A standardized definition of placental infection by SARS-CoV-2, a consensus statement from the National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development SARS-CoV-2 Placental Infection Workshop.

Pregnant individuals infected with SARS-CoV-2 have higher rates of intensive care unit admission, oxygen requirement, need for mechanical ventilation, and death than nonpregnant individuals. Increased COVID-19 disease severity may be associated with an increased risk of viremia and placental infection. Maternal SARS-CoV-2 infection is also associated with pregnancy complications such as preeclampsia and preterm birth, which can be either placentally mediated or reflected in the placenta. Maternal viremia followed by placental infection may lead to maternal-fetal transmission (vertical), which affects 1% to 3% of exposed newborns. However, there is no agreed-upon or standard definition of placental infection. The National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a group of experts to propose a working definition of placental infection to inform ongoing studies of SARS-CoV-2 during pregnancy. Experts recommended that placental infection be defined using techniques that allow virus detection and localization in placental tissue by one or more of the following methods: in situ hybridization with antisense probe (detects replication) or a sense probe (detects viral messenger RNA) or immunohistochemistry to detect viral nucleocapsid or spike proteins. If the abovementioned methods are not possible, reverse transcription polymerase chain reaction detection or quantification of viral RNA in placental homogenates, or electron microscopy are alternative approaches. A graded classification for the likelihood of placental infection as definitive, probable, possible, and unlikely was proposed. Manuscripts reporting placental infection should describe the sampling method (location and number of samples collected), method of preservation of tissue, and detection technique. Recommendations were made for the handling of the placenta, examination, and sampling and the use of validated reagents and sample protocols (included as appendices).

Introduction

Pregnant individuals infected with SARS-CoV-2 have higher rates of admission to the intensive care unit, requirement for mechanical ventilation, extracorporeal membrane oxygenation, and death than nonpregnant individuals1, 2, 3 but the risk of placental, fetal, and pregnancy complications have been less well described. Studies show that maternal SARS-CoV-2 infection is associated with an increased risk of preterm birth and preeclampsia,4, 5, 6 both of which may be placentally mediated or placentally-reflected complications. Moreover, published reports7, 8, 9 show that first- and second-trimester infections with SARS-CoV-2 are possible and can result in pregnancy loss. Recent reports from Ireland found second-trimester miscarriage and stillbirth associated with presumed placental SARS-CoV-2 infection, placentitis, and specifically the B.1.1.7 variant. , These reports suggest that placental infection does carry substantial perinatal morbidity and that clinical surveillance of exposed pregnancies and placental evaluation for infection are warranted. In addition, placental infection does not equate with vertical transmission but may cause placental damage, which leads to perinatal morbidity without infection. Many have noted the triad of histiocytic intervillositis, increased perivillous fibrin deposition, and trophoblast necrosis associated with placental infection. , 12, 13, 14, 15, 16, 17, 18, 19 Although these histologic findings are not specific for infection, they are characteristic enough to indicate a study to evaluate whether the placenta was infected.

Although early reports were focused on SARS-CoV-2 placental infection and in utero transmission,19, 20, 21, 22, 23, 24, 25, 26 larger studies and meta-analyses indicate that placental infection and vertical transmission were rare, , , 27, 28, 29, 30, 31, 32 particularly in comparison with other maternal viral infections in pregnancy (such as cytomegalovirus, Zika, rubella, and untreated HIV33, 34, 35, 36). Diagnostic criteria for confirming placental infection have been inconsistent. Early reports conflated placental infection with vertical transmission, highlighting the need for consensus definitions and nomenclature in this regard. One report found SARS-CoV-2 infection in 100% of placentas from mothers with COVID-19 based on positive immunohistochemistry studies, whereas others have rarely reported placental infection, if at all. , , , Investigators have employed a variety of techniques to diagnose placental infection, including reverse transcription polymerase chain reaction (RT-PCR) on placental homogenates, , , immunohistochemistry, , , RNA in situ hybridization (RNA-ISH), , , , and electron microscopy, , 42, 43, 44 making it difficult to compare across studies and establish definitive evidence regarding placental infection risks. The relative sensitivity and specificity of these techniques in the placenta have not been rigorously studied, but we have included what is known in the appendices. Given the patchy nature of infection of the placenta by SARS-CoV-2, legitimate concerns are raised about false positive and false negative results by various detection methods. Misclassification might lead to erroneous diagnoses with potential clinical and research implications. Indeed, knowing the true prevalence of placental infection depends on accurate diagnoses, and thus, these techniques are critically important to perform with validation and proper controls. Despite these caveats, a systematic review and meta-analysis of these disparate case reports and case series reported a placental infection rate of 7% (2 of 26 cases). Vertical transmission rates of 1.1% to 3% have been noted in cross-sectional studies ranging in size from 101 to 2399 mothers. , , , , There is clearly a need for standardization of methods to ensure robust and reproducible results and to facilitate cross-study comparisons.

