Endometrial Origins of Stillbirth (EOS), a case-control study of menstrual fluid to understand and prevent preterm stillbirth and associated adverse pregnancy outcomes: study protocol.

INTRODUCTION: Current research aimed at understanding and preventing stillbirth focuses almost exclusively on the role of the placenta. The underlying origins of poor placental function leading to stillbirth, however, remain poorly understood. There is evidence demonstrating that the endometrial environment in which the embryo implants impacts not only the establishment of pregnancy but also the development of some pregnancy outcomes. Menstrual fluid has recently been applied to the study of menstrual disorders such as heavy menstrual bleeding or endometriosis, however, it has great potential in the study of adverse pregnancy outcomes. This study aims to identify differences in menstrual fluid and menstrual cycle characteristics of women who have experienced preterm stillbirth and other associated adverse pregnancy outcomes, compared with those who have not. The association between menstrual fluid composition and menstrual cycle characteristics will also be determined. METHODS AND ANALYSIS: This is a case-control study of women who have experienced a late miscarriage, spontaneous preterm birth or preterm stillbirth or a pregnancy complicated by placental insufficiency (fetal growth restriction or pre-eclampsia), compared with those who have had a healthy term birth. Cases will be matched for maternal age, body mass index and gravidity. Participants will not currently be on hormonal therapy. Women will be provided with a menstrual cup and will collect their sample on day 2 of menstruation. Primary exposure measures include morphological and functional differences in decidualisation of the endometrium (cell types, immune cell subpopulations and protein composition secreted from the decidualised endometrium). Women will complete a menstrual history survey to capture menstrual cycle length, regularity, level of pain and heaviness of flow. ETHICS AND DISSEMINATION: Ethics approval was obtained from Monash University Human Research Ethics Committee (27900) on 14/07/2021 and will be conducted in accordance with these conditions. Findings from this study will be disseminated through peer-reviewed publications and conference presentations.

Radiotherapy exposure directly damages the uterus and causes pregnancy loss.

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.

New concepts on the etiology of endometriosis.

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.

Subtle changes in perivascular endometrial mesenchymal stem cells after local endometrial injury in recurrent implantation failure.

Improvements in reproductive techniques have resulted in the live birth rates from IVF procedures increasing from 5% to approximately 30% in recent decades but has plateaued since. Emerging preclinical and clinical data implicates endometrial receptivity deficiencies in patients with recurrent implantation failure (RIF) as the predominant factor hindering successful implantation. Mechanisms on how local endometrial injury (LEI) improves implantation rates in patients with RIF are currently unknown. We hypothesized that LEI may influence perivascular endometrial mesenchymal stem/progenitor cells (eMSCs) which are thought to regenerate the stromal vascular component of the functional layer every month. Here, we assessed the effect of LEI on the proportion and function of eMSCs present in consecutive LEI biopsies. Consecutive paired mid-luteal phase endometrial biopsies obtained from patients with RIF were digested to single cells and the proportion of SUSD2-expressing cells determined. Growth kinetics and decidualization were compared between the consecutive LEI samples. A mid-luteal LEI altered the decidualization capacity of SUSD2+ eMSCs in women with RIF, but not their proportion or clonogenicity. With the potential of LEI to improve IVF outcomes in women with RIF, additional investigations are needed to understand the impact of the altered decidualization response in eMSCs.

Endometrial Stem/Progenitor Cells: Prospects and Challenges.

The human endometrium is one of the most regenerative tissues in the body, undergoing over 400 cycles of menstrual shedding and regeneration during reproductive life [...].

ENDOCELL-Seud: a Delphi protocol to harmonise methods in endometrial cell culturing.

