SARS-CoV-2 can infect human embryos

Abstract

The spread of SARS-CoV-2 has led to a devastating pandemic, with infections resulting in a range of symptoms collectively known as COVID-19. The full repertoire of human tissues and organs susceptible to infection is an area of active investigation, and some studies have implicated the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood, and particularly the impact on early embryogenesis and establishment of a pregnancy are not known. In this work, we explore the susceptibility of early human embryos to SARS-CoV-2 infection. By using RNA-seq and immunofluorescence, we note that ACE2 and TMPRSS2, two canonical cell entry factors for SARS-CoV-2, are co-expressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos. For the purpose of viral entry studies, we used fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, and we observe robust infection of trophectoderm cells. This permissiveness could be attenuated with blocking antibodies targeting S or ACE2. When exposing human blastocysts to the live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath.

Figure 1

Expression of Genes Involved in SARS-CoV-2 Infection in Embryo Cells. Violin plots showing log10-normalized expression profiles obtained by RNA-seq performed on trophectoderm biopsies of blastocysts. Each data point represents one embryo (n = 24). Each trophectoderm biopsy consisted of 5–10 cells. (A) Canonical SARS-CoV-2 entry factors ACE2 and TMPRSS2. (B) Proposed alternative/ancillary mediators of SARS-CoV-2 entry, replication, traffic, and assembly.

Figure 2

Localization of ACE2 and TMPRSS2 in Embryos. Maximum intensity projections (MIPs) of confocal z-stacks of blastocysts, showing nuclei (blue) and ACE2 or TMPRSS2 (white). Pink arrowheads point to cell membranes. Scale bars represent 50 µm in low magnification panels, and 10 µm in high magnification panels. Representative images from independent biological replicates (n = 5 embryos for each factor).

Figure 3

Embryo Infection by Reporter Virions Pseudotyped with the S protein of SARS-CoV-2. Sample confocal MIP images of embryos infected with (A) HIV- based or (B) VSV∆G -based reporter virions pseudotyped with the S protein from SARS-CoV-2. Pink arrowheads point to cells displaying robust signal. Scale bars represent 20 µm. Representative images from independent biological replicates (n = 15 embryos with HIV- based virion, n = 7 embryos with VSV∆G -based virion).

Figure 4

Embryos Exposed to Live SARS-CoV-2 are Susceptible to Infection. Fluorescent signal indicates cells infected with icSARS-CoV-2-mNeonGreen. (A) Four sample blastocysts exposed to the virus. One blastocyst has not initiated hatching and shows no evidence of infection, two blastocysts are at early stages of hatching (one shows evidence of infection in the herniating cells), and one blastocyst is in advanced hatching phase showing high incidence of infected cells. (B) High magnification of blastocyst with high incidence of positive cells, with two panels showing the mNeonGreen channel at different focus depths (z-axis). White arrowheads point to positive herniating cells in hatching blastocysts, pink arrowhead points to positive cells in the zona pellucida compartment of a hatching blastocyst. Representative images from independent biological replicates (n = 19 embryos). icm = inner cell mass. bc = blastocoel cavity. zp = zona pellucida. Scale bar represents 100 µm.

Figure 5

The Zona Pellucida Might Confer Protection Against SARS-CoV-2 Infection. (A) Pie charts indicating the proportion of blastocysts displaying evidence of infection at three separate stages of development (before, during, and after hatching), when exposed to live SARS-CoV-2 expressing a fluorescent reporter (in the presence or absence of control IgG blocking antibody). Non-hatching blastocysts have an intact zona pellucida, hatching blastocysts have some cells herniating out of the zona pellucida opening as well as some cells confined in the zona pellucida compartment, and fully hatched blastocysts have emerged completely out of the zona pellucida. (B) Only example of a non-hatching blastocyst with zona pellucida-encapsulated positive cells (orange arrowhead). (C) Example of a hatching blastocyst with positive cells exclusively in the herniating compartment. (D,E) Examples of hatching blastocysts, with positive cells in the herniating compartment (white arrowheads) as well as in cells proximal to the zona pellucida opening (pink arrowheads). zp = zona pellucida. Scale bars represent 50 µm.

Figure 6

Embryo Health is Negatively Affected by SARS-CoV-2 Infection. Sample images of infected embryos with various degrees of cytopathic effects. (A,B) Embryo with cell damage (yellow arrowheads) in fluorescent (infected) cells in the herniating and zona pellucida compartments. (C) Infected herniating embryo (bottom) with severely affected health, and non-infected embryo (top) with good health. (D) Hatched infected embryo next to empty zona pellucida, displaying global cell death. (E,F) Examples of infected embryos displaying total collapse and death. (G) Summary of embryo health 12–16 h after exposure to SARS-CoV-2. Top bar includes all embryos with evidence of infection (fluorescent cells). Bottom bar includes all embryos with no evidence of infection (no fluorescent cells). Scale bars represent 50 µm.