methods-for-studying-uterine-contributions-to-pregnancy-establishment

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation ...

Monash University

Studies from five independent laboratories conclude that bone marrow stem cells transdifferentiate into endometrial stroma, epithelium, and endothelium. We investigated the nature of bone marrow-derived cells in the mouse endometrium by reconstituting irradiated wild type recipients with bone marrow containing transgenic mTert-green fluorescent protein (GFP) or chicken β-actin (Ch β-actin)-GFP reporters. mTert-GFP is a telomerase marker identifying hematopoietic stem cells and subpopulations of epithelial, endothelial, and immune cells in the endometrium. Ch β-actin-GFP is a ubiquitous reporter previously used to identify bone marrow-derived cells in the endometrium. Confocal fluorescence microscopy for GFP and markers of endometrial and immune cells were used to characterize bone marrow-derived cells in the endometrium of transplant recipients. No evidence of GFP bone marrow-derived stroma, epithelium, or endothelium was observed in the endometrium of mTert-GFP or Ch β-actin-GFP recipients. All GFP cells detected in the endometrium were immune cells expressing the pan leukocyte marker CD45, including CD3 T cells and F4/80 macrophages. Further examination of the Ch β-actin-GFP transplant model revealed that bone marrow-derived F4/80 macrophages immunostained weakly for CD45. These macrophages were abundant in the stroma, infiltrated the epithelial and vascular compartments, and could easily be mistaken for bone marrow-derived endometrial cells. We conclude that it is unlikely that bone marrow cells are able to transdifferentiate into endometrial stroma, epithelium, and endothelium. This result has important therapeutic implications, as the expectation that bone marrow stem cells contribute directly to endometrial regeneration is shaping strategies designed to regenerate endometrium in Asherman's syndrome and to control aberrant endometrial growth in endometriosis. Stem Cells 2018;36:91-102.

Bone Marrow Stem Cells Do Not Contribute to Endometrial Cell Lineages in Chimeric Mouse Models.

Is there a specific surface marker that identifies human endometrial epithelial progenitor cells with adult stem cell activity using in vitro assays?

N-cadherin identifies human endometrial epithelial progenitor cells by in vitro stem cell assays.

Endometrial organoids: in vitro models for endometrial research and personalized medicine.

In Reply to Letter to the Editor from Bhartiya: Transplantation of Whole Bone Marrow Indicates That Bone Marrow Very Small Embryonic-Like Cells Do Not Contribute to Endometrial Lineages.

Human endometrium regenerates on a cyclical basis each month, likely mediated by endometrial stem/progenitor cells. Several types of stem/progenitor cells have been identified: CD140bCD146 or SUSD2 endometrial mesenchymal stem cells (eMSCs), N-cadherin endometrial epithelial progenitor cells (eEPs), and side population (SP) cells, a heterogeneous population predominantly comprising endothelial cells. eMSCs reside in a perivascular niche and likely mediate angiogenesis and stromal regeneration. Human eEPs are located in the bases of glands in the basalis and are likely more primitive than SSEA-1 basalis epithelial cells. Endometrial stem/progenitor cells may contribute to the pathogenesis of endometriosis by their retrograde shedding into the pelvic cavity, either after menarche or as a result of neonatal uterine bleeding. eMSCs may have a role in the generation of progesterone-resistant phenotype of endometrial stromal fibroblasts (eSFs) in endometriosis. In future clinical practice, endometrial stem/progenitor cells may be used to establish diagnosis of endometriosis or as therapeutic targets.

Endometrial stem/progenitor cells and their role in the pathogenesis of endometriosis.

The regenerative capacity of the endometrium has been attributed to resident stem/progenitor cells. A number of stem/progenitor markers have been reported for human endometrial stem/progenitor cells; however, the lack of convenient markers in the mouse has made experimental investigation into endometrial regeneration difficult. We recently identified endometrial epithelial, endothelial, and immune cells, which express a reporter for the stem/progenitor marker, mouse telomerase reverse transcriptase (mTert). In this study, we investigate the expression pattern of a green fluorescent protein (GFP) reporter for mTert promoter activity (mTert-GFP) in endometrial regeneration following a menses-like event. mTert-GFP expression marks subepithelial populations of T cells and mature macrophages and may play a role in immune cell regulated repair. Clusters of mTert-GFP-positive epithelial cells were identified close to areas of reepithelialization and possibly highlight a role for mTert in the repair and regeneration of the endometrial epithelium.

Telomerase Reverse Transcriptase Expression in Mouse Endometrium During Reepithelialization and Regeneration in a Menses-Like Model.

