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Placental Sampling for Understanding Viral Infections - A Simplified Protocol for the COVID-19 Pandemic

Coleta placentária para entender infecções virais - Um protocolo simplificado para a pandemia de COVID-19

Abstract

Objective

The coronavirus disease 2019 (COVID-19) is a pandemic viral disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The impact of the disease among the obstetric population remains unclear, and the study of the placenta can provide valuable information. Adequate sampling of the placental tissue can help characterize the pathways of viral infections.

Methods

A protocol of placental sampling is proposed, aiming at guaranteeing representativity of the placenta and describing the adequate conservation of samples and their integrity for future analysis. The protocol is presented in its complete and simplified versions, allowing its implementation in different complexity settings.

Results

Sampling with the minimum possible interval from childbirth is the key for adequate sampling and storage. This protocol has already been implemented during the Zika virus outbreak.

Conclusion

A protocol for adequate sampling and storage of placental tissue is fundamental for adequate evaluation of viral infections on the placenta. During the COVID-19 pandemic, implementation of this protocol may help to elucidate critical aspects of the SARS-CoV-2 infection.

Keywords:
COVID-19; placenta; pregnancy; systematic sampling; viral infections

Resumo

Objetivo

A doença do novo coronavírus (COVID-19) é uma doença viral pandêmica causada pelo coronavírus da síndrome respiratória aguda 2 (SARS-CoV-2). O impacto da doença entre a população obstétrica ainda é incerto, e o estudo da placenta pode fornecer informações valiosas. Assim, a coleta adequada do tecido placentário pode ajudar a caracterizar algumas propriedades das infecções virais.

Métodos

Um protocolo de coleta placentária é proposto, objetivando a garantia de representatividade da placenta, descrevendo a maneira de conservação adequada das amostras, e visando garantir sua integridade para análises futuras. O protocolo é apresentado em suas versões completa e simplificada, permitindo sua implementação em diferentes configurações de infraestrutura.

Resultados

A amostragem com o intervalo mínimo possível do parto é essencial para coleta e armazenamento adequados. Esse protocolo já foi implementado durante a epidemia de vírus Zika.

Conclusão

Um protocolo para coleta e armazenamento adequados de tecido placentário é fundamental para a avaliação adequada de infecções virais na placenta. Durante a pandemia de COVID-19, a implementação deste protocolo pode ajudar a elucidar aspectos críticos da infecção por SARS-CoV-2.

Palavras-chave:
COVID-19; placenta; gestação; coleta sistemática; infecções virais

