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  1. Evaluation Summary:

    Yang et al. provide a scientifically sound and compelling manuscript characterizing mid-to-late gestation trophoblast and decidual organoids as ex vivo models to study vertically transmitted microbial infections, using human cytomegalovirus as a model pathogen. They demonstrate organoids have tissue-specific immunological responses and susceptibilities to viral infection.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Yang et al. provide a scientifically sound and compelling manuscript.

    -Very thorough study characterizing transcriptional and secretory responses of organoids derived from mid-to-late gestation at baseline and after viral infection.
    -Establishes organoids as an important model to study vertically transmitted microbial infections.

    -Discussion and characterization of what each organoid type is specifically modeling (orientation/cell types) is important for context to fully grasp inherent strengths and weakness of model.
    -Despite commonly indicating TO and DO are matched, the use of this matched specificity is not utilized either experimentally or in the interpretation of data.
    -Given gestational age can putatively impact outcome, a better understanding of the result of each organoid line in terms of specific gestational age derived is warranted.
    -There is only a single readout for viral infection with quantification being % infected organoid (which could include organoids with only a single cell or hundreds of cells infected). A more fine-tuned quantification seems necessary given the conclusions of the manuscript.

  3. Reviewer #2 (Public Review):

    This manuscript aims to better model infection at the maternal-fetal interface and describes the innate immune responses of trophoblast (TO) and decidual organoids (DO) in response to viral infection. The authors determined the antiviral responses of both organoids by bulk RNA sequencing and profiled secreted cytokines and chemokines without infection (basal levels), in response to RNA viruses as modeled by poly I:C treatment, and in response to human Cytomegalovirus (HCMV).

    The strengths and weaknesses of this manuscript are as follows:

    Strength 1) this is the first manuscript to show that trophoblast organoids can be derived from mid-late gestation time points (with slight modifications to the traditional TOM medium first described in Turco et al 2018). Until now, all organoids have been derived from first trimester and this limits the utilization of the model when later time points are desired. On the other hand, a weakness in this work is that there was no characterization of the resulting organoids proving that they indeed transcriptionally or epigenetically model a second-third trimester timepoint. It is possible that in vitro conditions could generate attracting forces (cell survival etc.) that make organoids derived from second or third trimesters similar to first trimester.

    Strength #2 is that they relate the TO model back to traditional 2-dimensional culture and show that their type III interferon secretion is similar, bolstering the validity of the model.

    Finally 3) the advantage of utilizing organoid model systems (as opposed to limited, artificial 2D and primary tissue explants) is that organoid model systems are composed of a more in vivo analogous system with biologically appropriate portions of one tissue or organ that can be systematically tested, as opposed to the highly artificial, flat, "hard" environment with limited diversity of cell types in traditional 2 dimensional culture and the highly variable, multiple tissue and cell type-contained primary tissue explant models.

    The largest weakness is that they conclude that EVTs contained in TOs are not susceptible to HCMV infection but their methodology is not ideal to investigate the question. TOs maintained in "TOM" (see Turco et al 2018) give rise to a few spontaneously differentiated early EVTs, but I am not convinced that these are mature, invasive EVTs (which could be susceptible as previously shown). The authors should repeat HCMV infections in EVT differentiated organoids (see Sheridan et al 2020) before concluding no infection of EVTs by HCMV.

    In terms of achieving the authors' aims, I believe they have made a significant discovery in the understanding of maternal-fetal viral infections. They have identified a number of transcriptional similarities and differences between DOs and TOs in response to a variety of viral stimuli. Additionally, they have characterized the secreted cytokine and chemokine responses of both organoid models. These datasets will be important to further understanding HCMV, a world-wide debilitating viral affront to women and developing fetuses and could inform future drug targeting and anti-viral interventions.