To develop a patient derived xenograft (PDX) model of cervical cancer and cervical dysplasia using the subrenal capsule.

A patient derived xenograft model of cervical cancer and cervical dysplasia.

Transvaginal meshes for the treatment of Pelvic Organ Prolapse (POP) have been associated with severe adverse events and have been banned for clinical use in many countries. We recently reported the design of degradable poly L-lactic acid-co-poly ε-caprolactone nanofibrous mesh (P nanomesh) bioengineered with endometrial mesenchymal stem/stromal cells (eMSC) for POP repair. We showed that such bioengineered meshes had high tissue integration as well as immunomodulatory effects . This study aimed to determine the key molecular players enabling eMSC-based foreign body response modulation.

Electrospun Nanofiber Meshes With Endometrial MSCs Modulate Foreign Body Response by Increased Angiogenesis, Matrix Synthesis, and Anti-Inflammatory Gene Expression in Mice: Implication in Pelvic Floor.

Endometriosis is a common and painful condition characterised by the formation of endometrial lesions within the peritoneal cavity. Previous studies have suggested a role for hedgehog signalling in the pathogenesis of endometriosis. We investigated the role of hedgehog signalling in the establishment of endometriosis lesions using 5E1, a hedgehog ligand neutralising antibody, and a mouse model of endometriosis. To mimic the initiation of endometriosis by retrograde menstruation, which is believed to occur in humans, donor mice underwent an artificial menstruation protocol. Fragments of menstrual endometrium were injected into the peritoneal cavity of estrogen primed recipients. Recipients received twice weekly injections of 5E1 or an isotype matched control antibody for three weeks. Lesions were collected and analysed for markers of epithelium, proliferation and apoptosis by immunofluorescence microscopy.

The effects of hedgehog ligand neutralising antibody 5E1 in a mouse model of endometriosis.

The human endometrium undergoes approximately 450 cycles of proliferation, differentiation, shedding and regeneration over a woman's reproductive lifetime. The regenerative capacity of the endometrium is attributed to stem/progenitor cells residing in the basalis layer of the tissue. Mesenchymal stem cells have been extensively studied in the endometrium, whereas endometrial epithelial stem/progenitor cells have remained more elusive. This review details the discovery of human and mouse endometrial epithelial stem/progenitor cells. It highlights recent significant developments identifying putative markers of these epithelial stem/progenitor cells that reveal their  identity, location in both human and mouse endometrium, raising common but also different viewpoints. The review also outlines the techniques used to identify epithelial stem/progenitor cells, specifically  functional assays and  lineage tracing. We will also discuss their known interactions and hierarchy and known roles in endometrial dynamics across the menstrual or estrous cycle including re-epithelialization at menses and regeneration of the tissue during the proliferative phase. We also detail their potential role in endometrial proliferative disorders such as endometriosis.

The Elusive Endometrial Epithelial Stem/Progenitor Cells.

Mesenchymal stem cells (MSCs) that meet the International Society for Cellular Therapy (ISCT) criteria are obtained from placental tissue by plastic adherence. Historically, no known single marker was available for isolating placental MSCs (pMSCs) from the decidua basalis. As the decidua basalis is derived from the regenerative endometrium, we hypothesised that SUSD2, an endometrial perivascular MSC marker, would purify maternal perivascular pMSC. Perivascular pMSCs were isolated from the maternal placenta using SUSD2 magnetic bead sorting and assessed for the colony-forming unit-fibroblasts (CFU-F), surface markers, and in vitro differentiation into mesodermal lineages. Multi-colour immunofluorescence was used to colocalise SUSD2 and α-SMA, a perivascular marker in the decidua basalis. Placental stromal cell suspensions comprised 5.1%SUSD2 cells. SUSD2 magnetic bead sorting of the placental stromal cells increased their purity approximately two-fold. SUSD2 pMSCs displayed greater CFU-F activity than SUSD2 stromal fibroblasts (pSFs). However, both SUSD2 pMSC and SUSD2 pSF underwent mesodermal differentiation in vitro, and both expressed the ISCT surface markers. Higher percentages of cultured SUSD2 pMSCs expressed the perivascular markers CD146, CD140b, and SUSD2 than SUSD2 pSFs. These findings suggest that SUSD2 is a single marker that enriches maternal pMSCs, suggesting they may originate from eMSC. Placental decidua basalis can be used as an alternative source of MSC for clinical translation in situations where there is no access to endometrial tissue.

Identification and characterisation of maternal perivascular SUSD2 placental mesenchymal stem/stromal cells.