Introduction

Coronavirus disease 2019 (COVID-19) is a severe and highly relevant viral disease in the global scenario. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (family Coronaviridae, genus Betacoronavirus), the etiological agent of the disease, causes asymptomatic or a mild respiratory infection in the majority of cases.11 Andersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF. The proximal origin of SARS-CoV-2. Nat Med. 2020;26(04):450-452. Doi: 10.1038/s41591-020-0820-9
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22 Neuman BW, Kiss G, Kunding AH, et al. A structural analysis of M protein in coronavirus assembly and morphology. J Struct Biol. 2011;174(01):11-22. Doi: 10.1016/j.jsb.2010.11.021
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33 Gorbalenya AE, Baker SC, Baric RS, et al; Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoVand naming it SARS-CoV-2. NatMicrobiol. 2020;5(04):536-544. Doi: 10.1038/s41564-020-0695-z
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44 Cheng ZJ, Shan J. 2019 Novel coronavirus: wherewe are andwhat we know. Infection. 2020;48(02):155-163. Doi: 10.1007/s15010-020-01401-y
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However, people with underlying risk factors, such as increased age, cardiovascular disease, and diabetes, present higher rates of clinical complications and severe acute respiratory syndrome (SARS).44 Cheng ZJ, Shan J. 2019 Novel coronavirus: wherewe are andwhat we know. Infection. 2020;48(02):155-163. Doi: 10.1007/s15010-020-01401-y
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In viruses of the same genus, such as SARS-CoV (subgenus Sarbecovirus) and Middle East respiratory syndrome coronavirus (MERS-CoV) (subgenus Merbecovirus), as well as in other respiratory disease viruses, there is an increased risk of morbidity and mortality during pregnancy.33 Gorbalenya AE, Baker SC, Baric RS, et al; Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoVand naming it SARS-CoV-2. NatMicrobiol. 2020;5(04):536-544. Doi: 10.1038/s41564-020-0695-z
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55 Rasmussen SA, Smulian JC, Lednicky JA, Wen TS, Jamieson DJ. Coronavirus Disease 2019 (COVID-19) and pregnancy: what obstetricians need to know. Am J Obstet Gynecol. 2020;222 (05):415-426. Doi: 10.1016/j.ajog.2020.02.017
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66 Wong SF, Chow KM, Leung TN, et al. Pregnancy and perinatal outcomes of women with severe acute respiratory syndrome. Am J Obstet Gynecol. 2004;191(01):292-297. Doi: 10.1016/j.ajog. 2003.11.019
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77 Jeong SY, Sung SI, Sung JH, et al.MERS-CoV infection in a pregnant woman in Korea. J Korean Med Sci. 2017;32(10):1717-1720. Doi: 10.3346/jkms.2017.32.10.1717
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88 Kourtis AP, Read JS, Jamieson DJ. Pregnancy and infection. N Engl J Med. 2014;370(23):2211-2218. Doi: 10.1056/NEJMra1213566
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The impact of COVID-19 on the obstetric population and the gestational consequences of SARS-CoV-2 are a great concern for investigation.99 Favre G, Pomar L, Musso D, Baud D. 2019-nCoV epidemic: what about pregnancies? Lancet. 2020;395(10224):e40. Doi: 10.1016/S0140-6736(20)30311-1
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1010 Breslin N, Baptiste C, Gyamfi-Bannerman C, et al. Coronavirus disease 2019 infection among asymptomatic and symptomatic pregnant women: two weeks of confirmed presentations to an affiliated pair of New York City hospitals. Am J Obstet Gynecol MFM. 2020;2(02):100118. Doi: 10.1016/j.ajogmf.2020.100118
https://doi.org/10.1016/j.ajogmf.2020.10...
1111 Breslin N, Baptiste C, Miller R, et al. Coronavirus disease 2019 in pregnancy: early lessons. Am J Obstet Gynecol MFM. 2020;2(02): 100111. Doi: 10.1016/j.ajogmf.2020.100111
https://doi.org/10.1016/j.ajogmf.2020.10...
Data from the United Kingdom show that most women admitted with SARS-CoV-2 infection during pregnancy were in the late second or third trimester, which replicates the pattern seen for other respiratory viruses, with women in later pregnancy being more severely affected, a third of whom had preexisting comorbidities.1212 Knight M, Bunch K, Vousden N, et al; UK Obstetric Surveillance System SARS-CoV-2 Infection in Pregnancy Collaborative Group. Characteristics and outcomes of pregnant women admitted to hospital with confirmed SARS-CoV-2 infection in UK: national population based cohort study. BMJ. 2020;369:m2107. Doi: 10.1136/bmj.m2107
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In United States, reports show higher rates of hospitalization (31.5%), intensive care unit (ICU) admission (1.5%), and mechanical ventilation (0.5%) in pregnant women, when compared with nonpregnant women (5.8%, 0.9%, 0.3%, respectively).1313 Ellington S, Strid P, Tong VT, et al. Characteristics of women of reproductive age with laboratory-confirmed SARS-CoV-2 infection by pregnancy status - United States, January 22-June 7, 2020. MMWR Morb Mortal Wkly Rep. 2020;69(25):769-775. Doi: 10.15585/mmwr.mm6925a1
https://doi.org/10.15585/mmwr.mm6925a1...
Thus, recent data from Brazil have demonstrated an increased risk of severity among pregnant women, with high numbers of maternal death, and significant cases without adequate respiratory support and with no intensive care admissions.1414 Takemoto MLS, Menezes MO, Andreucci CB, et al. The tragedy of COVID-19 in Brazil: 124 maternal deaths and counting. Int J Gynaecol Obstet. 2020;151(01):154-156. Doi: 10.1002/ijgo.13300
https://doi.org/10.1002/ijgo.13300...

