Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids

  1. Thomas M Rawlings
  2. Komal Makwana
  3. Deborah M Taylor
  4. Matteo A Molè
  5. Katherine J Fishwick
  6. Maria Tryfonos
  7. Joshua Odendaal
  8. Amelia Hawkes
  9. Magdalena Zernicka-Goetz
  10. Geraldine M Hartshorne
  11. Jan J Brosens
  12. Emma S Lucas

  • Curated by eLife

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    Evaluation Summary:

    This manuscript is of broad interest to scientists in the field of reproduction and has clinical relevance. It builds on innovative studies interrogating the impact of cell senescence on stromal cell differentiation and embryo implantation. It presents the development of a novel co-culture system taking advantage of organoid technology to study cell-specific interactions and outcomes.

    (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. Reviewers #1, #2, and #3 agreed to share their names with the authors.)

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Abstract

Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterized endometrial assembloids, consisting of gland-like organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by eliminating the emergence of senescent decidual cells. In co-culture experiments, accelerated decidualization resulted in entrapment of collapsed human blastocysts in a robust, static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings suggest that decidual senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

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  1. Version published to 10.7554/elife.69603 on eLife
  2. eLife

    Author Response:

    Reviewer #1:

    Understanding the underlying mechanisms of stromal cell decidualization and cellular interactions in the uterus is vital to improving women's reproductive health and pregnancy outcomes. This manuscript builds on a series of innovative studies interrogating the impact of cell senescence on decidualization and embryo implantation. A novel decidualization co-culture system containing endometrial epithelial organoids and stromal cells (assembloids) was established. The authors utilize this model in combination with single cell RNA-sequencing and receptor-ligand analysis to interrogate the mechanisms underlying decidual cell senescence and their subsequent roles in embryo implantation. Notably, the authors move beyond predictive bioinformatics and utilize pharmacological inhibition to alter the developmental trajectory of decidualizing cells, resulting in an altered assembloid environment and ultimately impeding human blastocyst development. Overall, this manuscript provides foundational information that will help design definitive and mechanistic studies in the future. The data from this paper will be of general interest to those studying cell-type-specific interactions including both reproductive scientists and clinicians.

    We are grateful for these supportive comments.

    Reviewer #2:

    In this interesting and well written paper, the authors employ organoid culture, single cell transcriptomics and cell-cell interaction mapping and embryo-co-culture to investigate the role of senescent endometrial cells in implantation biology. The organoids consist of primary uterine epithelial cells and stromal fibroblasts. Transition to luteal phase endometrium is induced by MPA (an artificial progestin) and a membrane permeable cAMP derivative as in in vitro stromal cell decidualization. The so treated organoids are subjected to single cell transcriptomic analysis to reveal the cellular diversity induced in these constructs. Most importantly the authors report an unexpected degree of cellular diversity, both in the epithelial as well as in the stromal compartment, both include cells interpreted as senescent cells, and in the stromal compartment also a clearly distinct pre-decidual cell population. A ligand - receptor analysis suggests that the latter two populations are characterized by a strong engagement of the receptor tyrosine kinase signaling pathways, which gave them a chance to specifically address these cells with a tyrosine kinase inhibitor. They were able to produce decidualized organoids without senescent cells which allowed them to demonstrate that embryo implantation into the endometrial organoids is impossible without senescent cells, while it is readily happening in the presence of senescent stromal cells. The lack of uNK cells, necessary to limit excessive senescence, probably limits the stability of these cultures. This is the most direct evidence to date for a physiological role of senescent cells in embryo implantation.

    Thank you; this is an accurate summary of our findings.

    The main strength of the paper consists of the creative combination of organoid culture and single cell technology, revealing both cell state/type heterogeneity and cell-cell communication networks and the experimental test of hypotheses derived from the latter. Naturally this study is a waypoint towards more complete in vitro models of the in vivo situation, by the lack of leukocytes and blood vessels. There are also some questions about the exact details of the experimental protocol, but the robust, biologically interesting and meaningful results speak for themselves.

    We agree with the Reviewer that our model is an intermediate step towards increased cellular complexity. There are, however, some important hurdles to overcome. For example, while it is possible to co-culture immune cells in our model, cell motility is greatly restricted by the gel properties. Technical issues like these will need to be overcome before additional cellular complexity can be achieved.