In vitro: culturing of endometrial cells obtained from the uterine mucosa or ectopic sites is used to study molecular and cellular signalling relevant to physiologic and pathologic reproductive conditions. However, the lack of consensus on standard operating procedures for deriving, characterising and maintaining primary cells in two- or three-dimensional cultures from eutopic or ectopic endometrium may be hindering progress in this area of research. Guidance for unbiased in vitro research methodologies in the field of reproductive science remains essential to increase confidence in the reliability of in vitro models. We present herein the protocol for a Delphi process to develop a consensus on in vitro methodologies using endometrial cells (ENDOCELL-Seud Project). A steering committee composed of leading scientists will select critical methodologies, topics and items that need to be harmonised and that will be included in a survey. An enlarged panel of experts (ENDOCELL-Seud Working Group) will be invited to participate in the survey and provide their ratings to the items to be harmonised. According to Delphi, an iterative investigation method will be adopted. Recommended measures will be finalised by the steering committee. The study received full ethical approval from the Ethical Committee of the Maastricht University (ref. FHML-REC/2021/103). The study findings will be available in both peer-reviewed articles and will also be disseminated to appropriate audiences at relevant conferences. Lay summary: Patient-derived cells cultured in the lab are simple and cost-effective methods used to study biological and dysfunctional or disease processes. These tools are frequently used in the field of reproductive medicine. However, the lack of clear recommendations and standardised methodology to guide the laboratory work of researchers can produce results that are not always reproducible and sometimes are incorrect. To remedy this situation, we define here a method to ascertain if researchers who routinely culture cells in the lab agree or disagree on the optimal laboratory techniques. This method will be used to make recommendations for future researchers working in the field of reproductive biology to reproducibly culture endometrial cells in the laboratory.

Vaginal pressure sensor measurement during maximal voluntary pelvic floor contraction correlates with vaginal birth and pelvic organ prolapse-A pilot study.

AIMS: To measure the force applied along the anterior and posterior vaginal walls in a cohort of 46 patients measured by a fiber-optic pressure sensor and determine if this correlates with vaginal parity and pelvic organ prolapse (POP). METHODS: An intravaginal fiber-optic sensor measured pressure at nine locations along the anterior and posterior vaginal walls during a maximal voluntary pelvic floor muscle contraction (MVC). An automated probe dilation cycle measured the tissue resistance incorporating the vagina and surrounding anatomy. MVC and resting tissue resistance (RTR) were assessed between subjects grouped by the number of vaginal births and prolapse stage. RESULTS: A previous vaginal birth was associated with a significant threefold decrease in the overall anterior pressure measurement during MVC. Decreased anterior pressure measurements were observed at Sensors 1 and 3 (distal vagina) and, posteriorly at Sensors 4-6 (midvagina). Women with Stage 2 posterior prolapse exhibited a decreased MVC pressure in the midvagina than those with Stage 0/1. In this pilot study, there was no difference in the vaginal wall RTR according to previous vaginal birth or stage of prolapse. CONCLUSION: This pilot study found that a decrease in vaginal pressure measured during MVC is associated with vaginal birth and with posterior POP. Greater sample size is required to assess the role of resting tissue pressure measurement.

The fate of human SUSD2+ endometrial mesenchymal stem cells during decidualization.

Regeneration of the endometrial stromal compartment in premenopausal women is likely maintained by the perivascular endometrial mesenchymal stem/stromal cells (eMSC) expressing sushi domain containing 2 (SUSD2). The fate of SUSD2+ eMSC during pregnancy and their role in decidualization is not fully known. The aim of our study was to determine the effect of progesterone on the stemness of the SUSD2+ eMSC isolated from non-pregnant uterine samples. Secondary objectives were to characterize the functional capacity including differentiation and clonogenicity assays of SUSD2+ eMSC isolated from decidua at full term and compare it to the capacity of those isolated from non-pregnant uterine samples. Progesterone treatment induced changes in the decidual gene expression profile in non-pregnant SUSD2+ eMSC. Data analysis of a publicly available single cell RNA-seq data set revealed differential expression of several mesenchymal and epithelial signature genes between the SUSD2+ eMSC and the decidual stromal cells, suggesting mesenchymal-to-epithelial transition occurs during decidualization. Histological analysis revealed a significantly lower abundance of SUSD2+ eMSC in 1st trimester and full term samples compared to non-pregnant samples, p = 0.0296 and 0.005, respectively. The differentiation and the colony forming capacity did not differ significantly between the cells isolated from non-pregnant and pregnant uterine samples. Our results suggest that SUSD2+ eMSC undergo decidualization in vitro, while maintaining MSC plasma membrane phenotype. Human eMSC seem to play an important role in the course of endometrial decidualization and embryo implantation. Pregnancy reduced the abundance of SUSD2+ eMSC, however eMSC function remains intact.