To understand the different facets of COVID-19 during pregnancy, the placenta can serve as a valuable source of information about maternal and fetal conditions. The placenta is a complex and unique interface between maternal and fetal vascular beds, mediating the exchange of nutrients and others residues, allowing the fetal uterine existence and maintaining a highly reliable homeostasis.1515 Maltepe E, Fisher SJ. Placenta: the forgotten organ. Annu Rev Cell Dev Biol. 2015;31(01):523-552. Doi: 10.1146/annurev-cellbio-100814-125620
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1616 Proenca-Modena JL, Milanez GP, Costa ML, Judice CC, Maranhão Costa FT. Zika virus: lessons learned in Brazil. Microbes Infect. 2018;20(11-12):661-669. Doi: 10.1016/j.micinf.2018.02.008
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The broad spectrum of placental functions depends on its tissues and cellular stratification, which form a selective biological barrier, called the blood-placental barrier.1717 Pereira L. Congenital viral infection: traversing the uterine-placental interface. Annu Rev Virol. 2018;5(01):273-299. Doi: 10.1146/annurev-virology-092917-043236
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Those tissues may be affected by viral infections, such as parvovirus B19, rubella virus, cytomegalovirus, herpes simplex viruses, and Zika virus (ZIKV), and the consequences of the placental tissues' immune response and destruction during different periods of pregnancy can lead to severe consequences on gestational and neonatal outcomes.1818 Costa ML, de Moraes Nobrega G, Antolini-Tavares A. Key infections in the placenta. Obstet Gynecol Clin North Am. 2020;47(01): 133-146. Doi: 10.1016/j.ogc.2019.10.003
https://doi.org/10.1016/j.ogc.2019.10.00...

The current evidences about vertical transmission are uncertain and the preponderance of evidence so far does not indicate a significant role for vertical transmission.55 Rasmussen SA, Smulian JC, Lednicky JA, Wen TS, Jamieson DJ. Coronavirus Disease 2019 (COVID-19) and pregnancy: what obstetricians need to know. Am J Obstet Gynecol. 2020;222 (05):415-426. Doi: 10.1016/j.ajog.2020.02.017
https://doi.org/10.1016/j.ajog.2020.02.0...
1010 Breslin N, Baptiste C, Gyamfi-Bannerman C, et al. Coronavirus disease 2019 infection among asymptomatic and symptomatic pregnant women: two weeks of confirmed presentations to an affiliated pair of New York City hospitals. Am J Obstet Gynecol MFM. 2020;2(02):100118. Doi: 10.1016/j.ajogmf.2020.100118
https://doi.org/10.1016/j.ajogmf.2020.10...
1919 Verma S, Carter EB, Mysorekar IU. SARS-CoV2 and pregnancy: An invisible enemy? Am J Reprod Immunol. 2020;84(05):e13308. Doi: 10.1111/aji.13308
https://doi.org/10.1111/aji.13308...
However, to understand the impact of COVID-19 on maternal morbidity and mortality is crucial, and the evaluation of the placental tissue may provide data about pathways related to the viral infection within the placenta.2020 Shanes ED, Mithal LB, Otero S, Azad HA, Miller ES, Goldstein JA. Placental Pathology in COVID-19. AmJ Clin Pathol. 2020;154(01): 23-32. Doi: 10.1093/ajcp/aqaa089
https://doi.org/10.1093/ajcp/aqaa089...
Recent placental histopathology results from SARS-CoV-2-positive women did not demonstrate a specific pathology or pathological pattern; however, nonspecific histomorphologic changes suggestive of maternal/fetal vascular malperfusion have been reported.2121 He M, Skaria P, Kreutz K, et al. Histopathology of third trimester placenta from SARS-CoV-2-Positive women. Fetal Pediatr Pathol. 2020:1-10. Doi: 10.1080/15513815.2020.1828517[ahead of print]
https://doi.org/10.1080/15513815.2020.18...
Viral particles in the organ has been detected, although aspects of the effects and pathways of infection by SARS-CoV-2 and how it occurs on placental tissues remain largely unknown up to this date.2121 He M, Skaria P, Kreutz K, et al. Histopathology of third trimester placenta from SARS-CoV-2-Positive women. Fetal Pediatr Pathol. 2020:1-10. Doi: 10.1080/15513815.2020.1828517[ahead of print]
https://doi.org/10.1080/15513815.2020.18...
2222 Algarroba GN, Rekawek P, Vahanian SA, et al. Visualization of severe acute respiratory syndrome coronavirus 2 invading thehuman placenta using electron microscopy. Am J Obstet Gynecol. 2020;223(02):275-278. Doi: 10.1016/j.ajog.2020.05.023
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2323 Hecht JL, Quade B, Deshpande V, et al. SARS-CoV-2 can infect the placenta and is not associatedwith specific placental histopathology: a series of 19 placentas from COVID-19-positive mothers. Mod Pathol. 2020;33(11):2092-2103. Doi: 10.1038/s41379-020-0639-4
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2424 Hosier H, Farhadian SF, Morotti RA, et al. SARS-CoV-2 infection of the placenta. J Clin Invest. 2020;130(09):4947-4953. Doi: 10.1172/JCI139569
https://doi.org/10.1172/JCI139569...