    One aspect that should be justified in the paper is the use of the MPA/cAMP protocol to decidualize the organoids. This is the standard protocol for decidualizing stromal fibroblasts, and circumvents the lack of epithelial cells in standard stromal culture, essentially replacing the effects of epithelial signals with a downstream second messenger, cAMP. In this context it is not clear what this treatment is supposed to be simulating. In humans receptivity is reached with systemic progesterone. A treatment with proteases and/or IL1 could simulate the presence of the embryo. To properly interpret the results using the MPA/cAMP protocol a discussion of this point would be helpful to the reader.

    For initial characterisation of decidualizing assembloids, we decided to use our standard differentiation protocol (cAMP/MPA), first developed over a quarter of a century ago, partly because of its demonstrable robustness and partly because it has been used in other endometrial organoid studies (e.g. Fitzgerald et al., PMID: 31666317). However, we acknowledge the Reviewer’s point that there is a need to revisit the physiological drivers of decidualization, especially those activating the cAMP/PKA pathway in stromal cells. A great candidate is PGE2 and, in a preliminary experiment, PGE2 production by gland-like organoids was found markedly induced by relaxin, suggesting a potential stromal-epithelial feedback loop. However, we respectfully wish to argue that such complex studies are outside the scope of the present investigations.

    The authors interpret the epithelial compartment of their organoids as representing uterine gland epithelium. It is not clear why the authors do not also expect luminal epithelium (LE) identity to be present, and in particular since some key changes constituting the window of implantation are affecting the luminal epithelium. Is it possible that some of the epithelial diversity revealed in their single cell classification are actually LE cells? In particular the cells called "transitional" could be seen as LE cells, as a loss of polarity towards a mesenchymal phenotype is part of their biology during the window of receptivity.

    We refer the Reviewer to our previous response (Essential Revisions, point 2). However, the Reviewer raises an important point regarding the origins of luminal epithelial cells. In our opinion, luminal epithelium in cycling human endometrium is likely of mixed origins, although we do not have direct experimental evidence in support of this statement. However, rapid epithelization is the principal mechanism to limit menstrual blood loss and while this could involve rapid proliferation from remnant epithelium, compelling evidence have implicated ‘transitional’ cells (MET). We previously reported that mid-luteum endometrial surface epithelium is characterised by ‘stretches’ of P16INK4+ cells interspersed by P16INK4- cells. Hence, it is conceivable that cell turnover in luminal epithelium is greater and more dynamic than currently appreciated, even during the implantation window. Further, we recently reported the presence of similarly ‘ambiguous’ cells expressing both epithelial and stromal/mesenchymal genes in single- cell RNA-seq analysis of fresh luteal phase endometrial biopsies (PMID: 31965050), as stated in the Discussion of the current paper.

    The experimental protocol also needs a little interpretation: the authors grow their organoids for four days in "expansion media" [simulating the proliferative phase of the menstrual cycle] and some of those samples are then subjected to SC analysis. Another set of cultures is also subjected to differentiation media for an additional 4 days simulating transition to luteal phase and receptivity. Comparing the expansion media organoids only with the treated ones allows us to see what happens in this simulation of the transition to luteal phase, and as such this is OK. However, the result is never the less confounded by the fact that the treated organoids are older than the "expansion media only" samples. A control comparison with organoids treated 4 days with expansion media and then four days with differentiation media but without the MPA and cAMP would be helpful to disentangle the hormone/cAMP effects and the age related changes in culture.

    We appreciate the comments about the experimental design. However, in our opinion, maintaining undifferentiated assembloids for a further 4 days in the absence of differentiation stimuli would make comparison with decidualized assembloids potentially less informative than in the approach we chose to take. Our aim was to mimic the temporal progression from proliferative phase to secretory phase endometrium, at which point cell division ceases and transformation of the cells ensues. Arguably, maintaining undifferentiated assembloids in minimal differentiation medium without differentiation signals would lead to continued proliferation and growth, heightened stress responses, and potentially erroneous observations. However, we will keep this suggestion in mind for future experiments.

    Reviewer #3:

    In their study, authors designed a novel human endometrium research model, which they refer to as assembloids, containing not only the endometrium epithelial cells (as standard organoid models do), but also the tissue stromal cells. Once developed and well characterized using, among others, state-of-the-art single-cell RNA-sequencing, the authors showed its application potential by dipping into a candidate cause of endometrial declined function and receptivity, i.e. dysbalanced senescence. Culminating in the study is the addition of human (spare IVF) embryos to the developed endometrial assembloid with and without senescence perturbation, which is a first step toward in vitro deciphering human embryo-endometrium interaction in health and disease.