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

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.

Endometrial SUSD2+ Mesenchymal Stem/Stromal Cells in Tissue Engineering: Advances in Novel Cellular Constructs for Pelvic Organ Prolapse.

Cellular therapy is an emerging field in clinical and personalised medicine. Many adult mesenchymal stem/progenitor cells (MSC) or pluripotent derivatives are being assessed simultaneously in preclinical trials for their potential treatment applications in chronic and degenerative human diseases. Endometrial mesenchymal stem/progenitor cells (eMSC) have been identified as clonogenic cells that exist in unique perivascular niches within the uterine endometrium. Compared with MSC isolated from other tissue sources, such as bone marrow and adipose tissue, eMSC can be extracted through less invasive methods of tissue sampling, and they exhibit improvements in potency, proliferative capacity, and control of culture-induced differentiation. In this review, we summarize the potential cell therapy and tissue engineering applications of eMSC in pelvic organ prolapse (POP), emphasising their ability to exert angiogenic and strong immunomodulatory responses that improve tissue integration of novel surgical constructs for POP and promote vaginal tissue healing.

Cyclical endometrial repair and regeneration.

Uniquely among adult tissues, the human endometrium undergoes cyclical shedding, scar-free repair and regeneration during a woman's reproductive life. Therefore, it presents an outstanding model for study of such processes. This Review examines what is known of endometrial repair and regeneration following menstruation and parturition, including comparisons with wound repair and the influence of menstrual fluid components. We also discuss the contribution of endometrial stem/progenitor cells to endometrial regeneration, including the importance of the stem cell niche and stem cell-derived extracellular vesicles. Finally, we comment on the value of endometrial epithelial organoids to extend our understanding of endometrial development and regeneration, as well as therapeutic applications.

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

Endometrial stem/progenitor cells in menstrual blood and peritoneal fluid of women with and without endometriosis.

RESEARCH QUESTION: Are endometrial stem/progenitor cells shed into uterine menstrual blood (UMB) and the peritoneal cavity in women with and without endometriosis during menstruation? DESIGN: Women with (n = 32) and without endometriosis (n = 29) at laparoscopy (total 61), carried out during the menstrual (n = 41) and non-menstrual phase (n = 20) were recruited. The UMB, peritoneal fluid and peripheral blood were analysed by clonogenicity assay and flow cytometry to quantify the concentrations of endometrial clonogenic cells, SUSD2+ mesenchymal stem cells (eMSC) and N-cadherin+ epithelial progenitor cells (eEPC). RESULTS: Clonogenic endometrial cells, eMSC and eEPC were found in most UMB samples at similar concentrations in women with and without endometriosis. In contrast, 62.5% of women with endometriosis and 75.0% without (controls) had clonogenic cells in peritoneal fluid samples during menses. The eMSC were present in the peritoneal fluid of 76.9% of women with endometriosis and 44.4% without, and eEPC were found in the peritoneal fluid of 60.0% of women with and 25.0% without endometriosis during menses. Median clonogenic, eMSC and eEPC concentrations in peritoneal fluid were not significantly different between groups. More clonogenic cells persisted beyond the menstrual phase in the peritoneal fluid of women with endometriosis (menstrual 119/ml [0-1360/ml] versus non-menstrual 8.5/ml [0-387/ml]; P = 0.277) compared with controls (menstrual 76.5/ml [1-1378/ml] versus non-menstrual 0/ml [0-14/ml]; P = 0.0362). No clonogenic endometrial cells were found in peripheral blood. CONCLUSIONS: Clonogenic endometrial cells, SUSD2+ eMSC and N-cadherin+ eEPC are present in UMB and the peritoneal fluid of women with and without endometriosis. Further study of the function of these cells may shed light on the cellular origins of endometriosis.

The Elusive Endometrial Epithelial Stem/Progenitor Cells.

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 in vivo 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 in vitro functional assays and in vivo 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.

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

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 in vitro 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 PDGFRß 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 in vivo.

Comparing the Effect of TGF-ß Receptor Inhibition on Human Perivascular Mesenchymal Stromal Cells Derived from Endometrium, Bone Marrow and Adipose Tissues.