Although a definition of transplacental infection has been proposed (World Health Organization reference number: WHO/2019-nCoV/mother-to-child transmission/2021.1), the criteria for placental infection have not. Although not all viruses infect the placenta before infecting the fetus, , placental infection seems to be a necessary intermediate step for in utero transmission of SARS-CoV-2 to the fetus. Objective, robust, consensus-driven criteria for the documentation of placental infection are needed to provide guidelines for research and clinical care. Herein, we present consensus definitions, tiered by rigor, formulated by a team of experts on the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)–assembled task force, for what constitutes placental infection by SARS-CoV-2 for clinical and research applications. We include, in appendices, protocols for implementation with specifics and references.

Methods

A group of active investigators with expertise in placental pathology, virology, obstetrics, infectious diseases, immunology, and molecular biology convened virtually to discuss and critique methods used to diagnose placental infection with SARS-CoV-2. In this multidisciplinary virtual workshop, experts in each field presented the following methods for documenting placental infection by SARS-CoV-2 (highlighting the strengths and limitations of each method): RNA RT-PCR or quantitative RT-PCR from placental homogenates, immunohistochemistry, RNA-ISH, electron microscopy, and histopathology. A round-table discussion followed the presentations and dialogue continued over several days. A 3-person team (D.J.R., A.G.E., R.J.R.) then devised a ranked template of specific diagnostic techniques and procedures in descending order of rigor (Table 1 ). This was then distributed to the larger committee for further discussion until consensus was achieved on the recommendations that follow.

Table 1

Definition categories of placental infection with SARS-CoV-2

Definite: evidence of active replicating virus with location in the placental tissues

Probable: evidence of viral RNA or protein located in placental tissues

Possible: evidence of viral RNA in placental homogenates or viral-like particles by electron microscopy in placental tissues

Unlikely: no evidence of any of the above

No testing: testing not done

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

Recommendations for Definition of Placental Infection

Recognizing that there may be a difference between what represents the most stringent criteria for defining placental infection and what can be done in the greatest number of real-world settings, we seek to offer guidance on definitions of placental SARS-CoV-2 infection in clinical settings and in biomedical investigation.

As such, we offer 5 levels of rigor for detecting placental infection with SARS-CoV-2 (both acute and current or persistent) (Table 1), followed by a pragmatic approach for defining placental infection. For each individual investigation, we suggest choosing 1 (or more) of the 5 categories based on the level of rigor desired by the investigator or clinician and what tools are available for implementation. Our recommended diagnostic criteria are provided in Table 2 . In addition, we provide instruction for handling, processing, and examination of the placenta (with additional sample collection, handling, and storage information in NICHD, National Institutes of Health, and United States Department of Health and Human Services (2021). “Promoting Data Harmonization to Accelerate COVID-19 Pregnancy Research,” Biospecimens Common Data Elements section. Retrieved from: https://www.niehs.nih.gov/research/programs/disaster/database/nihpromotin_dataharmonizationacceleratecovid19pregnancyresearchbiomedicalpsychosocialbiospecimens_vf.pdf.) The recommendations for reporting in scientific manuscripts are presented in Table 3 .