    We thank the Reviewer for this accurate interpretation of our results.

    The study has multiple strengths. Central are the design and detailed characterization of this new endometrium assembloid model, and the demonstration of its applicability for endometrial deficiency studies regarding biology and embryo interaction. The finding that cellular complexity is recapitulated in the assembloid culture and that cell types and states (in particular, senescence) mimic in vivo decidualization are major achievements. The benchmarking with in vivo data is highly interesting for the field. Limitations may be situated in the use of a rather crude method to inhibit cellular stress and senescence (i.e. using a generally acting tyrosine kinase inhibitor) and the premature immediate-generalization of findings to multiple fertility problems without the needed grounds so far. Although definitely a strong advancement in the field, adding still other endometrial cells, most importantly of luminal epithelial cells but also innate immune cells, will in the future further perfect the model.

    We agree that dasatinib has broad actions and more specific inhibitors are currently being tested in the lab. However, as detailed below, the expression profiles of senescent cells pointed towards dasatinib as a relevant inhibitor. Further, dasatinib has been shown to be effective in preventing uterine ageing in mice (PMID: 3195515).

    Adding complexity to the assembloid model is also an ongoing focus of our work but, as outlined in our response to Reviewer 1, major technical hurdles related to gel properties will need to be overcome first.

    The authors achieved their aims of establishing and characterizing a new, straightforward endometrium tissue model. Moreover, they achieved to applying this new tool to start unraveling causes of endometrium non-receptivity or ill-performance, in particular regarding (dys-)balanced senescence. Together, the study presents a promising path along which human (in-)fertility research can develop, to provide basic and translational insights in reproductive biology and into (deficient) fertility which may eventually be taken to the clinic to improving pregnancy chances.

    We thank the Reviewer for these supportive comments.

    Reviewer #4:

    Rawlings et al. investigated endometrial mechanisms that may underlie reproductive disorders such as recurrent implantation failure and pregnancy loss. The proper decidualization of the endometrium is essential for correct embryo implantation and this process is tightly controlled by hormone action during the menstrual cycle. The authors hypothesized that acute senescence in the decidualizing endometrium is necessary for successful embryo implantation. They also sought to characterize gene expression changes that occur in stromal and epithelial compartments as a result of decidualization.

    The authors used a novel assembloid model of endometrial culture to investigate how endometrial epithelial and stromal cells respond to decidualization and found that both epithelial and stromal cells displayed distinct gene expression signature groups before and after decidualization. They successfully showed that the cellular stress that occurs during decidualization can directly affect the degree to which decidual endometrial cells undergo senescence. By culturing their endometrial assembloids with human embryos, they were able to convincingly demonstrate that endometrial decidual senescence is necessary to allow the embryo to grow and invade after implantation.

    This work will be important to the endometrial biology field as well as to clinicians. In addition to offering a mechanism for some types of implantation failure and recurrent pregnancy loss, the authors showed that treatment with a tyrosine kinase inhibitor can modulate pre-decidual stress and decidual senescence, suggesting that endometrial receptivity could one day be manipulated pharmacologically.

    We greatly appreciate this endorsement of our study and indeed consider assembloids as an informative model to evaluate new therapeutics to modulate endometrial receptivity.

    Strengths:

    The major strength of this paper is their assembloid-embryo coculture model, which provides strong evidence that a lack of endometrial decidual senescence results in a lack of embryo growth and failure of the embryo to invade into the endometrial assembloid. Although endometrial organoid models containing both epithelial and stromal cells already exist, the assembloid model allows the visualization of embryo growth and invasion. The single-cell RNA-sequencing of the assembloids convincingly demonstrates the existence of different populations of endometrial epithelial and stromal cells displaying different gene expression profiles before and after decidualization. The separation into these groups is very clean and the gene expression correlates with endometrial gene expression across the cycle remarkably well.

    We thank the Reviewer.

    Weaknesses:

    Given that the women who contributed endometrial tissue to this study were attending an Implantation Research Clinic and were mostly nulliparous with past first trimester pregnancy loss, it is unclear whether the endometrial samples used in this study is representative of a healthy condition.