Rare perivascular mesenchymal stromal cells (MSCs) with therapeutic properties have been identified in many tissues. Their rarity necessitates extensive in vitro expansion, resulting in spontaneous differentiation, cellular senescence and apoptosis, producing therapeutic products with variable quality and decreased potency. We previously demonstrated that A83-01, a transforming growth factor beta (TGF-ß) receptor inhibitor, maintained clonogenicity and promoted the potency of culture-expanded premenopausal endometrial MSCs using functional assays and whole-transcriptome sequencing. Here, we compared the effects of A83-01 on MSCs derived from postmenopausal endometrium, menstrual blood, placenta decidua-basalis, bone marrow and adipose tissue. Sushi-domain-containing-2 (SUSD2+) and CD34+CD31-CD45- MSCs were isolated. Expanded MSCs were cultured with or without A83-01 for 7 days and assessed for MSC properties. SUSD2 identified perivascular cells in the placental decidua-basalis, and their maternal origin was validated. A83-01 promoted MSC proliferation from all sources except bone marrow and only increased SUSD2 expression and prevented apoptosis in MSCs from endometrial-derived tissues. A83-01 only improved the cloning efficiency of postmenopausal endometrial MSCs (eMSCs), and expanded adipose tissue MSCs (adMSCs) underwent significant senescence, which was mitigated by A83-01. MSCs derived from bone marrow (bmMSCs) were highly apoptotic, but A83-01 was without effect. A83-01 maintained the function and phenotype in MSCs cultured from endometrial, but not other, tissues. Our results also demonstrated that cellular SUSD2 expression directly correlates with the functional phenotype.

Cellular Origins of Endometriosis: Towards Novel Diagnostics and Therapeutics.

Endometriosis remains an enigmatic disease of unknown etiology, with delayed diagnosis and poor therapeutic options. This review will discuss the cellular, physiological, and genomic evidence of Sampson's hypothesis of retrograde menstruation as a cause of pelvic endometriosis and as the basis of phenotypic heterogeneity of the disease. We postulate that collaborative research at the single cell level focused on unlocking the cellular, physiological, and genomic mechanisms of endometriosis will be accompanied by advances in personalized diagnosis and therapies that target unique subtypes of endometriosis disease. These advances will address the clinical conundrums of endometriosis clinical care-including diagnostic delay, suboptimal treatments, disease recurrence, infertility, chronic pelvic pain, and quality of life. There is an urgent need to improve outcomes for women with endometriosis. To achieve this, it is imperative that we understand which cells form the lesions, how they arrive at distant sites, and what factors govern their ability to survive and invade at ectopic locations. This review proposes new research avenues to address these basic questions of endometriosis pathobiology that will lay the foundations for new diagnostic tools and treatment pathways.

Impact of Sustained Transforming Growth Factor-ß Receptor Inhibition on Chromatin Accessibility and Gene Expression in Cultured Human Endometrial MSC.

Endometrial mesenchymal stem cells (eMSC) drive the extraordinary regenerative capacity of the human endometrium. Clinical application of eMSC for therapeutic purposes is hampered by spontaneous differentiation and cellular senescence upon large-scale expansion in vitro. A83-01, a selective transforming growth factor-ß receptor (TGFß-R) inhibitor, promotes expansion of eMSC in culture by blocking differentiation and senescence, but the underlying mechanisms are incompletely understood. In this study, we combined RNA-seq and ATAC-seq to study the impact of sustained TGFß-R inhibition on gene expression and chromatin architecture of eMSC. Treatment of primary eMSC with A83-01 for 5 weeks resulted in differential expression of 1,463 genes. Gene ontology analysis showed enrichment of genes implicated in cell growth whereas extracellular matrix genes and genes involved in cell fate commitment were downregulated. ATAC-seq analysis demonstrated that sustained TGFß-R inhibition results in opening and closure of 3,555 and 2,412 chromatin loci, respectively. Motif analysis revealed marked enrichment of retinoic acid receptor (RAR) binding sites, which was paralleled by the induction of RARB, encoding retinoic acid receptor beta (RARß). Selective RARß inhibition attenuated proliferation and clonogenicity of A83-01 treated eMSC. Taken together, our study provides new insights into the gene networks and genome-wide chromatin changes that underpin maintenance of an undifferentiated phenotype of eMSC in prolonged culture.