The human uterus is a formidable organ. From puberty to menopause, it completely sheds off its internal lining every 28 days or so, creating what is in effect a large open wound. Unlike the skin or other parts of the body, however, this tissue can quickly repair itself without scarring. This fascinating process remains poorly understood, partly because human samples and animal models that mimic human menstruation are still lacking. This makes it difficult to grasp how various types of uterine cells get mobilised for healing.

To fill this gap, Kirkwood et al. focused on fibroblasts, a heterogenous cell population which helps to support the epithelial cells lining the inside of the uterus. How these cells responded to the advent of menstruation was examined in female mice genetically manipulated to have human-like periods. A method known as single-cell RNAseq was used to track which genes were active in each of these cells before, one day and two days after period onset. This revealed the existence of a subpopulation of cells which only appeared when wound healing was most needed.

These ‘repair-specific’ fibroblasts expressed a mixture of genes; those typical of fibroblasts but also some known to be active in the epithelial cells lining the uterus. This suggests that the cells were in the process of changing their identity so they could remake the uterine layer lost during a period. And indeed, labelling these fibroblasts with a fluorescent tag showed that, during healing, they had migrated from within the uterine tissue to become part of its newly restored internal surface. These results represent the first evidence that fibroblasts play a direct role in repairing the uterus during menstruation.

From endometriosis to infertility, the lives of millions of people around the world are impacted by disorders which affect the uterine lining. A better understanding of how the uterus can fix itself month after month could help to find new treatments for these conditions. This knowledge could also be useful for to address abnormal wound healing in the skin and other tissues, as this process often involves fibroblasts.

Efficient wound repair and restoration of tissue homeostasis is essential for reproductive and general health. Repetitive injury, inflammation, and fibrosis resulting in disordered tissue architecture is a major cause of morbidity and mortality due to organ failure (Greenhalgh et al., 2015). The endometrium, a complex hormone responsive multicellular tissue, exhibits remarkable resilience in its ability to respond to the monthly wound of ‘menstruation’ without fibrosis and with a response that is both rapid and scar-free (Garry et al., 2009; Ludwig and Spornitz, 1991). A critical ‘trigger’ for menstruation in women is the rapid fall in circulating concentrations of progesterone that occurs as a result of the involution of the ovarian corpus luteum in a non-fertile cycle (Maybin and Critchley, 2011). Menstruation is considered an inflammatory event with a ‘cascade’ of changes including an increase in synthesis of pro-inflammatory factors such as prostaglandins, cytokines, and chemokines which is accompanied by infiltration of immune cells of the myeloid lineage (neutrophils, monocyte/macrophages) (Evans and Salamonsen, 2012). Increased production of prostaglandins is associated with arteriole vasoconstriction which results in local hypoxia, stabilisation of HIF1alpha and increased expression of genes such as VEGF (Critchley et al., 2006b). Whilst we have a good appreciation of the mechanisms that contribute to the tightly regulated breakdown and shedding of a portion of the endometrium at menses gaps remain in our understanding of the processes that contribute to rapid, fibrosis/scar-free restoration of an intact luminal epithelium over the tissue surface. It is also important to separate the mechanisms responsible for menstrual repair, which occurs at a time in the cycle when ovarian-derived steroids are low, from endometrial regeneration/proliferation which occurs subsequently in response to rising levels of follicle-derived oestrogens during the proliferative phase of the cycle (Maybin and Critchley, 2012).

Several different mechanisms have already been proposed as contributing to the luminal repair process including, activation of progenitor/stem cells in the stroma and/or epithelium, epithelial cell proliferation/migration and transformation of stromal cells into epithelial cells (mesenchyme to epithelial transition, MET) each of which are discussed briefly below.

The ability of the endometrium to rapidly regenerate during each menstrual cycle has prompted researchers to explore a role for putative stem/progenitor cells residing in either the stroma and/or the epithelial compartments in endometrial repair (Gargett et al., 2016; Cousins et al., 2021). The Gargett group have identified populations of endometrial mesenchymal stem cells; eMSC with clonogenic and broad multilineage potential in both human endometrial tissue and menstrual fluid and characterised them as PDGFRB+/CD146+/ SUSD2+ (Gargett et al., 2016). Notably in human endometrial tissue sections these cells had a perivascular location and a putative pericyte identity. A role for progenitor cells residing in the basal glandular epithelium has also been proposed with a recent review highlighting a number of marker proteins employed to establish their identity including SOX9 and SSEA1 (Cousins et al., 2021). Another source of putative stem cells that may contribute to endometrial repair processes and/or endometrial pathology is the bone marrow (Kaitu’u-Lino et al., 2012) although evidence for the contribution of bone marrow-derived stem cells is somewhat limited (Deane et al., 2019; Aghajanova et al., 2010).

