The Essential Role of PGF2α/PTGFR in Molding Endometrial Breakdown and Vascular Dynamics, Regulated by HIF-1α in a Mouse Menstrual-like Model.

In women of childbearing age, extensive decidualization, shedding and remodeling of the endometrium during the menstrual cycle are fundamental for successful pregnancy. The role of prostaglandins (PGs) in menstruation has long been proposed in humans, and the rate-limiting enzyme cyclooxygenase was shown to play a key role in endometrial breakdown and shedding in a mouse menstrual-like model in our previous study. However, the specific types of PGs involved and their respective roles remain unclear. Therefore, our objective was to investigate the mechanism through which PGs regulate endometrial disintegration. In this study, the microscopy was observed by HE; the protein levels of prostaglandins E1 (PGE1), prostaglandins E2 (PGE2), prostaglandin F2α (PGF2α) and Prostaglandin I2 (PGI2) were detected by ELISA; the mRNA level of Pfgfr2, Vascular Endothelial Growth Factor(Vegf), Angiostatin and Hypoxia inducible factor-1α (Hif1α) were examined by real-time PCR; PTGFR Receptor (PTGFR), VEGF, Angiostatin and HIF-1α protein levels were investigated by western blotting; the locations of protein were observed by Immunohistochemistry; HIF-1α binding PTGFR promoter was detected by Chromatin Immunoprecipitation (ChIP) and real-time PCR. We found that the concentrations of PGE1, PGE2, and PGF2α all increased significantly during this process. Furthermore, Ptgfr mRNA increased soon after Progesterone (P4) withdrawal, and PTGFR protein levels increased significantly during abundant endometrial breakdown and shedding processes. PTGFR inhibitors AL8810 significantly suppressed endometrial breakdown and shedding, promoted Angiostatin expression, and reduced VEGF-A expressions and vascular permeability. And HIF-1α and PTGFR were mainly located in the luminal/gland epithelium, vascular endothelium, and pre-decidual zone. Interestingly, HIF-1α directly bound to Ptgfr promoter. Moreover, a HIF-1α inhibitor 2-methoxyestradiol (2ME) significantly reduced PTGFR expression and suppressed endometrial breakdown which was in accord with PTGFR inhibitor's effect. Similar changes occurred in human stromal cells relevant to menstruation in vitro. Our study provides evidence that PGF2α/PTGFR plays a vital role in endometrial breakdown via vascular changes that are regulated by HIF-1α during menstruation.

HGF Secreted by Menstrual Blood-Derived Endometrial Stem Cells Ameliorates Non-Alcoholic Fatty Liver Disease Through Downregulation of Hepatic Rnf186.

Mesenchymal stem cells (MSCs) have been demonstrated to protect against fatty liver diseases, but the mechanism is still not clear. Menstrual blood-derived endometrial stem cells (MenSCs) are a substantial population of MSCs that can be obtained in a noninvasive manner. In the present study, we investigated the therapeutic effects and underlying mechanisms of MenSC transplantation in mouse models of diet-induced nonalcoholic fatty liver disease (NAFLD). The results revealed that MenSCs markedly promoted hepatic glycogen storage and attenuated lipid accumulation after transplantation. We further identified Rnf186 as a novel regulator involved in MenSC-based therapy for NAFLD mice. Rnf186 deficiency substantially inhibited high-fat diet-induced insulin resistance and abnormal hepatic glucose and lipid metabolism in mice. Mechanistically, Rnf186 regulated glucose and lipid metabolism through the AMPK-mTOR pathway. More importantly, hepatocyte growth factor (HGF) is identified as the key functional cytokine secreted by MenSCs and decreases the expression of hepatic Rnf186. HGF deficient MenSCs cannot attenuate glucose and lipid accumulation after transplantation in NAFLD mice. Collectively, our results provide preliminary evidence for the protective roles of HGF secreted by MenSCs in fatty liver diseases through downregulation of hepatic Rnf186 and suggest that MenSCs or Rnf186 may be an alternative therapeutic approach/target for the treatment of NAFLD.

Single-cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation.

