Over 150,000 pregnant women in the US have been infected with SARS-CoV-2, resulting in variable outcomes ranging from asymptomatic to severe/critical disease. While most experience mild or no symptoms, pregnant women with SARS-CoV-2 infection were initially, and subsequently shown to be at increased risk for hospitalization, ventilation, ICU admission, and preterm birth. Maternal mortality rates, however, were reported to be similar to non-pregnant women. Most neonates born to infected mothers test negative for SARS-CoV-2, and those testing positive generally have mild symptoms. The timing of mother-to-child transmission remains unclear, with possibilities including in utero, intrapartum, or postnatal transmission. While rare, in utero vertical transmission has been documented, likely via the hematogenous route requiring the virus to cross the maternal-fetal interface by infecting the syncytiotrophoblast layer of the placenta. The mechanisms of placental cell infection are under investigation; however, the co-expression of ACE-2 and TMPRSS2, canonical entry mediators, is negligible in placental cells. Placental cells can express non-canonical mediators, and vascular damage associated with SARS-CoV-2 infection could facilitate viral entry. While placental infection has been reported, it isn't confirmatory evidence of in utero transmission. The maternal inflammatory response, however, could have adverse effects on the offspring, prompting investigation of host immune responses in umbilical cord blood and the maternal-fetal interface.

Existing literature highlights the varied clinical presentations of COVID-19 in pregnant women, ranging from asymptomatic to severe illness. Studies have shown increased risks of adverse pregnancy outcomes like preterm birth and hospitalization in SARS-CoV-2-infected pregnant women. However, data on maternal mortality rates have been inconsistent. There's also conflicting evidence regarding the frequency and severity of neonatal SARS-CoV-2 infections, and the mechanisms of vertical transmission remain largely unknown. Previous research has investigated the expression of SARS-CoV-2 entry receptors in placental tissues, revealing limited expression of canonical receptors but potential roles for non-canonical entry pathways. The maternal inflammatory response to SARS-CoV-2 infection and its potential impact on the fetus are also areas of ongoing research.

This multidisciplinary study enrolled 23 pregnant women: 12 SARS-CoV-2 positive (eight asymptomatic, one mild, three severe) and 11 controls. Maternal blood was collected upon admission. Several analytical techniques were used, including: 1. **Serological analysis:** Measurement of SARS-CoV-2 IgM and IgG levels in maternal and cord blood. 2. **Multiplex cytokine assays:** Measurement of 20 cytokines in maternal and cord blood plasma. 3. **Immunophenotyping:** Analysis of leukocyte subpopulations and reactive oxygen species production by neutrophils and monocytes. 4. **Single-cell RNA sequencing (scRNA-seq):** Analysis of placental tissues (chorioamniotic membranes (CAM) and placental villi and basal plate (PVBP)) to identify cell types and differentially expressed genes. 5. **Bulk transcriptomics:** RNA sequencing of maternal and cord blood to identify differentially expressed genes. 6. **Viral RNA and protein detection:** RT-qPCR and immunohistochemistry to detect SARS-CoV-2 RNA and proteins in placental tissues. 7. **Microbiome diversity assessment:** 16S rRNA gene qPCR and sequencing to characterize the bacterial DNA load and profiles in placental tissues. 8. **Placental histopathology:** Examination of placental tissues for inflammatory lesions and other abnormalities. Statistical analyses included Mann-Whitney U-tests, linear mixed-effects models, logistic regression, Spearman's correlation, and PERMANOVA, depending on the specific data type.

The study revealed several key findings: 1. **Serological Response:** Pregnant women with SARS-CoV-2 infection had significantly higher levels of both IgM and IgG in their maternal blood compared to controls. However, only IgG was detected in the cord blood of neonates, suggesting a lack of acute fetal infection. 2. **Cytokine Response:** SARS-CoV-2-infected mothers showed increased levels of IL-8, IL-10, and IL-15. Importantly, both mothers and neonates exhibited increased IL-8 levels in their circulation. 3. **T-cell Response:** Pregnant women with SARS-CoV-2 infection showed reduced T-cell numbers, including specific subsets like CD4+ TCM, Th1-like, CD8+ TEM, and Tc17-like cells. This reduction was not observed in neonates. 4. **Placental Immune Response (scRNA-seq):** Single-cell RNA sequencing of placental tissues revealed significant alterations in the immune repertoire of placentas from infected women. These changes were primarily observed in maternal T cells and macrophages infiltrating the CAM, as well as their fetal stromal cells. Fetal T cells, however, were minimally altered. 5. **Blood Transcriptomics (Bulk RNA-seq):** Maternal blood and cord blood showed distinct transcriptomic changes in response to SARS-CoV-2 infection. Upregulated genes in maternal blood were associated with humoral responses, while downregulated genes were linked to phagocytosis and extracellular matrix organization. Cord blood showed upregulation of genes involved in defense against fungi and bacteria. Notably, correlations were observed between the blood transcriptomes and scRNA-seq data from the placenta. 6. **Absence of SARS-CoV-2 in Placenta:** Extensive testing (RT-qPCR and immunohistochemistry) failed to detect SARS-CoV-2 RNA or proteins in placental tissues from infected women, even those with severe COVID-19, indicating that placental infection is rare in largely asymptomatic populations. 7. **Placental Sterility:** Analysis of the placental microbiome showed no evidence that SARS-CoV-2 infection compromises placental sterility. Bacterial loads and profiles were primarily influenced by the mode of delivery (vaginal vs. cesarean).

This study demonstrates that SARS-CoV-2 infection during pregnancy primarily elicits maternal immune responses, both systemically and at the maternal-fetal interface. The absence of detectable IgM in the cord blood strongly suggests that in utero vertical transmission is uncommon in largely asymptomatic populations. The mild neonatal inflammatory response, however, warrants further investigation into potential long-term effects. The alteration of specific maternal T-cell subsets could affect maternal-fetal tolerance, though more research is needed to fully understand this impact. The significant changes in gene expression in fetal stromal cells are novel and highlight the need for further investigation into the functions of these cells. The consistent absence of viral RNA and proteins in placental tissue from our study population further emphasizes the rarity of placental infection by SARS-CoV-2. The finding that SARS-CoV-2 infection does not seem to compromise placental sterility challenges traditional views. The results have implications for clinical management and counseling of pregnant women during the COVID-19 pandemic.

This study provides comprehensive insight into the maternal-fetal immune responses to SARS-CoV-2 infection during pregnancy. The findings highlight the primarily maternal nature of the immune response, the rarity of placental infection, and the lack of evidence for significant compromise of placental sterility. Future research could focus on longitudinal studies to evaluate the long-term effects on both mother and child, further investigate the impact of different viral loads and disease severities, and explore potential therapeutic interventions to mitigate the maternal immune response.

The cross-sectional design limits inference on disease progression and severity timelines. The limited number of severe COVID-19 cases might restrict the generalizability of findings regarding severe disease. The study population was predominantly asymptomatic, potentially limiting the generalizability of findings to cases with more severe disease manifestations. The reliance on nasopharyngeal swabs to confirm infection could have introduced some level of misclassification.