During the first 2 days of menstruation rapid shedding of the luminal portion of the tissue (functional layer) leaves a denuded (basal) stroma interspersed with glandular ‘stumps’. This phenomenon was beautifully documented in the elegant studies of Ludwig and Spornitz, 1991. In the 1970’s Ferenczy proposed that new epithelial cells arose from proliferation of intact surface epithelium bordering denuded stroma and the basal glands that were exposed when the functional layer was shed (Ferenczy, 1976b; Ferenczy, 1976a). He noted that the re-epithelialisation of the human endometrium was rapid (~48 hr) and did not appear to involve mitosis of the epithelial cells (migration?). These mechanisms of epithelial repair were later endorsed by Ludwig and Spornitz, 1991 who also observed fibrin mesh formation on the denuded surface in the early days of menstruation and growth of an epithelial monolayer in spirals which would appear to be consistent with them being associated with the residual stumps of endometrial glands. When Garry and colleagues used a more dynamic approach involving pressure-controlled, continuous flow hysteroscopy they found evidence that loss and repair of the surface occur synchronously at different places within the endometrial cavity a process they called ‘piecemeal’ (Garry et al., 2009). They also reported epithelial cells of the glands underwent apoptosis and were shed with the decidual tissue mass. Apoptosis of glandular epithelial cells in carefully dated human endometrium has also been documented using staining for cleaved caspase 3 and shown to be high in both late secretory and menstrual phases (Armstrong et al., 2017). Taken together the data from Garry et al and Armstrong et al challenges the earlier papers citing the glands as the source of new luminal cells.

There is also evidence that cells within the stromal compartment may change their identity to adopt an epithelial phenotype (mesenchyme to epithelial transition, MET) and thereby contribute to the luminal surface. This was first proposed in the 1960’s by Baggish et al who evaluated sections of menstruating human endometria and noted hyperchromatic stromal cells between gland stumps that appeared to take part in re-epithelisation by a process simulating metaplasia (Baggish et al., 1967). In the 1970’s the involvement of the stroma was challenged (Ferenczy, 1976b) but has been supported by more recent studies (Garry et al., 2009). Specifically in 2009 the authors reported that the denuded basalis endometrium was rapidly covered with a fibrinous mesh within which new surface epithelial cells were present which appeared to arise from the underlying stroma.

To complement studies on human tissue samples and studies in primates (Brenner et al., 2002; Critchley et al., 2006a) we have used a highly reproducible mouse model that recapitulates key features of human menstruation in response to withdrawal of progesterone including vascular breakdown (vaginal bleeding), spatial and temporal hypoxia, cell apoptosis, expression of matrix metalloproteinases and influx of myeloid immune cells (Armstrong et al., 2017; Maybin et al., 2018; Cousins et al., 2016a; Cousins et al., 2016b; Cousins et al., 2014). Importantly, as in the human endometrium (Garry et al., 2009), restoration of an intact luminal epithelial layer occurs within two days (Cousins et al., 2014). Consistent with some of the reports on repair processes in human endometrium we found evidence for epithelial cell migration and proliferation of residual epithelial cells (Ki67+) but no significant proliferation of basal glands (Cousins et al., 2014). Other groups have also used similar mouse models to explore the dynamic changes during a menstrual-like event. Kaitu’u-Lino et al used a mouse model in combination with pulse labelling of cells with BrdU, and reported that a population of epithelial progenitor cells residing in the basal glands might contribute to postmenstrual repair (Kaitu’u-Lino et al., 2010, Kaitu’u-Lino et al., 2012).

In our endometrial repair/menstrual model one of our novel findings was the existence of stromal cells that co-expressed vimentin (stromal cell marker) and cytokeratin (epithelial cell marker) specifically residing in areas denuded of epithelium which suggested to us that stromal cells might be changing their phenotype via MET (Cousins et al., 2014).

