Presence of SARS-CoV-2 in fetal organs via intraamniotic infection

Categories: Disease & Virus

November 21, 2025

Donor recruitment and sample collection

All autopsies were conducted with written informed consent from donors and the sample collections were approved by the Ethics Committee of the Shanghai First Maternity and Infant Hospital affiliated with Tongji University (permission numbers: KS2317, KS21343).

This study enrolled pregnant women from May 12, 2022 through June 6, 2023, who either had COVID-19 history during pregnancy (N = 18) or had never been infected (N = 6; Control donors). None of donors had COVID-19 history prior to her pregnancy. The COVID-19 history was determined by periodic PCR test ensured by local government. Donors’ pregnancies were terminated in the second trimester due to voluntary induction of labor, chromosomal abnormalities, anatomical structural abnormalities or other reasons. At the time of pregnancy termination, donors were negative on COVID-19 PCR test. The demographics of the donors were summarized in Supplementary Table 1. Detailed macroscopic dissections were performed on ice to collect tissue samples from multiple organs within 6 h after induction of labor (stored on ice), including proximal and distal sides of fetal membrane, fetal and maternal sides of placenta, umbilical cord, skin, heart, thymus, lung, gallbladder, liver, stomach, spleen, pancreas, small intestine, large intestine, ureter, bladder, kidney, adrenal gland, thyroid, trachea, esophagus, parotid gland, submandibular gland, tongue, cerebrum, cerebellum, skeletal muscle, uterus (female fetuses), ovary (female fetuses), and testis (male fetuses). Fresh tissues were snap frozen in liquid nitrogen and then stored at −80 °C for subsequent RNA extraction and data-independent acquisition proteomics assay. Tissues used for histopathologic/ISH analysis and transmission electron microscopy were freshly fixed with 10% neutral buffered formalin and 2.5% pentanediol for 24 h, respectively. All experiments were conducted by researchers who were negative on COVID-19 PCR test at the moment.

Digital droplet PCR (ddPCR) and subgenomic RNA analysis

Total RNA was isolated using a RNAprep Pure Tissue Kit (Tiangen Biotech, China) according to the manufacturer’s protocols. The QuantStudio 3D Digital PCR System and QuantStudio 3D Digital Chip Kit v2 (Life Technologies, Carlsbad, CA, USA, A26316) were used to detect and quantify SARS-CoV-2 nucleocapsid RNA (forward primer: 5′-TTACAAACATTGGCCGCAAA-3′; reverse primer 5′-GCGCGACATTCCGAAGAA-3′; probe 5′-FAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ1-3′)⁴⁸ and human GAPDH RNA (forward primer 5′-CAAGGCTGTGGGCAAGGT-3′; reverse primer 5′-VIC-GGAAGGCCATGCCAGTGA-MGB-3′; probe 5′-ATCCCTGAGCTGAACG-3′; designed using Primer Express Software v3.0.1) of samples. The raw data was collected and analyzed using QuantStudio 3D Analysis Suite (version 1.0, Thermo Fisher Scientific). Results were then normalized to copies of the N gene per nanogram of RNA input. The sample must be positive for the human GAPDH gene under the manufacturer’s limit of detection (LOD) with ≥4 copies per microliter to ensure successful RNA extraction and to be reported. After the background was removed according to the negative control samples, LOD of ≥5 copies per ng RNA input was considered to be SARS-CoV-2 N gene positive. To further detect the replicating virus generated during productive infection, we conducted one-step RT-qPCR assay targeting subgenomic RNA of the envelope (E) gene (Forward primer 5′-CGATCTCTTGTAGATCTGTTCTC-3′; Reverse primer 5′-ATATTGCAGCAGCACACA-3′; probe 5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1-3′)⁴⁹ using the One step RT-qPCR mix (Accurate Biotechnology, China, catalog no. RM0042) on a QuantStudio 5 Real-Time PCR system (version 1.3.1, Thermo Fisher Scientific). Primers and probes were synthesized by Thermo Fisher Scientific.

RNAscope in situ hybridization (ISH)

Tissues with SARS-CoV-2 N gene abundance higher than 10 copies per ng RNA were further submitted for RNA in situ hybridization using the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, USA) according to manufacturer protocols. Briefly, formalin-fixed and paraffin-embedded (FFPE) tissue blocks were cut at 4 μm. After the sections were mounted on slides, the slides were air dried at room temperature overnight, then baked and deparaffinized. After deparaffinization, the slides were applied with RNAscope hydrogen peroxide followed by target retrieval using the steamer. Then the slides were incubated with protease at 40 °C for the amount of time specified by the tissue pretreatment recommendation provided by manufacturer. After the digestion, the slides were hybridized for 2 h at 40 °C in the HybEZOven with probe-V-nCov2019-S (Advanced Cell Diagnostics, catalog no. 848561), probe-V-nCoV2019-ORF1ab (Advanced Cell Diagnostics, catalog no. 895661) or probe-V-nCoV2019-N (Advanced Cell Diagnostics, catalog no. 846081). In addition, 3-plex Negative Control Probe (Advanced Cell Diagnostics, catalog no. 320871) and 3-plex Positive Control Probe (Advanced Cell Diagnostics, catalog no. 320861) were used as a negative and positive control, respectively. Then the slides were counterstained with DAPI and mounted. Images were acquired with an automatic digital slide scanner (PANNORAMIC MIDI, 3DHISTECH, Hungary) and presented in the form of merge layers.