Our group has previously shown, during the ZIKV outbreak in Brazil, that the placenta is a possible site for viral persistence and that viral detection relies on adequate and appropriate sampling and storage.2525 Venceslau EM, Guida JPS, Nobrega GM, et al; Zika-Unicamp Network. Adequate placental sampling for the diagnosis and characterization of placental infection by Zika Virus. FrontMicrobiol. 2020;11:112. Doi: 10.3389/fmicb.2020.00112
https://doi.org/10.3389/fmicb.2020.00112...
Thus, here we detail sampling procedures and also propose a simple protocol that can be performed in the delivery room, to guarantee representative tissues of placenta, allowing further investigation consequences of viral infection in pregnancy, including SARS-CoV-2 infection.

Methods

Placental Sampling Protocol

The placental sampling protocol aims to represent the various tissues that constitute the placenta, and also the umbilical cord, at the time of childbirth. The sampling includes 4 regions of the placenta: the basal plate, the chorionic villus, the chorionic plate, and the amniotic membrane (Fig. 1). To preserve the best sampling quality, collection should be performed in the shortest possible interval from childbirth. Due to different conditions for sampling in different facilities, two versions of the protocol are proposed, the complete (Fig. 1A) and the simplified (Fig. 1B). All procedures must be performed following the local biosafety rules. The current manuscript is a protocol description. Each study that implements it must necessarily undergo appropriate ethical approval. The protocol was approved by the ethics committee of the coordinating center (#4.047.168) and of each participating center, with implementation in 5 obstetric reference centers of the Brazilian Network of COVID-19 during Pregnancy (REBRACO, in the Portuguese acronym) up to now.2626 Costa ML, Pacagnella RC, Guida JP, et al; Brazilian Network for Studies on Reproductive and Perinatal Research. Call to action for a South American network to fight COVID-19 in pregnancy. Int J Gynaecol Obstet. 2020;150(02):260-261. Doi: 10.1002/ijgo.13225
https://doi.org/10.1002/ijgo.13225...
The latest World Health Organization (WHO) recommendations for sampling COVID-19 patients include a biosafety level 2 (BSL2) facility with all adequate caution.2727 World Health Organization. Laboratory biosafety manual. 3rd ed. Geneva: WHO; 2004 Specific procedures with high viral load, like viral isolation, must be conducted in biosafety level 3 (BSL3) facilities.2828 World Health Organization. Laboratory biosafety guidance related to coronavirus disease 2019 (COVID-19): interim recommendations [Internet]. GenevaWHO2020 [cited 2020 May 12]. Available from: https://apps.who.int/iris/rest/bitstreams/1277819/retrieve
https://apps.who.int/iris/rest/bitstream...

Fig. 1
Cross section of the placenta showing its components in different versions of the protocol—complete (A) and simplified (B) placental protocol version.

Complete Placental Sampling

After childbirth, the placenta should be immediately prepared for sampling or preserved in a cool refrigerator (4°C) in a sterile container for a maximum of 2 hours after childbirth until sampling is possible. The processing of the placenta must be done in an adequate sterile hood; the materials and equipment necessary for adequate placental sampling are described in Supplementary Data S1 (online only). Placental samples will be stored in cryotubes and histology cassettes. All storage materials must be properly identified before the procedure, with patient identification and corresponding placental region.

The first step is to have the placenta washed, with sterile saline or sterile phosphate buffer saline, inside a tray, and any solid residues or visible blood clots must be removed. After cleaning, the placenta is placed on a surface with sterile absorbent paper with the basal plate facing up. The choice of the sampling locals to ensure representativeness is based on the insertion of the umbilical cord (Fig. 2). In placentas with umbilical cord centrally inserted, three imaginary concentric circles (one coincident with the placental disc borders, one marginal to the umbilical cord, and a third placed between those two previously described) should be projected, and the sampling places are positioned in the intermediate circle. Four points are chosen in the intermediate circle for sampling, equidistant from each other (Fig. 2A). In placentas with peripheral cord insertion, three concentric semi-circles starting from the cord insertion site should be considered, and sampling will be performed in the intermediate circle (Fig. 2B). The areas where the sampling take place must not contain macroscopic anomalies, such as areas of detachment or extensive calcification.

Fig. 2
Placental sampling based on umbilical cord insertion site — central (A) and peripheral (B) insertion.

