Keywords
COVID-19 - placenta - pregnancy - systematic sampling - viral infections
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.[1]
[2]
[3]
[4] 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).[4]
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.[3]
[5]
[6]
[7]
[8] The impact of COVID-19 on the obstetric population and the gestational consequences
of SARS-CoV-2 are a great concern for investigation.[9]
[10]
[11] 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.[12] 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).[13] 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.[14]
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.[15]
[16] The broad spectrum of placental functions depends on its tissues and cellular stratification,
which form a selective biological barrier, called the blood-placental barrier.[17] 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.[18]
The current evidences about vertical transmission are uncertain and the preponderance
of evidence so far does not indicate a significant role for vertical transmission.[5]
[10]
[19] 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.[20] 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.[21] 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.[21]
[22]
[23]
[24]
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.[25] 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.[26] The latest World Health Organization (WHO) recommendations for sampling COVID-19
patients include a biosafety level 2 (BSL2) facility with all adequate caution.[27] Specific procedures with high viral load, like viral isolation, must be conducted
in biosafety level 3 (BSL3) facilities.[28]
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.[27]
[28] 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.[27]
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.[29] 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.[30]
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.[25] 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.[25] However, inadequate placental sampling can be distracting and generate misleading
results; for that reason, all studies involving placental samples should detail the
procedure.[31]
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.[10]
[11]
[32]
[33]
[34] 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.[35]
[36]
[37] 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.[38]
[39] 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.[40]
[41] 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.[22]
[23]
[24] 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.[23]
As well as the ZIKV, SARS-CoV-2 contains a positive-sense single-stranded RNA genome.[1]
[3]
[42] Given the previous experience to detect the ZIKV genome in placentas sampled by
this protocol,[25] 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.[14]
[26]
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.