Immunofluorescence

FFPE slides of different organs were technically supported by the Manual IHC kit (Akoya, NEL861001KT) according to manufacturer protocols. The following dilutions of primary antibody were used: SARS-CoV-2 nucleocapsid antibody (Cell Signaling Technology, 26369S, USA), 1:1000; SARS-CoV2 orf3a antibody (Novus Biologicals, NBP3-15985, USA), 1:1000; SARS-CoV-2 Spike Glycoprotein S2 antibody (Novus Biologicals, NB100-56578SS), 1:1000; ACE2 antibody (Proteintech, 21115-1-AP), 1:1000; TMPRSS2 antibody (Proteintech, 14437-1-AP), 1:1000; HMGB1 antibody (Proteintech, 10829-1-AP), 1:1000; CD11B antibody (Novus Biologicals, NB110-89474SS), 1:1000; CD86 antibody (Affinity Bioscience, DF6332, China), 1:1000; Fibronectin (FN) antibody (Proteintech, 66042-1-Ig), 1:1000 for IF staining. After incubated with secondary antibodies, the signal was amplified using the fluorophore-conjugated tyramide amplification system (PerkinElmer, USA), followed by counterstaining with DAPI. Slides were scanned by an automatic digital slide scanner (PANNORAMIC MIDI). Positive cells were automatically counted using the Halo software (version 3.4.2986, Lndica Labs, USA).

Transmission electron microscopy (TEM)

A transmission electron microscope was used to detect virus-like particles in fetal organs. Tissues fixed in 2.5% pentanediol were washed, dehydrated, embedded and polymerized. Then the tissue blocks were cut to 70 nm thin and fished out onto the copper screen. Images were observed and captured by a H7650TEM (HITACHI, Japan) using RADIUS software (version 2.1, EMSIS GmbH, Germany).

Hematoxylin and Eosin (H&E) staining

FFPE slides of different organs were subjected to standard Hematoxylin and Eosin (H&E) staining for the detection of histological changes. Slides were deparaffinized, stained with H&E, and then subjected to a digital pathological section scanner (KFBIO, KF-PRO-400-HI). K-Viewer was used to determine lesion area in different sections.

Astral-DIA proteomic profiling

A total of 12 samples were analyzed (n = 2 vs 2 in each of three organs). Proteins were extracted by SDT (4% SDS, 100 mM Tris-HCl, pH 7.6) lysis buffer and quantified using a BCA Protein Assay Kit (Beyotime, China). DTT (final concentration at 40 mM) was added to each sample and mixed at 600 rpm for 1.5 h (37 °C). After the samples cooled to room temperature, IAA (final concentration at 20 mM) was added into the mixture to block reduced cysteine residues and the samples were incubated for 30 min in darkness. Next, the samples were transferred to the filters (Microcon units, 10 kDa). The filters were washed with 100 μl UA buffer three times and then 100 μl 25 mM NH₄HCO₃ buffer twice. Finally, trypsin was added to the samples (trypsin: protein (wt/wt) ratio was 1:50) and incubated at 37 °C for 15–18 h (overnight), and the resulting peptides were collected as a filtrate. The peptides of each sample were desalted on C18 Cartridges (Empore SPE Cartridges MCX, 30 μm, Waters), concentrated by vacuum centrifugation and reconstituted in 40 µl of 0.1% (v/v) formic acid. The peptide content was estimated by UV light spectral density at 280 nm. The iRT standard peptide was added to peptide segments. The peptides from each sample were analyzed by Orbitrap Astral mass spectrometer (Thermo Scientific) connected to an Vanquish Neo system liquid chromatography (Thermo Scientific) in the data independent acquisition (DIA) mode. Precursor ions were scanned at a mass range of 380–980 m/z, MS1 resolution was 240,000 at 200 m/z, Normalized AGC Target: 500%, Maximum IT: 5 ms. 299 windows were set for DIA mode in MS2 scanning, Isolation Window: 2 m/z, HCD Collision Energy: 25 eV, Normalized AGC Target: 500%, Maximum IT: 3 ms. DIA data was analyzed with DIA-NN 1.8.1. Main software parameters were set as follows: the enzyme as trypsin, the max miss cleaning site as 1, the fixed modification as Carbammimethyl (C), the dynamic modification was set to Oxidation (M) and Acetyl (Protein N-term), and the protein identified by database retrieval must pass the set filtering parameter FDR < 1%. The database was downloaded at website: http://www.uniprot.org. The input of GSEA is a protein expression matrix, in which the samples are divided into two groups, and the differentially expressed proteins between the two groups are found, and then sorted according to fold change to indicate the changing trend of protein expression between the two groups. The cluster profile package in R package (v4.4.4) was used in analysis.

Statistical analysis

Statistical analysis was performed using Graphpad Prism (version 9.0, GraphPad Software, USA). The Student’s t test was applied for comparison between independent groups. All data are presented as mean ± s.e.m. P < 0.05 was considered statistically significant.