After the sampling areas are defined, tissues are sampled in the following order: basal plate, chorionic villus, amniotic membrane, chorionic plate, and umbilical cord (Fig. 1A). The basal plate corresponds to the maternal face of the placenta; at the sampling places, an incision should be made with the scalpel 5 mm deep, seeking to avoid contamination with villi. Chorionic villus corresponds to the tissue underlying the basal plate; superficial tissue must be despised because it may contain traces of the basal plate. The amniotic membrane corresponds to the thin and transparent membrane that lines the chorionic plate; to acquire it, this layer must be detached from the chorionic plate. The chorionic plate corresponds to the fetal face of the placenta; it is necessary to dissect the amniotic membrane previously and collect the tissue below, ∼ 2 mm thick, and visible calibrated blood vessels should be avoided. Finally, for the umbilical cord, samples are obtained sectioning it transversely, to obtain two samples.

Samples of ∼15 × 15 × 15 mm (except for the amniotic membrane, where the sample is ∼10 × 5 × 2 mm) are initially obtained, from each placental region, and, further, each of these pieces are divided in 3 equal parts (technical triplicate). Umbilical cord samples ∼10 mm thick and divided in 3 equal parts are also obtained.

Each tissue sampling has two storage destinations: histology cassettes for formalin fixation and cryotubes for cryopreservation. One of three parts of each sample replica will be stored in a cassette, that will in the end contain four samples, one from each previously selected region — again focusing on representativeness (Fig. 3A). Two of three parts of each replica will be placed in two cryotubes, each containing 4 samples, one from each previously selected local, guaranteeing the representativeness of the placenta (Fig. 3B). The umbilical cord samples follow the same storage method: the histology cassette and each cryotube present two fragments of total section of the tissue.

Fig. 3
Tissue sample storage process for each sampling site. (A). Histology cassettes storage. (B). Cryotube storage.

Simplified Placental Sampling

After childbirth, the placenta can be immediately sampled in the operating room, by the responsible delivery team, after adequate training. This can mitigate any concerns regarding biosafety standards, materials usage, and adequate use of appropriate personal protective equipment (PPE) (Supplementary Data 1). All storage material must be properly identified with the patient coding, as previously proposed for the complete placental sampling.

The selection of sampling areas to ensure representativeness is the same as that applied for the complete placental sampling, and it is based on the insertion of the umbilical cord, defining four places (Fig. 2). When obtaining such samples, there is no detail on the different regions, and the samples must contain almost the full thickness of the placenta, removing the more superficial maternal tissue and focusing on collecting the villous tissue in deeper regions (Fig. 1B). Samples of ∼15 × 15 × 15 mm are obtained from each local.

The samples collected are stored in cryotubes, each containing a sample from a previously selected area, and properly preserved after childbirth. To guarantee representativeness of the placenta, analysis with samples obtained with simplified placental sampling must use material from the four sampling areas for each assay (Fig. 4). After sampling in the operating room, the placenta should be sent for routine pathological analysis.

Fig. 4
Placenta sample storage process for simplified protocol version.

Storage and Cautions

Placenta samples in histology cassettes must be placed in a fixative solution, such as 10% buffered formalin, and then be processed for embedding, which can be made in paraffin or other material (Fig. 3A). Cryotubes containing samples must be preserved immediately at very low temperatures to maintain sample integrity. Cryotubes must be stored in a liquid nitrogen (N2) container or directly in -80°C freezer. Low temperatures must be maintained in the final accommodation (Fig. 3B). Biosafety guidelines must be followed during all manipulation of samples: sampling, freezing, storing, and processing.2727 World Health Organization. Laboratory biosafety manual. 3rd ed. Geneva: WHO; 20042828 World Health Organization. Laboratory biosafety guidance related to coronavirus disease 2019 (COVID-19): interim recommendations [Internet]. GenevaWHO2020 [cited 2020 May 12]. Available from: https://apps.who.int/iris/rest/bitstreams/1277819/retrieve
https://apps.who.int/iris/rest/bitstream...
All disposable materials used during the sampling process must be considered as infectious waste. Other materials must be cleaned and sterilized, preferably in an initial 10% sodium hypochlorite solution.2727 World Health Organization. Laboratory biosafety manual. 3rd ed. Geneva: WHO; 2004