Endometrial organoids (EMO) are an important tool for gynecological research but have been limited by generation from (1) invasively acquired tissues and thus advanced disease states and (2) from women who are not taking hormones, thus excluding 50% of the female reproductive-aged population. We sought to overcome these limitations by generating organoids from (1) menstrual fluid (MF; MFO) using a method that enables the concurrent isolation of menstrual fluid supernatant, stromal cells, and leukocytes and (2) from biopsies and hysterectomy samples from women taking hormonal medication (EMO-H). MF was collected in a menstrual cup for 4-6 h on day 2 of menstruation. Biopsies and hysterectomies were obtained during laparoscopic surgery. Organoids were generated from all sample types, with MFO and EMO-H showing similar cell proliferation rates, proportion and localization of the endometrial basalis epithelial marker, Stage Specific Embryonic Antigen-1 (SSEA-1), and gene expression profiles. Organoids from different disease states showed the moderate clustering of epithelial secretory and androgen receptor signaling genes. Thus, MFO and EMO-H are novel organoids that share similar features to EMO but with the advantage of (1) MFO being obtained non-invasively and (2) EMO-H being obtained from 50% of the women who are not currently being studied through standard methods. Thus, MFO and EMO-H are likely to prove to be invaluable tools for gynecological research, enabling the population-wide assessment of endometrial health and personalized medicine.

Comparison of Organoids from Menstrual Fluid and Hormone-Treated Endometrium: Novel Tools for Gynecological Research.

The human endometrium is a remarkable tissue, undergoing ~450 cycles of proliferation, differentiation, shedding (menstruation), repair, and regeneration over a woman's reproductive lifespan. Post-menstrual repair is an extremely rapid and scar-free process, with re-epithelialization of the luminal epithelium completed within 48 h of initiation of shedding. Following menstruation, the functionalis grows from the residual basalis layer during the proliferative phase under the influence of rising circulating estrogen levels. The regenerative capacity of the endometrium is attributed to stem/progenitor cells which reside in both the epithelial and stromal cell compartments of the basalis layer. Finding a definitive marker for endometrial epithelial progenitors (eEPCs) has proven difficult. A number of different markers have been suggested as putative progenitor markers including, N-cadherin, SSEA-1, AXIN2, SOX-9 and ALDH1A1, some of which show functional stem cell activity in  assays. Each marker has a unique location(s) in the glandular epithelium, which has led to the suggestion that a differentiation hierarchy exists, from the base of epithelial glands in the basalis to the luminal epithelium lining the functionalis, where epithelial cells express different combinations of markers as they differentiate and move up the gland into the functionalis away from the basalis niche. Perivascular endometrial mesenchymal stem cells (eMSCs) can be identified by co-expression of PDGFRβ and CD146 or by a single marker, SUSD2. This review will detail the known endometrial stem/progenitor markers; their identity, location and known interactions and hierarchy across the menstrual cycle, in particular post-menstrual repair and estrogen-driven regeneration, as well as their possible contributions to menstruation-related disorders such as endometriosis and regeneration-related disorder Asherman's syndrome. We will also highlight new techniques that allow for a greater understanding of stem/progenitor cells' role in repair and regeneration, including 3D organoids, 3D slice cultures and gene sequencing at the single cell level. Since mouse models are commonly used to study menstruation, repair and regeneration we will also detail the mouse stem/progenitor markers that have been investigated .

Endometrial Stem/Progenitor Cells-Their Role in Endometrial Repair and Regeneration.

Endometriosis is a serious, chronic disorder where endometrial tissue grows outside the uterus, causing severe pelvic pain and infertility. It affects 11% of women. Endometriosis is a multifactorial disorder of unclear etiology, although retrograde menstruation plays a major role. It has a genetic component with over 40 genetic risk factors mapped, although their mechanism of action is still emerging. New evidence suggests a role for retrograde menstruation of endometrial stem/progenitor cells, now that identifying markers of these cells are available. Recent lineage tracing and tissue clearing microscopy and 3D reconstruction has provided new understanding of endometrial glandular structure, particularly the horizontal orientation and interconnection of basalis glands. New sequencing technologies, particularly whole genome DNA sequencing are revealing somatic mutations, including in cancer driver genes, in normal and eutopic endometrium of patients with endometriosis, as well as ectopic endometriotic lesions. Methylome sequencing is offering insight into the regulation of genes and the role of the environmental factors. Single cell RNA sequencing reveals the transcriptome of individual endometrial cells, shedding new light on the diversity and range of cellular subpopulations of the major cell types present in the endometrium and in endometriotic lesions. New endometrial epithelial organoid cultures replicating glandular epithelium are providing tractable models for studying endometriosis. Organoids derived from menstrual fluid offer a non-invasive source of endometrial tissue and a new avenue for testing drugs and developing personalized medicine for treating endometriosis. These new approaches are rapidly advancing our understanding of endometriosis etiology.