The human endometrium experiences repetitive cycles of tissue wounding characterised by piecemeal shedding of the surface epithelium and rapid restoration of tissue homeostasis. In this study, we used a mouse model of endometrial repair and three transgenic lines of mice to investigate whether epithelial cells that become incorporated into the newly formed luminal epithelium have their origins in one or more of the mesenchymal cell types present in the stromal compartment of the endometrium. Using scRNAseq, we identified a novel population of PDGFRb + mesenchymal stromal cells that developed a unique transcriptomic signature in response to endometrial breakdown/repair. These cells expressed genes usually considered specific to epithelial cells and in silico trajectory analysis suggested they were stromal fibroblasts in transition to becoming epithelial cells. To confirm our hypothesis we used a lineage tracing strategy to compare the fate of stromal fibroblasts (PDGFRa+) and stromal perivascular cells (NG2/CSPG4+). We demonstrated that stromal fibroblasts can undergo a mesenchyme to epithelial transformation and become incorporated into the re-epithelialised luminal surface of the repaired tissue. This study is the first to discover a novel population of wound-responsive, plastic endometrial stromal fibroblasts that contribute to the rapid restoration of an intact luminal epithelium during endometrial repair. These findings form a platform for comparisons both to endometrial pathologies which involve a fibrotic response (Asherman's syndrome, endometriosis) as well as other mucosal tissues which have a variable response to wounding.

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.

Endometrial macrophages in health and disease.

Macrophages are present in the endometrium throughout the menstrual cycle and are most abundant during menstruation. Endometrial macrophages contribute to tissue remodeling during establishment of pregnancy and are thought to play key roles in mediating tissue breakdown and repair during menstruation. Despite these important roles, the phenotype and function of endometrial macrophages remains poorly understood. In this review, we summarize approaches used to characterize endometrial macrophage phenotype, current understanding of the functional role of macrophages in normal endometrial physiology as well as the putative contribution of macrophage dysfunction to women's reproductive health disorders.

Effect of estrogen/progesterone ratio on the differentiation and the barrier function of epidermal keratinocyte and three-dimensional cultured human epidermis.

AIMS: Estrogen (E) and progesterone (P) are the major female hormones and are secreted with changing concentration ratios throughout the menstrual cycle. These hormones have been studied individually regarding their physiological function in the skin, but their concentration ratio (E/P) and its effect on the skin has not yet been investigated. The purpose of this study was to clarify the effect of the E/P ratio on skin barrier function. The menstrual cycle was divided into the menstrual, follicular, ovulation, and luteal phases. MATERIALS AND METHODS: The E/P concentration ratios corresponding with each phase were added to a three-dimensional epidermal model or normal human epidermal keratinocytes for 5 days. Gene and protein expression levels of several markers of cell differentiation, including loricrin (LOR) and transglutaminase (TGase), were quantified by real-time PCR and western blotting, respectively. Transepidermal water loss (TEWL) of the three-dimensional epidermal model was measured, and ceramide content was quantified by thin-layer chromatography. KEY FINDINGS: Gene expression of the epidermal differentiation markers, LOR and TGase, increased when applying the concentration ratio of E/P associated with the menstrual and luteal phases. The LOR protein level decreased from menstrual to luteal phases, and the TGase protein level increased from menstrual to luteal phases. During the same phases, ceramide NS increased and TEWL decreased. SIGNIFICANCE: Skin barrier function was improved by culturing cells at specific E/P concentration ratios, which would, therefore, be considered beneficial for female skin. This suggests that dysregulated E/P concentration ratios may be the cause of certain skin problems.

A Comparative Study of Gene Expression in Menstrual Blood-Derived Stromal Cells between Endometriosis and Healthy Women.