    When it comes to implantation and pregnancy, a binary classification of ‘heatlhy’ and ‘unhealthy’ subjects is not particularly useful or easily definable. Women who have had a miscarriage, even multiple consecutive losses, often achieve successful pregnancies; and a successful pregnancy, even multiple successful pregnancies, does not preclude a future miscarriage(s). This clinical reality also applies for implantation failure after IVF treatment. This should not be surprising as the endometrium is a cycling tissue that leads to a midluteal implantation environment under external homeostatic control of NK cells and bone marrow- derived progenitors (both recruited from the circulation). Thus, the likelihood of reproductive failure caused by an endometrial defect is much more likely to reflect the stringency of homeostatic control and the frequency of cycles resulting in an abnormal peri-implantation environment. Put differently, the notion that the endometrium ‘carbon-copies’ itself in each cycle, leading to a permanent ‘normal’ or ‘abnormal’ state, is mostly for the birds and not grounded in either clinical reality or biology.

    The rationale for using a minimal differentiation medium rather than the differentiation medium that has been established in the literature is unclear. In particular, the induction of glandular differentiation in endometrial assembloids by NAC, an antioxidant, deserves some discussion.

    The established medium for endometrial organoids (based on Turco et al., PMID: 28394884 but also highly similar to Boretto et al., PMID: 28442471) contains various mitogens and pathway inhibitors and modulators, designed to allow the establishment of epithelial organoids and facilitate the proliferation in the absence of supporting cells (i.e. stroma). We reasoned that (i) some of these factors may interfere with differentiation, and (ii) that the addition of the stromal cells to the culture should negate the need to provide additional stroma- derived growth factors. As described above, in response to Reviewer 2, the concentration of NAC added to the medium is low and does not appear to interfere with differentiation responses in either the gland-like epithelial structures or stroma.

    Additionally, some explanation as to why the authors chose to treat their assembloid cultures with decidualization cocktail for only four days, when decidualization in vivo occurs over a much longer period, would be helpful.

    As stated in the manuscript, this timepoint was chosen based on previous reconstruction of the decidual pathway in 2D cultures at single-cell level, demonstrating the emergence on day 4 of both decidual and senescent decidual populations (PMID: 31965050). Because assembloids do not as yet harbour uterine NK cells, extending the cultures with several days beyond this time-point will lead to progressive disintegration because of unopposed SASP.

    The authors state that EpS5 is a population of senescent epithelial cells producing SASP based on gene expression data. It would be more convincing if the authors could provide other evidence to characterize these cells as senescent.

    As shown in Figure 3-figure supplement 1 of the manuscript, the EpS5 population highly express CDKN1A (p21) and CDKN2A (p16) as well as numerous genes encoding for canonical SASP components (i.e. meeting the widely accepted criteria of cellular senescence). We also demonstrated that SASP produced by senescent epithelial cells is distinct from that of senescent decidual cells. We previously reported that P16-positive glandular and luminal epithelial cells emerge in mid-luteal endometrium, along with a modest but discernible rise P16-positive stromal cells, before rising sharply in late-luteal phase samples (PMID: 29227245).

    The authors draw many conclusions based on data from the CellPhoneDB computational tool, but it is unclear how the authors chose the input for this tool and whether the output of this program was validated in any way.

    CellPhoneDB is an online, publicly available repository of ligands, receptors and their interactions (https://github.com/Teichlab/cellphonedb), which integrates various existing datasets and new manually reviewed information. In order to use this computational tool, expression counts and cell metadata were extracted from our single-cell data for decidualizing cells (i.e. those populations present in D4 cultures) according to pipelines provided by the CellPhoneDB vignette. The CellphoneDB package then derives enriched receptor–ligand interactions between two cell types based on expression of a receptor by one cell type and a ligand by another cell type (as described here: https://www.cellphonedb.org/explore-sc-rna-seq). We chose to exclude integrin-interactions from our analysis and focus on cell-cell interactions rather than cell-ECM, but future investigation of these is certainly of interest to the progression of the model.

  3. eLife

    Evaluation Summary:

    This manuscript is of broad interest to scientists in the field of reproduction and has clinical relevance. It builds on innovative studies interrogating the impact of cell senescence on stromal cell differentiation and embryo implantation. It presents the development of a novel co-culture system taking advantage of organoid technology to study cell-specific interactions and outcomes.