Table 2

Definition specifics of placental infection with SARS-CoV-2 in order of rigor

RNA-ISH positive signal in syncytial and cytotrophoblast (10× original)

RNA-ISH, RNA in situ hybridization.

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

Antispike protein IHC with signal in syncytial and cytotrophoblast (20× original)

IHC, immunohistochemistry.

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

RNA-ISH positive signal in syncytial and cytotrophoblast (10× original)

RNA-ISH, RNA in situ hybridization.

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

Antispike protein IHC with signal in syncytial and cytotrophoblast (20× original)

IHC, immunohistochemistry.

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

Definite: documentation of viral presence, location in the placenta tissues, and replication, by:

1) RNA probe to the antisense strand, showing a positive signal in placental tissues with appropriate positive and negative controls (validated reagents and sample protocols are detailed in Appendix A)

or

2) Positive staining by RNA-ISH for double-stranded RNA, produced as replication intermediate for positive-sense RNA virus in placental tissues with appropriate positive and negative controls (validated reagents and sample protocols are detailed in Appendix A)

Probable: documentation of viral proteins or RNA within placental tissues, without evidence of active replication via:

1) RNA probe to the positive-sense strand, showing a positive signal in placental tissues with appropriate positive and negative controls (Figure 3 ).Figure 3

or

2) Positive staining by immunohistochemistry in placental tissues with antibodies directed to viral proteins, with appropriate positive and negative controls (validated reagents and sample protocols are detailed in Appendix B) (Figure 4 ).Figure 4

Possible: less specific detection of virus. These approaches could be detecting viral particles engulfed by macrophages rather than actively replicating virus. RT-PCR of placental homogenates theoretically may have a positive result owing to maternal viremia (although this is a rare entity), rather than placental involvement.

1) RT-PCR detection or quantification of viral RNA in PBS-rinsed placental homogenates, no tissue localization (validated reagents and sample protocols are summarized in Appendix C).

Note that an alternative approach is a 2-step approach, in which RT-PCR is used as a screen and then followed up with one of the methods recommended to confirm “definite” or “probable” infection. This hybrid/2-step approach would be more rigorous than RT-PCR alone and potentially more sensitive than the “definite” and “probable” approaches.

2) Electron microscopic detection of viral-like particles in placental tissues.

Unlikely: Negative results from any of the above tests

No testing: placenta not tested

PBS, phosphate-buffered saline; RNA-ISH, RNA in situ hybridization; RT-PCR, reverse transcription polymerase chain reaction.

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

Table 3

Recommended reporting guidelines for scientific manuscripts

Scientific manuscripts that report on placental detection of SARS-CoV-2 should report on:

(1) Sampling method (including location and number of sites sampled)

(2) Time from delivery to sample preservation

(3) Method of sample preservation

(4) Method of detection used (and in the discussion reflect strengths and limitations of that method)

Other issues of importance to report:

(1) The timing of the maternal infection in relationship to the delivery

(2) Maternal disease severity, recommend using the National Institutes of Health51 criteria

(3) The strain of virus, if known

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

RNA probe to the positive-sense strand, showing a positive signal in placental tissues with appropriate positive and negative controls (Figure 3 ).

Figure 3

RNA-ISH positive signal in syncytial and cytotrophoblast (10× original)

RNA-ISH, RNA in situ hybridization.

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

Positive staining by immunohistochemistry in placental tissues with antibodies directed to viral proteins, with appropriate positive and negative controls (validated reagents and sample protocols are detailed in Appendix B) (Figure 4 ).

Figure 4

Antispike protein IHC with signal in syncytial and cytotrophoblast (20× original)

IHC, immunohistochemistry.