In the current study, we have used single cell RNA sequencing and lineage tracing to specifically address which, if any, of the stromal mesenchymal cell subpopulations we previously identified in the mouse endometrium (Kirkwood et al., 2021) might contribute to the rapid restoration of the intact luminal epithelial cell layer observed in our mouse model (Cousins et al., 2014; Cousins et al., 2016a). To the best of our knowledge this is the first time that inducible-Cre lineage tracing approaches in adult mice have been used to explore MET in a model of menstruation.

In this study, we used a combination of single cell RNA sequencing and cell lineage tracing to investigate the contribution of the stromal mesenchyme to rapid restoration of the endometrial luminal epithelium at the time of menstruation. The human endometrium is unusual in its ability to experience cyclical episodes of breakdown/shedding (wounding) which occur in parallel with repair processes that leave the tissue intact without an obvious scar. We used a previously validated mouse model that mimics the key features of human menstruation in which we made some preliminary observations that aligned with studies suggesting MET of stromal cells might contribute to the rapid repair process (Cousins et al., 2014). In the work presented here we have identified a new population of repair-specific PDGFRb + cells that responded to changes in the endometrial environment induced by progesterone withdrawal by upregulating expression of genes associated with MET/epithelial cell identify. To best understand how this population might influence our understanding of endometrial repair we designed further experiments to answer two specific questions. First – what was the originating cell type of this transient population? Second – what was its fate?

The primary objective of the study was to explore whether we could substantiate our hypothesis that MET was occurring in stromal cells exposed by shedding of the decidual mass in our mouse model of menstruation (Cousins et al., 2014). Therefore we decided to use Pdgfrb-BAC-eGFP reporter mice in our menstrual model as we had previously shown eGFP was only expressed in stromal cell populations during the oestrus cycle (Kirkwood et al., 2021). Comparison between scRNAseq datasets from cycling mice identified three populations of mesenchyme cells that were unique to endometrial tissue recovered 24 or 48 hr after withdrawal of progesterone at a time when the tissue in the process of active breakdown/repair. One population was very closely associated with the previously identified pericyte cell population with sequence analysis suggesting it might represent a subgroup of cells that had some minor changes in function possibly associated with loss of some of the vascular compartment. A second population was only present at 48 hr and based on expression of several genes associated with apoptosis we concluded that it represented stromal cells that were not going to survive in repairing tissue and that cell death was one potential fate of the GFP + cells. The most interesting population of GFP + cells (F4) were unique to the 24 hr tissue with a transcriptome that included expression of both canonical mesenchymal and epithelial cell markers as well as a number of genes such as Ctnnb1 (beta-catenin) and Twist which have been implicated cell transitions between mesenchyme and epithelium. Notably it has previously been reported that mice with stabilisation of Ctnnb1, a gene that plays an important role in Wnt signalling, had smaller uteri, endometrial gland defects, and were infertile (Stewart et al., 2013). Further analysis using flow cytometry and immunohistochemistry confirmed the existence of GFP + EPCAM + cells in the 24 hr samples.

These results backed up our previous observation that MET occurred in regions of tissue associated with endometrial repair and are in agreement with studies exploring the fate of mesenchyme cells after parturition (Huang et al., 2012; Patterson et al., 2013). For example, Huang et al used a knock-in strategy with cells expressing LacZ induced by Amhr2-Cre: in virgin females the reporter was specific to stromal cells but post-partum they found labelled cells in both luminal and glandular epithelium. In another study using a SM22a-Cre driver, expression of which was reported to be restricted to a subset of CD34 +mesenchymal cells, reporter gene expression (GFP) was also detected in the epithelium (Yin et al., 2019). Whilst authors of all these papers concluded their results were consistent with stromal MET this interpretation has been challenged (Ghosh et al., 2020). Specifically, Ghosh et al conducted a comprehensive examination of embryonic and adult reproductive tracts using LacZ reporter lines driven by promoters for the genes Amhr2, Sm22, Cspg4, Thy1 and Pdgfrß all of which are expressed in mesenchyme. They specifically questioned whether epithelial cells expressing reporter proteins in the adult endometrium arose from MET or were induced at a time when cells had meso-epithelial characteristics. In all cases, they attributed epithelial expression in adult epithelial cells to activation of the promoters during embryonic life ruling out MET in adult cycling mice (Ghosh et al., 2020).