    (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. Reviewers #1, #2, and #3 agreed to share their names with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Understanding the underlying mechanisms of stromal cell decidualization and cellular interactions in the uterus is vital to improving women's reproductive health and pregnancy outcomes. This manuscript builds on a series of innovative studies interrogating the impact of cell senescence on decidualization and embryo implantation. A novel decidualization co-culture system containing endometrial epithelial organoids and stromal cells (assembloids) was established. The authors utilize this model in combination with single cell RNA-sequencing and receptor-ligand analysis to interrogate the mechanisms underlying decidual cell senescence and their subsequent roles in embryo implantation. Notably, the authors move beyond predictive bioinformatics and utilize pharmacological inhibition to alter the developmental trajectory of decidualizing cells, resulting in an altered assembloid environment and ultimately impeding human blastocyst development. Overall, this manuscript provides foundational information that will help design definitive and mechanistic studies in the future. The data from this paper will be of general interest to those studying cell-type-specific interactions including both reproductive scientists and clinicians.

  5. eLife

    Reviewer #2 (Public Review):

    In this interesting and well written paper, the authors employ organoid culture, single cell transcriptomics and cell-cell interaction mapping and embryo-co-culture to investigate the role of senescent endometrial cells in implantation biology. The organoids consist of primary uterine epithelial cells and stromal fibroblasts. Transition to luteal phase endometrium is induced by MPA (an artificial progestin) and a membrane permeable cAMP derivative as in in vitro stromal cell decidualization. The so treated organoids are subjected to single cell transcriptomic analysis to reveal the cellular diversity induced in these constructs. Most importantly the authors report an unexpected degree of cellular diversity, both in the epithelial as well as in the stromal compartment, both include cells interpreted as senescent cells, and in the stromal compartment also a clearly distinct pre-decidual cell population. A ligand - receptor analysis suggests that the latter two populations are characterized by a strong engagement of the receptor tyrosine kinase signaling pathways, which gave them a chance to specifically address these cells with a tyrosine kinase inhibitor. They were able to produce decidualized organoids without senescent cells which allowed them to demonstrate that embryo implantation into the endometrial organoids is impossible without senescent cells, while it is readily happening in the presence of senescent stromal cells. The lack of uNK cells, necessary to limit excessive senescence, probably limits the stability of these cultures. This is the most direct evidence to date for a physiological role of senescent cells in embryo implantation.

    The main strength of the paper consists of the creative combination of organoid culture and single cell technology, revealing both cell state/type heterogeneity and cell-cell communication networks and the experimental test of hypotheses derived from the latter. Naturally this study is a waypoint towards more complete in vitro models of the in vivo situation, by the lack of leukocytes and blood vessels. There are also some questions about the exact details of the experimental protocol, but the robust, biologically interesting and meaningful results speak for themselves.

    One aspect that should be justified in the paper is the use of the MPA/cAMP protocol to decidualize the organoids. This is the standard protocol for decidualizing stromal fibroblasts, and circumvents the lack of epithelial cells in standard stromal culture, essentially replacing the effects of epithelial signals with a downstream second messenger, cAMP. In this context it is not clear what this treatment is supposed to be simulating. In humans receptivity is reached with systemic progesterone. A treatment with proteases and/or IL1 could simulate the presence of the embryo. To properly interpret the results using the MPA/cAMP protocol a discussion of this point would be helpful to the reader.

    The authors interpret the epithelial compartment of their organoids as representing uterine gland epithelium. It is not clear why the authors do not also expect luminal epithelium (LE) identity to be present, and in particular since some key changes constituting the window of implantation are affecting the luminal epithelium. Is it possible that some of the epithelial diversity revealed in their single cell classification are actually LE cells? In particular the cells called "transitional" could be seen as LE cells, as a loss of polarity towards a mesenchymal phenotype is part of their biology during the window of receptivity.

    The experimental protocol also needs a little interpretation: the authors grow their organoids for four days in "expansion media" [simulating the proliferative phase of the menstrual cycle] and some of those samples are then subjected to SC analysis. Another set of cultures is also subjected to differentiation media for an additional 4 days simulating transition to luteal phase and receptivity. Comparing the expansion media organoids only with the treated ones allows us to see what happens in this simulation of the transition to luteal phase, and as such this is OK. However, the result is never the less confounded by the fact that the treated organoids are older than the "expansion media only" samples. A control comparison with organoids treated 4 days with expansion media and then four days with differentiation media but without the MPA and cAMP would be helpful to disentangle the hormone/cAMP effects and the age related changes in culture.

  6. eLife

    Reviewer #3 (Public Review):

    In their study, authors designed a novel human endometrium research model, which they refer to as assembloids, containing not only the endometrium epithelial cells (as standard organoid models do), but also the tissue stromal cells. Once developed and well characterized using, among others, state-of-the-art single-cell RNA-sequencing, the authors showed its application potential by dipping into a candidate cause of endometrial declined function and receptivity, i.e. dysbalanced senescence. Culminating in the study is the addition of human (spare IVF) embryos to the developed endometrial assembloid with and without senescence perturbation, which is a first step toward in vitro deciphering human embryo-endometrium interaction in health and disease.