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

Maternal disease severity, recommend using the National Institutes of Health criteria

Recommended placental handling, processing, and examination

For RT-PCR testing, we recommend at least 2 fresh placental samples (0.5 cm3) taken within 20 minutes of placental delivery (and not >1 hour after placental delivery to avoid RNA degradation), best obtained in the delivery room by appropriately protected and trained individuals, from the fetal side of the placenta (Figures 1 and 2 ) rinsed in sterile normal saline or phosphate-buffered saline, and then either snap-frozen (in liquid nitrogen or on dry ice until placed in −80°C) or placed in 5 to 10 volumes of RNAlater (Thermo Fisher Scientific, Waltham, MA) for subsequent freezing and storage at −80°C. Biopsies should be taken from a midpoint between the chorionic and basal plates and midway between the cord insertion and the placental edge (at least 3 cm from the cord insertion and 3 cm from the placental edge) to ensure viable villous parenchyma is collected (Figures 1 and 2). The fetal membranes should be dissected off the fetal surface and not included in the biopsy. Notably, 2 samples are recommended for protection against failure of RNA extraction and for biological sampling diversity of different placental regions, given that patchy infection is possible. Snap-frozen biopsies may permit single-cell RNA-Seq/nuc-Seq approaches, other RNA analyses, and protein analyses, whereas biopsies preserved in RNAlater can be used for RNA/DNA analyses and protein analyses, but not for scRNA-Seq/nuc-Seq. RNA quality is likely better for longer periods of time with the use of RNAlater. For RNA-ISH and immunohistochemistry studies, formalin-fixed paraffin-embedded full-thickness placental parenchyma cut at 5 μm onto glass slides should be used. All studied placentas should be examined and reported promptly by pathology following the Amsterdam Criteria for sampling and histologic diagnoses.

Figure 1

Gross photograph of the chorionic plate (fetal side) of a normal (uninfected) placenta

The white line is cut site for Figure 2 slab section. The white zeroes are possible biopsy sites.

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

Figure 2

Gross photograph of the full-thickness slab section through the white line in Figure 1

The white brace highlights chorionic plate—fetal membranes should be excluded from the biopsy. The white bracket indicates fetal parenchyma as target for sampling; approximately 0.5 cm depth is recommended. The white arrows highlight the brace and the bracket.

Roberts et al. Consensus definition of SARS-CoV-2 placental infection. Am J Obstet Gynecol 2021.

Conclusion

We submit this set of recommendations to standardize the definition of SARS-CoV-2 infection of the placenta. These recommendations are made by a consensus panel of experts in the fields of obstetrics, virology, placental pathology, infectious disease, immunology, and molecular biology. The definitions are tiered by rigor of the diagnostic technique. Investigators and clinical care providers are encouraged to use the most rigorous method available for documenting placental infection in studies and in clinical diagnosis. Comparisons across studies are more valuable when the studies use the same standardized methods. We understand that not all, or perhaps not any, of these methods will be available at all institutions, but we predict that most institutions will have at least one recommended method at their disposal, and collaboration or consultation is advised for those that do not have any recommended method available. We anticipate that utilization of these recommended definitions of placental infection by SARS-COV-2 will facilitate comparisons of results among studies and that improved interpretation will follow, allowing for maximum impact of research in this area and optimization of patient care.

Immunohistochemistry (IHC): antibody-mediated localization of proteins in the tissue.

Negative-sense strand: provides evidence of viral replication in a positive-sense RNA virus (eg, SARS-CoV-2).

N-protein: the nucleocapsid RNA binding protein of the SARS-CoV-2 virus.

RNA in situ hybridization (RNA-ISH): molecular localization of the RNA transcript in the tissue.

RT-PCR: reverse transcription polymerase chain reaction for amplification of RNA.

Positive-sense strand: provides evidence of viral presence but not necessarily viral replication in a positive-sense RNA virus as in SARS-CoV-2.

RT-qPCR: quantitative reverse transcription polymerase chain reaction for amplification of RNA.

S-protein: the spike surface glycoprotein of the SARS-CoV-2 virus.

Viral replication: active/infectious viral reproduction.