One limitation of the results obtained using Pdgfrb-BAC-eGFP reporter mice was that the knockin strategy used to generate the line meant GFP was only expressed in cells when the Pgdfrb promoter was active. We have previously established that the expression of GFP in the endometrium of adult female mice was identical to the native PDGFRβ protein and was stroma-specific (Kirkwood et al., 2021). A comparison between GFP expression in cycling mice those using uteri recovered after induction of endometrial shedding/repair led us to conclude that the presence of GFP in the epithelial cells was most likely because the turnover of the GFP was slow enough for it to persist after the Pgdfrb promoter was no longer active. This would be consistent with reports that eGFP protein was engineered to have increased fluorescence and slower turnover in vivo Zhao et al., 1999; although the half-life of the protein varies in different cell models a paper exploring in vivo expression in cancer reported it to be ~15 hr (Danhier et al., 2015). These results were also considered consistent with detection of less GFP in EPCAM + epithelial cells at 48 than 24 hr when this was evaluated using flow cytometry.

Therefore, to make a more robust case for MET of endometrial stromal cells we expanded our scRNAseq datasets by analysing the epithelial cells (EPCAM+) from tissue recovered from both cycling mice and those in which endometrial repair was well advanced (48 hr). We then complemented these findings by undertaking lineage tracing studies in two new lines of mice using a strategy that induced expression of a tdTm reporter protein in adulthood to avoid any confounding effects of MET that might occur during development of the uterus. Analysis of EPCAM + cells resulted in identification of nine subpopulations of epithelial cells based on cluster analysis and comparisons to genes previously identified as being expressed in different locations including the lumen (Yang et al., 2021) and glands (Foxa2, Kelleher et al., 2017).

When we combined our eGFP + and EPCAM + scRNAseq datasets three broad groups of cell types was present: perivascular, fibroblast and epithelial with the repair-specific transient population of cells (24 hr specific, F4) appearing as a distinct fibroblast subpopulation which on trajectory analysis appeared to form a ‘bridge’ between the previously identified fibroblast clusters (Kirkwood et al., 2021) and the new epithelial datasets. Based on previous reports that identified cells with stem-like properties in a perivascular location (Gargett et al., 2016) we had initially anticipated pericytes might have been mobilised to undergo MET however the trajectory data suggested we might be wrong. To complement and extend the transcriptional profiling and to gain a fuller picture of the source of the putative MET cells we compared results from two new lines of mice. One in which we targeted fibroblasts using a Cre under the control of Pdgfra and a second line in which all perivascular/vSMC were targeted using the Cspg4 (NG2) promoter. We used a TAM-dependent induction schedule that we refined so that there was robust transgene induction but no residual impact on the response of the tissue to decidualisation and induction of endometrial injury. The results obtained were unequivocal and supported not only MET of fibroblasts but also their incorporation into the restored luminal epithelium in a layer adjacent to epithelial cells which had not arisen by MET but were likely to have migrated from residual epithelial cell remnants left at time of shedding. These novel findings provide robust evidence that the endometrium can use multiple mechanisms including recruitment of mesenchyme cells to rapidly restore its integrity a feature of the tissue that is important for its normal functioning and for fertility. In future studies, it would be interesting to use cell ablation strategies specifically targeting F2 to investigate whether repair is delayed if they are not present, whether any other fibroblasts replace them and the relative contributions of the other mechanisms previously identified (epithelial proliferation and migration Cousins et al., 2014) to the acute phase of restoration of luminal epithelial integrity.