    The study has multiple strengths. Central are the design and detailed characterization of this new endometrium assembloid model, and the demonstration of its applicability for endometrial deficiency studies regarding biology and embryo interaction. The finding that cellular complexity is recapitulated in the assembloid culture and that cell types and states (in particular, senescence) mimic in vivo decidualization are major achievements. The benchmarking with in vivo data is highly interesting for the field. Limitations may be situated in the use of a rather crude method to inhibit cellular stress and senescence (i.e. using a generally acting tyrosine kinase inhibitor) and the premature immediate-generalization of findings to multiple fertility problems without the needed grounds so far. Although definitely a strong advancement in the field, adding still other endometrial cells, most importantly of luminal epithelial cells but also innate immune cells, will in the future further perfect the model.

    The authors achieved their aims of establishing and characterizing a new, straightforward endometrium tissue model. Moreover, they achieved to applying this new tool to start unraveling causes of endometrium non-receptivity or ill-performance, in particular regarding (dys-)balanced senescence. Together, the study presents a promising path along which human (in-)fertility research can develop, to provide basic and translational insights in reproductive biology and into (deficient) fertility which may eventually be taken to the clinic to improving pregnancy chances.

  7. eLife

    Reviewer #4 (Public Review):

    Rawlings et al. investigated endometrial mechanisms that may underlie reproductive disorders such as recurrent implantation failure and pregnancy loss. The proper decidualization of the endometrium is essential for correct embryo implantation and this process is tightly controlled by hormone action during the menstrual cycle. The authors hypothesized that acute senescence in the decidualizing endometrium is necessary for successful embryo implantation. They also sought to characterize gene expression changes that occur in stromal and epithelial compartments as a result of decidualization.

    The authors used a novel assembloid model of endometrial culture to investigate how endometrial epithelial and stromal cells respond to decidualization and found that both epithelial and stromal cells displayed distinct gene expression signature groups before and after decidualization. They successfully showed that the cellular stress that occurs during decidualization can directly affect the degree to which decidual endometrial cells undergo senescence. By culturing their endometrial assembloids with human embryos, they were able to convincingly demonstrate that endometrial decidual senescence is necessary to allow the embryo to grow and invade after implantation.

    This work will be important to the endometrial biology field as well as to clinicians. In addition to offering a mechanism for some types of implantation failure and recurrent pregnancy loss, the authors showed that treatment with a tyrosine kinase inhibitor can modulate pre-decidual stress and decidual senescence, suggesting that endometrial receptivity could one day be manipulated pharmacologically.

    Major strengths and weaknesses of method and results.

    Strengths:

    The major strength of this paper is their assembloid-embryo coculture model, which provides strong evidence that a lack of endometrial decidual senescence results in a lack of embryo growth and failure of the embryo to invade into the endometrial assembloid. Although endometrial organoid models containing both epithelial and stromal cells already exist, the assembloid model allows the visualization of embryo growth and invasion. The single-cell RNA-sequencing of the assembloids convincingly demonstrates the existence of different populations of endometrial epithelial and stromal cells displaying different gene expression profiles before and after decidualization. The separation into these groups is very clean and the gene expression correlates with endometrial gene expression across the cycle remarkably well.

    Weaknesses:

    Given that the women who contributed endometrial tissue to this study were attending an Implantation Research Clinic and were mostly nulliparous with past first trimester pregnancy loss, it is unclear whether the endometrial samples used in this study is representative of a healthy condition. The rationale for using a minimal differentiation medium rather than the differentiation medium that has been established in the literature is unclear. In particular, the induction of glandular differentiation in endometrial assembloids by NAC, an antioxidant, deserves some discussion. Additionally, some explanation as to why the authors chose to treat their assembloid cultures with decidualization cocktail for only four days, when decidualization in vivo occurs over a much longer period, would be helpful. The authors state that EpS5 is a population of senescent epithelial cells producing SASP based on gene expression data. It would be more convincing if the authors could provide other evidence to characterize these cells as senescent. The authors draw many conclusions based on data from the CellPhoneDB computational tool, but it is unclear how the authors chose the input for this tool and whether the output of this program was validated in any way.

  8. Version published to 10.1101/2021.03.02.433560v1 on bioRxiv