New concepts on the etiology of endometriosis.

Female cancer survivors are significantly more likely to experience infertility than the general population. It is well established that chemotherapy and radiotherapy can damage the ovary and compromise fertility, yet the ability of cancer treatments to induce uterine damage, and the underlying mechanisms, have been understudied. Here, we show that in mice total-body γ-irradiation (TBI) induced extensive DNA damage and apoptosis in uterine cells. We then transferred healthy donor embryos into ovariectomized adolescent female mice that were previously exposed to TBI to study the impacts of radiotherapy on the uterus independent from effects to ovarian endocrine function. Following TBI, embryo attachment and implantation were unaffected, but fetal resorption was evident at midgestation in 100% of dams, suggesting failed placental development. Consistent with this hypothesis, TBI impaired the decidual response in mice and primary human endometrial stromal cells. TBI also caused uterine artery endothelial dysfunction, likely preventing adequate blood vessel remodeling in early pregnancy. Notably, when pro-apoptotic protein Puma-deficient (Puma-/-) mice were exposed to TBI, apoptosis within the uterus was prevented, and decidualization, vascular function, and pregnancy were restored, identifying PUMA-mediated apoptosis as a key mechanism. Collectively, these data show that TBI damages the uterus and compromises pregnancy success, suggesting that optimal fertility preservation during radiotherapy may require protection of both the ovaries and uterus. In this regard, inhibition of PUMA may represent a potential fertility preservation strategy.

Article	Year
Bone Marrow Stem Cells Do Not Contribute to Endometrial Cell Lineages in Chimeric Mouse Models. Stem cells (Dayton, Ohio)\| PubMed ID: 28913973	2018
N-cadherin identifies human endometrial epithelial progenitor cells by in vitro stem cell assays. Human reproduction (Oxford, England)\| PubMed ID: 29040564	2017
Endometrial organoids: in vitro models for endometrial research and personalized medicine. Biology of reproduction\| PubMed ID: 29106468	2017
In Reply to Letter to the Editor from Bhartiya: Transplantation of Whole Bone Marrow Indicates That Bone Marrow Very Small Embryonic-Like Cells Do Not Contribute to Endometrial Lineages. Stem cells (Dayton, Ohio)\| PubMed ID: 29345012	2018
Endometrial stem/progenitor cells and their role in the pathogenesis of endometriosis. Best practice & research. Clinical obstetrics & gynaecology\| PubMed ID: 29503126	2018
Telomerase Reverse Transcriptase Expression in Mouse Endometrium During Reepithelialization and Regeneration in a Menses-Like Model. Stem cells and development\| PubMed ID: 30358490	2019
A patient derived xenograft model of cervical cancer and cervical dysplasia. PloS one\| PubMed ID: 30365542	2018
Electrospun Nanofiber Meshes With Endometrial MSCs Modulate Foreign Body Response by Increased Angiogenesis, Matrix Synthesis, and Anti-Inflammatory Gene Expression in Mice: Implication in P... Frontiers in pharmacology\| PubMed ID: 32265721	2020
The effects of hedgehog ligand neutralising antibody 5E1 in a mouse model of endometriosis. BMC research notes\| PubMed ID: 32977859	2020
The Elusive Endometrial Epithelial Stem/Progenitor Cells. Frontiers in cell and developmental biology\| PubMed ID: 33898428	2021
Identification and characterisation of maternal perivascular SUSD2 placental mesenchymal stem/stromal cells. Cell and tissue research\| PubMed ID: 33961124	2021
Comparison of Organoids from Menstrual Fluid and Hormone-Treated Endometrium: Novel Tools for Gynecological Research. Journal of personalized medicine\| PubMed ID: 34945786	2021
Endometrial Stem/Progenitor Cells-Their Role in Endometrial Repair and Regeneration. Frontiers in reproductive health\| PubMed ID: 36304009	2021
New concepts on the etiology of endometriosis. The journal of obstetrics and gynaecology research\| PubMed ID: 36746607	2023
Radiotherapy exposure directly damages the uterus and causes pregnancy loss. JCI insight\| PubMed ID: 36946464	2023

Fiona L. Cousins

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