Histopathological Analysis

The placenta should be further considered for histopathological examination. After the sampling protocol process is finished, the placenta should be placed in a container with an adequate volume of buffered formalin and sent for histopathological analysis. The sampling and analysis of normal and abnormal findings should follow the Amsterdam Placental Workshop Group Consensus Statements, to enable comparison and international standardization of report results.2929 Khong TY, Mooney EE, Ariel I, et al. Sampling and Definitions of Placental Lesions: Amsterdam Placental Workshop Group Consensus Statement. Arch Pathol Lab Med. 2016;140(07):698-713. Doi: 10.5858/arpa.2015-0225-CC
https://doi.org/10.5858/arpa.2015-0225-C...
A consistent understanding, with basic gross examination and histologic patterns of injury is important to maximize the diagnostic, prognostic, and therapeutic benefit of placental examination.3030 Ravishankar S, Redline RW. What obstetricians need to know about placental pathology. Obstet Gynecol Clin North Am. 2020; 47(01):29-48. Doi: 10.1016/j.ogc.2019.10.007
https://doi.org/10.1016/j.ogc.2019.10.00...

Results and Discussion

Adequate placental sampling is key to the evaluation of different insults that may affect the placenta, the woman, and the fetus. We described a placental sampling protocol, which has already been implemented in our setting and allowed us to provide some evidence regarding ZIKV infection during pregnancy.2525 Venceslau EM, Guida JPS, Nobrega GM, et al; Zika-Unicamp Network. Adequate placental sampling for the diagnosis and characterization of placental infection by Zika Virus. FrontMicrobiol. 2020;11:112. Doi: 10.3389/fmicb.2020.00112
https://doi.org/10.3389/fmicb.2020.00112...
Conserving the integrity of placental samples enables future analysis, using molecular biology and biochemistry techniques.

Immunohistochemistry, immunofluorescence, and a series of different stains, such as the commonly used hematoxylin and eosin stain, can be performed in the histological samples obtained from paraffin-embedded tissue cassettes. Using samples preserved in cryotubes, after specific treatments and extractions, experiments and assays based on proteins (such as Western-blot and proteomic analyzes) or nucleic acids (such as qPCR and next-generation sequencing), or even lipids and other biomolecules, can be implemented. We highlight that some samples are more appropriate than others for specific assays. As a relevant example, formalin-fixated and paraffin-embedded samples could lead to some methodological difficulties for the detection and testing of ribonucleic acid (RNA) viruses, while cryogenic stored samples are more adequate for such experiments.

In addition to maintaining the placental characteristics most similar to those at the moment of childbirth, the sampling is also representative of the placenta as an organ. The samples are collected from areas where the thickness of the placenta is regular, according to its distance from the umbilical cord's spot of insertion. By sampling in random equidistant regions, representativeness of the entire placenta is obtained, thus reducing the interference of specific site features (outliers) and bias in future analyses.

Due to the recommended sampling and subsequent adequate storage, the samples maintain the integrity of the biomolecules. In a previous study from our research group, in which the detailed placental protocol sampling was applied, it was possible to extract whole RNA molecules from samples preserved in -80°C freezers for periods of up to 2 years. This research made it possible to identify the ZIKV genome in placenta samples. This study suggested that, a simplified protocol, mainly with villous tissue samples, if respecting representativeness and adequate storage of the material, could be effective for viral detection.2525 Venceslau EM, Guida JPS, Nobrega GM, et al; Zika-Unicamp Network. Adequate placental sampling for the diagnosis and characterization of placental infection by Zika Virus. FrontMicrobiol. 2020;11:112. Doi: 10.3389/fmicb.2020.00112
https://doi.org/10.3389/fmicb.2020.00112...
However, inadequate placental sampling can be distracting and generate misleading results; for that reason, all studies involving placental samples should detail the procedure.3131 Burton GJ, Sebire NJ, Myatt L, et al. Optimising sample collection for placental research. Placenta. 2014;35(01):9-22. Doi: 10.1016/j.placenta.2013.11.005
https://doi.org/10.1016/j.placenta.2013....

