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Review 1: "Single-cell RNA sequencing highlights a reduced function of natural killer and cytotoxic T cell in recovered COVID-19 pregnant women"

This study characterizes the immune landscape during active infection phase and recovery in pregnant females from COVID-19. Reviewers find the study potentially informative, with concerns over variability in gestation stage in different groups, and lack of functional follow-ups.

Published onSep 28, 2022
Review 1: "Single-cell RNA sequencing highlights a reduced function of natural killer and cytotoxic T cell in recovered COVID-19 pregnant women"
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Single-cell RNA sequencing highlights a reduced function of natural killer and cytotoxic T cell in recovered COVID-19 pregnant women

AbstractPregnancy is a complex phenomenon during which women undergo immense immunological change throughout this period. Having an infection with the SARS-CoV-2 virus leads to an additional burden on the highly stretched immune response. Some studies suggest that age-matched pregnant women are more prone to SARS-CoV-2 infection compared with normal healthy (non-pregnant) women, while alternative evidence proposed that pregnant women are neither susceptible nor develop severe symptoms. This discrepancy in different findings regarding the immune responses of pregnant women infected with SARS-CoV-2 virus is not well understood. In this study, we investigated how SARS-CoV-2 viral infection could modulate the immune landscape during the active infection phase and recovery in pregnant females. Using flow cytometry, we identified that intermediate effector CD8+ T cells were increased in pregnant women who had recovered from COVID-19 as opposed to those currently infected. Similarly, an increase in CD4+ T helper cells (early or late) during the recovered phase was observed during the recovery phase compared with infected pregnant women or healthy pregnant women, whilst infected pregnant women had a reduced number of late effector CD4+ T cells. CD3+CD4- CD8-NKT cells that diminished during active infection in contrast to healthy pregnant women were significant increase in recovered COVID-19 recovered pregnant women. Further, our single-cell RNA sequencing data revealed that infection of SARS-CoV-2 had changed the gene expression profile of monocytes, CD4+ effector cells and antibody producing B cells in convalescent as opposed to healthy pregnant women. Additionally, several genes with cytotoxic function, interferon signalling type I & II, and pro- and anti-inflammatory functions in natural killer cells and CD8+ cytotoxic T cells were compromised in recovered patients compared with healthy pregnant women. Overall, our study highlights that SARS-CoV-2 infection deranged the adaptive immune response in pregnant women and could be implicated in pregnancy complications in ongoing pregnancies.

RR:C19 Evidence Scale rating by reviewer:

  • Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.



Does the manuscript confirm previous work or refute the current understanding?

The study confirms previous work and further broadens COVID-19 effects in recovered pregnant women's immune system dysregulation. The number of CD4+ and CD8+ effector T cells was increased in pregnant women who recovered from COVID-19 infection compared to pregnant infected or healthy women. However, mature Natural Killer (NK) T cells percentage was decreased in recovered women compared to healthy women. Single-cell RNA-seq gene expression analysis between recovered and healthy pregnant women showed an aberrant expression in these cell populations suggesting a reduced cytotoxic function in recovered pregnant women.

How well does the manuscript position the work within the current literature/understanding?

This manuscript needs work before publishing. The results show that 19 pregnant women were enrolled in the study, but Table 1 and Methods Section present 21 pregnant women. This discrepancy should be explained or corrected in the results and in Table 1. The authors show that gene expression profiles were downregulated in COVID-19 recovered from pregnant women compared to healthy women. Furthermore, the Gene Ontology (GO) enrichment pathways analyzed relates to the downregulation of cytotoxic function pathways—for example, in Natural Killer (NK) and CD8 T cells. However, no validation results of gene expression profiles were performed in this work, and no functional assays were done to correlate with the functionality of the cells. The authors conclude that the functionality of NK and cytotoxic T cells are reduced only by gene expression analysis. This limits the interpretation of the results.

Also, the authors should discuss the possibility that the results could be affected by the time of recovery from COVID-19 when the sample was taken. This is important since infection in one recovered patient was three months before the sample collection. However, infection in the other three patients was 2, 10, or 17 days after the positive PCR test result. Pregnancy shows dynamic immune system changes at different gestational ages, which might affect the recovery response. There is also a possible bias in the study since three healthy women out of eleven were in the early second trimester of pregnancy (13-15 weeks), while 1/4 of recovered women were in the third trimester (>28 weeks).

Further, the single-cell gene expression between infected pregnant women versus recovered pregnant women was not included in the study and analysis. The work presents gene expression results for healthy pregnant women vs. recovered pregnant women. Information is also lacking in the figure legends.

Is there clarity regarding the recommended actions that result from the findings?

The results are generally clearly presented and well-structured, but modifications need to be done to enhance the quality of the manuscript.

Do authors pay attention to ethics, diversity, and inclusion?

Malaysian pregnant women were enrolled in this study; thus, race and ethnicity are not well represented, and therefore the generalizability of the results is not well supported.

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