other pathological findings we noted were chorioamnionitis, stem villous vascular obliteration, significant calcification, villitis, villous atrophy, and villous edema.
Limitations
Limited conclusions about the effect of maternal SARS-CoV-2 infection on placental pathology can be drawn as the present study lacks appropriate gestational age and clinical condition matched controls. Maximizing the number of placenta samples examined will increase the reliability and generalizability of correlation findings.
CONCLUSION
This study is an attempt to understand how the SARS-CoV-2 infection affects the pregnancy, particularly in the last trimester by doing a histopathological examination of the placenta. From our study based on 50 placentas of pregnant women affected with SARS-CoV-2 infection, it is clear that there is a significant impact on fetal and maternal circulations causing features of fetal and maternal malperfusion such chorangiosis, villous crowding, and agglutination, suggesting that the infection could cause a potential rise in the risk of adverse perinatal outcomes such as intrauterine fetal growth retardation, preterm birth, or stillbirth. Therefore, it is imperative to do thorough antenatal tests for women diagnosed with SARS-CoV-2 so that adequate treatment may be given to the mother.
Ethics committee
The present proposal has been reviewed and approved by the ethics committee (Research Review Board) of Government Doon Medical College and Hospital, Dehradun, India.
Declaration of patient consent
Informed patient consent was obtained from all the patients before the study.
Financial support and sponsorship
Department of Pathology, Government Doon Medical College and Hospital, Dehradun, India.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
The authors would like to acknowledge the contribution of Ms. Divya for the technical support.
REFERENCES
1. Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia N Engl J Med. 2020;382:1199–207
2. Di Mascio D, Khalil A, Saccone G, Rizzo G, Buca D, Liberati M, et al Outcome of coronavirus spectrum infections (SARS, MERS, COVID-19) during pregnancy: A systematic review and meta-analysis Am J Obstet Gynecol MFM. 2020;2:100107
3. Vivanti AJ, Vauloup-Fellous C, Prevot S, Zupan V, Suffee C, Cao JD, et al Transplacental transmission of SARS-CoV-2 infection Nat Commun. 2020;11:3572
4. Bourne T, Kyriacou C, Coomarasamy A, Al-Memar M, Leonardi M, Kirk E, et al ISUOG consensus statement on rationalization of early-pregnancy care and provision of ultrasonography in context of SARS-CoV-2 Ultrasound Obstet Gynecol. 2020;55:871–8
5. Poon LC, Yang H, Kapur A, Melamed N, Dao B, Divakar H, et al Global interim guidance on coronavirus disease 2019 (COVID-19) during pregnancy and puerperium from FIGO and allied partners: Information for healthcare professionals Int J Gynaecol Obstet. 2020;149:273–86
6. Shanes ED, Mithal LB, Otero S, Azad HA, Miller ES, Goldstein JA. Placental pathology in COVID-19 Am J Clin Pathol. 2020;154:23–32
7. World Health Organization. . Laboratory biosafety guidance related to coronavirus disease 2019 (COVID-19) Interim guidance February 12. 2020 Available from:
https://apps.who.int/iris/handle/10665/332076. License: CC BY-NC-SA 3.0 IGO
8. Burton GJ, Sebire NJ, Myatt L, Tannetta D, Wang YL, Sadovsky Y, et al Optimising sample collection for placental research Placenta. 2014;35:9–22
9. Akbulut M, Sorkun HC, Bir F, Eralp A, Duzcan E. Chorangiosis: The potential role of smoking and air pollution Pathol Res Pract. 2009;205:75–8
10. Altshuler G. Chorangiosis. An important placental sign of neonatal morbidity and mortality Arch Pathol Lab Med. 1984;108:71–4
11. Zhou YY, Ravishankar S, Luo G, Redline RW, et al Predictors of high grade and other clinically significant placental findings by indication for submission in singleton placentas from term births Pediatr Dev Pathol. 2020;23:274–84
12. Baergen RN, Heller DS. Placental pathology in Covid-19 positive mothers: Preliminary findings Pediatr Dev Pathol. 2020;23:177–80
13. Khong TY, Mooney EE, Ariel I, Balmus NC, Boyd TK, Brundler MA, et al Sampling and definitions of placental lesions: Amsterdam placental workshop group consensus statement Arch Pathol Lab Med. 2016;140:698–713
14. Redline RW. Severe foetal placental vascular lesions in term infants with neurologic impairment Am J Obstet Gynecol. 2005;192:452–7
15. Chen A, Roberts DJ. Placental pathologic lesions with a significant recurrence risk-what not to miss! APMIS. 2018;126:589–601