The infection routes of SARS-CoV-2 regarding vertical transmission remain unclear, and there is limited information about COVID-19 during pregnancy and its consequences.1010 Breslin N, Baptiste C, Gyamfi-Bannerman C, et al. Coronavirus disease 2019 infection among asymptomatic and symptomatic pregnant women: two weeks of confirmed presentations to an affiliated pair of New York City hospitals. Am J Obstet Gynecol MFM. 2020;2(02):100118. Doi: 10.1016/j.ajogmf.2020.100118
https://doi.org/10.1016/j.ajogmf.2020.10...
1111 Breslin N, Baptiste C, Miller R, et al. Coronavirus disease 2019 in pregnancy: early lessons. Am J Obstet Gynecol MFM. 2020;2(02): 100111. Doi: 10.1016/j.ajogmf.2020.100111
https://doi.org/10.1016/j.ajogmf.2020.10...
3232 Zeng L, Xia S, Yuan W, et al. Neonatal early-onset infection with SARS-CoV-2 in 33 neonates born to mothers with COVID-19 in Wuhan, China. JAMA Pediatr. 2020;174(07):722-725. Doi: 10.1001/jamapediatrics.2020.0878
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3333 Xu H, Zhong L, Deng J, et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int J Oral Sci. 2020;12(01):8. Doi: 10.1038/s41368-020-0074-x
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3434 Chen Y, Peng H,Wang L, et al. Infants born tomothers with a New Coronavirus (COVID-19). Front Pediatr. 2020;8:104. Doi: 10.3389/fped.2020.00104
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Some cellular components have been considered as putative binding receptors for viral entry, such as the membrane protein angiotensin-converting enzyme 2 (ACE2), which is widely expressed in the surface of trophoblasts and endothelial cells.3535 JiaHP, LookDC, Shi L, et al.ACE2 receptorexpressionandsevere acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia. J Virol. 2005;79(23):14614-146213636 Tai W, He L, Zhang X, et al. Characterization of the receptorbinding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol. 2020;17(06):613-620. Doi: 10.1038/s41423-020-0400-4
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3737 Levy A, Yagil Y, Bursztyn M, Barkalifa R, Scharf S, Yagil C. ACE2 expression and activity are enhanced during pregnancy. Am J Physiol Regul Integr Comp Physiol. 2008;295(06):R1953-R1961. Doi: 10.1152/ajpregu.90592.2008
https://doi.org/10.1152/ajpregu.90592.20...
Recent studies suggest the ACE2 as part of the viral adsorption, and due to its expression in placental cells, it could possibly lead to a placental infection.3838 Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 2020;181(02):271-280.e8. Doi: 10.1016/j.cell.2020.02.052
https://doi.org/10.1016/j.cell.2020.02.0...
3939 Lukassen S, Chua RL, Trefzer T, et al. SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells. EMBO J. 2020;39(10):e105114. Doi: 10.15252/embj.20105114
https://doi.org/10.15252/embj.20105114...
Early studies published had not reported detection of the SARS-CoV-2 genome by reverse transcription-polymerase chain reaction (RT-PCR) assays in placental samples; however, details regarding the methodological process (sampling method, processing time, sample storage) are not clear and, therefore, did not rule out the possibility of viral presence at the maternal-fetal interface, which has now been shown.4040 Schwartz DA. An analysis of 38 pregnant women with COVID-19, their newborn infants, and maternal-fetal transmission of SARSCoV-2: maternal coronavirus infections and pregnancy outcomes. Arch Pathol Lab Med. 2020. Doi: 10.5858/arpa.2020-0901-SA [ahead of print]
https://doi.org/10.5858/arpa.2020-0901-S...
4141 Li Y, Zhao R, Zheng S, et al. Lack of Vertical Transmission of Severe Acute Respiratory Syndrome Coronavirus 2, China. Emerg Infect Dis. 2020;26(06):1335-1336. Doi: 10.3201/eid2606.200287
https://doi.org/10.3201/eid2606.200287...
Recent results demonstrated the presence and infection of the virus in the placental tissue, mainly in the chorionic villi, an area emphasized in the current protocol.2222 Algarroba GN, Rekawek P, Vahanian SA, et al. Visualization of severe acute respiratory syndrome coronavirus 2 invading thehuman placenta using electron microscopy. Am J Obstet Gynecol. 2020;223(02):275-278. Doi: 10.1016/j.ajog.2020.05.023
https://doi.org/10.1016/j.ajog.2020.05.0...
2323 Hecht JL, Quade B, Deshpande V, et al. SARS-CoV-2 can infect the placenta and is not associatedwith specific placental histopathology: a series of 19 placentas from COVID-19-positive mothers. Mod Pathol. 2020;33(11):2092-2103. Doi: 10.1038/s41379-020-0639-4
https://doi.org/10.1038/s41379-020-0639-...
2424 Hosier H, Farhadian SF, Morotti RA, et al. SARS-CoV-2 infection of the placenta. J Clin Invest. 2020;130(09):4947-4953. Doi: 10.1172/JCI139569
https://doi.org/10.1172/JCI139569...
A study involving 19 pregnant women infected with SARS-CoV-2 indicated the viral infection in villi syncytiotrophoblast and cytotrophoblasts by in-situ hybridization technique (nucleic-acid based technique), with a specific target for the SARS-CoV-2 RNA.2323 Hecht JL, Quade B, Deshpande V, et al. SARS-CoV-2 can infect the placenta and is not associatedwith specific placental histopathology: a series of 19 placentas from COVID-19-positive mothers. Mod Pathol. 2020;33(11):2092-2103. Doi: 10.1038/s41379-020-0639-4
https://doi.org/10.1038/s41379-020-0639-...