The generation of these scRNAseq datasets have allowed us to make some preliminary comparisons to results from studies using this technique to explore cell heterogeneity in human endometrium and human endometrial cells (Wang et al., 2020; Queckbörner et al., 2021; Cao et al., 2021; Garcia-Alonso et al., 2021; Shih et al., 2022; Wu et al., 2022). Queckborner and colleagues focused their analysis cells from 3 donors obtained during in the proliferative phase of their menstrual cycle (6864 cells) using a protocol that enriched for stromal cells (Queckbörner et al., 2021). They subtyped two populations of perivascular/pericyte cells with different expression levels of MY11D and CSPG4 both of which we have previously mapped to perivascular populations in the mouse endometrium (Kirkwood et al., 2021). They subtyped the stromal fibroblasts into 10 closely related clusters of fibroblasts and concluded that the markers for progenitor cells were not specific enough. They didn’t analyse any tissues/cells from the menstrual phase so there are no comparable data to ours. In contrast, Wang et al., 2020 incorporated tissues from across the menstrual cycle in their analysis including 6 that were assigned to the menstrual phase according to their supplementary data although these were identified as days 4–11 of the cycle which might be after the most rapid phase of repair (anticipated to be on days 1–3). They reported a previously uncharacterised ciliated epithelial subtype and changes in luminal epithelium and stroma that were discrete to the window of implantation but nothing specific to repair mechanisms. Cao et al focused their attention on the perivascular cell population performing sequence analysis of CD140b+CD146+ (eMSC) cells isolated from menstrual phase (days 2–3) and secretory phases (n=3 women in each group Cao et al., 2021). They identified two subclusters in cultured cells from the menstrual phase with the larger cluster having high expression of PDGFRβ/PDGFRα consistent with a fibroblast identity. When they compared their data to the Wang dataset they noted significant changes in gene expression patterns and concluded that the cell transcriptomes had been influenced by cell culture which may compromise attempts to compare between in vitro and in vivo data.

In a recent study, Shih et al., 2022 used a single-cell approach to interrogate tissue recovered from menstrual effluent from patients with an without a diagnosis of endometriosis 9 of which were classified as ‘controls’ (8/9 had normal cycle length 26-31d). Cluster analysis showed a high proportion of immune cells, five closely related stromal cell populations and three epithelial cell clusters (Shih et al., 2022). Comparison between the genes that defined each subpopulation of stromal cells (GFBP1, MGP, IL11, SRGN, HSPA6) with our current dataset did not find any that corresponded to F4 which is probably unsurprising as these signatures were based on shed cells not those that remained in situ and therefore able to contribute to the new luminal epithelium. In contrast, we did find some homologies within the dataset published by Wu et al., 2022 whose analysis included single-cell sequencing of full thickness endometrium from the proliferative and secretory phases (no menstrual tissue). Their stromal cell subclusters included one enriched for SFRP4 which they also showed provided a positive stimulus to regeneration in a rat endometrial injury model. Comparison with our own datasets identified Sfrp4 expression was highest in our F2 and F4 populations, very low in F1/F3 and absent from the epithelial cells: two of the factors secreted by the SFRP4 cells were also higher in the mouse F4 than other cell clusters (PENK, GLIPR1). Whilst further work is needed to validate whether a population equivalent to F4 is present in the menstrual endometrium looking for SFRP4 cells might provide a useful starting point and it is also notable that the authors found the SFRP4 cells to be distinct to putative progenitors identified as SUSD2 + which were enriched in the perivascular population.

In summary, we describe novel data and new datasets that show MET can contribute to the rapid scar-free healing of the endometrial luminal epithelium when this occurs in response to an endometrial ‘wound’ at the time of menstruation. The cell type involved in this MET is a PDGFRα+stromal cell and not the NG2 + pericytes. Further studies on the origin of these cells and their contribution to repair will require new studies using CRE lines which are specific to F2 for lineage tracing and cell ablation. The current findings also provide a platform for further analysis of the fibrosis-resistant phenotype of endometrial fibroblasts, comparisons to conditions where their function is abnormal (Asherman’s syndrome, endometriosis) and comparison to other mucosal barrier tissues such as that found in the oral cavity as recently described by Williams et al., 2021. We postulate that manipulation of putative MET progenitors/specific cell types in the disease setting may provide novel strategies in the management and/or treatment of disorders associated with poor response to healing or excess fibrosis.

Single cell RNAseq datasets have been deposited in GEO under accession codes GSE198556.

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Author details

1. Phoebe M Kirkwood

   Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Douglas A Gibson

   Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   Conceptualization, Supervision, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

3. Isaac Shaw

   Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

4. Ross Dobie

   Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Olympia Kelepouri

   Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Neil C Henderson

   1. Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom
   2. MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – review and editing

   Competing interests

   No competing interests declared

7. Philippa TK Saunders

   Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing – review and editing

   For correspondence

   p.saunders@ed.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9051-9380

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