As well as the ZIKV, SARS-CoV-2 contains a positive-sense single-stranded RNA genome.11 Andersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF. The proximal origin of SARS-CoV-2. Nat Med. 2020;26(04):450-452. Doi: 10.1038/s41591-020-0820-9
https://doi.org/10.1038/s41591-020-0820-...
33 Gorbalenya AE, Baker SC, Baric RS, et al; Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoVand naming it SARS-CoV-2. NatMicrobiol. 2020;5(04):536-544. Doi: 10.1038/s41564-020-0695-z
https://doi.org/10.1038/s41564-020-0695-...
4242 Liu ZY, ShiWF, Qin CF. The evolution of Zika virus from Asia to the Americas. Nat Rev Microbiol. 2019;17(03):131-139. Doi: 10.1038/s41579-018-0134-9
https://doi.org/10.1038/s41579-018-0134-...
Given the previous experience to detect the ZIKV genome in placentas sampled by this protocol,2525 Venceslau EM, Guida JPS, Nobrega GM, et al; Zika-Unicamp Network. Adequate placental sampling for the diagnosis and characterization of placental infection by Zika Virus. FrontMicrobiol. 2020;11:112. Doi: 10.3389/fmicb.2020.00112
https://doi.org/10.3389/fmicb.2020.00112...
investigation of the COVID-19 virus could benefit from this protocol. Therefore, the implementation of the simplified protocol focusing on the chorionic villi can enable a greater scope of biological material sampling in different reference centers, mainly in countries with a severe pandemic scenario in the obstetric population, such as Brazil.1414 Takemoto MLS, Menezes MO, Andreucci CB, et al. The tragedy of COVID-19 in Brazil: 124 maternal deaths and counting. Int J Gynaecol Obstet. 2020;151(01):154-156. Doi: 10.1002/ijgo.13300
https://doi.org/10.1002/ijgo.13300...
2626 Costa ML, Pacagnella RC, Guida JP, et al; Brazilian Network for Studies on Reproductive and Perinatal Research. Call to action for a South American network to fight COVID-19 in pregnancy. Int J Gynaecol Obstet. 2020;150(02):260-261. Doi: 10.1002/ijgo.13225
https://doi.org/10.1002/ijgo.13225...

Conclusion

The placenta has a key role in the understanding of maternal-fetal complications. The implementation of the protocol in different settings would standardize placental sampling and storage, improving techniques description and results, and even providing the exchange of samples through different settings and global locations. The adequate storage of the samples would allow accurate and biologically relevant results in future studies to understand possible critical aspects of viral infections, such as pathogenesis, transmission routes, and functional changes related to infection by SARS-CoV-2 in the placenta.

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  • Funding G. M. N. is supported by Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (grant number 19/18720-6) and Coordenação de Aperfeiçoamento Pessoal de Nível Superior - CAPES (grant number 88887.600190/2021-00). I. M. is supported in part by a grant from the National Institutes of Health (grant number R01HD091218). M. L. C. has support from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant number 409605/2016-6) and FAEPEX (grant number 2300/20). I. M. and M. L. C. have a McDonnell International Scholars Academy seed grant for research on infectious diseases and the impact of COVID-19. The funders had no role in the present study’s design, data collection and analysis, decision to publish, or preparation of the manuscript. Figures were created with BioRender. com. We also acknowledge the medical team at the REBRACO institutions, especially all themedical residents involved, for the great help in sample collection during the childbirth of COVID-19 positive patients, proving that the implementation of a research protocol is possible, even facing a pandemic.

Publication Dates

  • Publication in this collection
    30 July 2021
  • Date of issue
    May 2021

History

  • Received
    12 May 2020
  • Accepted
    04 Feb 2021
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