BACKGROUND: Research into the pathogenesis of endometriosis would substantially promote its effective treatment and early diagnosis. Currently, accumulating evidence has shed light on the importance of endometrial stem cells within the menstrual blood which are involved in the establishment and progression of endometriotic lesions in a retrograde manner. OBJECTIVES: We aimed to identify the differences in some genes' expression between menstrual blood-derived mesenchymal stem cells (MenSCs) isolated from endometriosis patients (E-MenSCs) and MenSCs from healthy women (NE-MenSCs). METHODS: Menstrual blood samples (2-3 mL) from healthy and endometriosis women in the age range of 22-35 years were collected. Isolated MenSCs by the Ficoll-Paque density-gradient centrifugation method were characterized by flow cytometry. MenSCs were evaluated for key related endometriosis genes by real-time-PCR. RESULTS: E-MenSCs were morphologically different from NE-MenSCs and showed, respectively, higher and lower expression of CD10 and CD9. Furthermore, E-MenSCs had higher expression of Cyclin D1 (a cell cycle-related gene) and MMP-2 and MMP-9 (migration- and invasion-related genes) genes compared with NE-MenSCs. Despite higher cell proliferation in E-MenSCs, the BAX/BCL-2 ratio was significantly lower in E-MenSCs compared to NE-MenSCs. Also, the level of inflammatory genes such as IL1ß, IL6, IL8, and NF-κB and stemness genes including SOX2 and SALL4 was increased in E-MenSCs compared with NE-MenSCs. Further, VEGF, as a potent angiogenic factor, showed a significant increase in E-MenSCs rather than NE-MenSCs. However, NE-MenSCs showed increased ER-α and ß-catenin when compared with E-MenSCs. CONCLUSION: Here, we showed that there are gene expression differences between E-MenSCs and NE-MenSCs. These findings propose that MenSCs could play key role in the pathogenesis of endometriosis and further support the menstrual blood retrograde theory of endometriosis formation. This could be of great importance in exploiting promising therapeutic targets and new biomarkers for endometriosis treatment and prognosis.

Prevalence, impact, and management perception of dysmenorrhea among university students: A cross-sectional study

Abstract Dysmenorrhea is a common condition among females that is characterized by painful cramps before or during menstruation. It is considered as a common gynecological complaint that affects the quality of women's life. The study evaluated prevalence of dysmenorrhea, its impact, associated risk factors, and the management strategies adopted by female university students in Taif city, Saudi Arabia. A cross-sectional study was conducted among 562 female students aged 18-30 years at the university level. The results showed a high prevalence rate of dysmenorrhea (79.4%) among the students. The most common risk factors were family history (87.4%) and length of menstruation (79%). Half (50.2%) of the respondents were absent at the university at least 1 day every month. The most widely used medications by the respondents were ibuprofen (42%) and paracetamol (40%), whereas only 3% used mefenamic acid, despite that they experienced complete pain relief with mefenamic acid. High prevalence rate of dysmenorrhea associated with risk factors such as family history and length of menstruation, was found among university students. However, pain and associated symptoms affect the quality of life.

Mouse model of menstruation: An indispensable tool to investigate the mechanisms of menstruation and gynaecological diseases (Review).

Abnormal menstruation may result in several pathological alterations and gynaecological diseases, including endometriosis, menstrual pain and miscarriage. However, the pathogenesis of menstruation remains unclear due to the limited number of animal models available to study the menstrual cycle. In recent years, an effective, reproducible, and highly adaptive mouse model to study menstruation has been developed. In this model, progesterone and oestrogen were administered in cycles following the removal of ovaries. Subsequently, endometrial decidualisation was induced using sesame oil, followed by withdrawal of progesterone administration. Vaginal bleeding in mice is similar to that in humans. Therefore, the use of mice as a model organism to study the mechanism of menstruation and gynaecological diseases may prove to be an important breakthrough. The present review is focussed ond the development and applications of a mouse model of menstruation. Furthermore, various studies have been described to improve this model and the research findings that may aid in the treatment of menstrual disorders in women are presented.

Effect of Tahiti lime (Citrus latifolia) juice on the Production of the PGF2α/PGE2 and Pro-Inflammatory Cytokines involved in Menstruation.

Tahiti lemon juice (Citrus latifolia) (TLJ), as a natural source of flavonoids, has been used as an alternative to anti-inflammatory drugs for the treatment of dysmenorrhea and menstrual excessive bleeding, often associated with an imbalance of the prostaglandins (PG) levels. However, despite the positive effects, the mechanisms that rule menstruation control are still unknown. Therefore, the objectives were to characterize the TLJ and analyze its effect on the production of PGF2α, PGE2 and pro-inflammatory cytokines involved inmenstruation. Flavonoids from TLJ were discriminated by UPLC-DAD-MS/MS (Qq-TOF) and the effects of TLJ were studied in vitro by quantification of the contraction of myoblasts in culture and PGF2α and PGE2 productions. Further, the systemic and menstrual fluid levels of PGF2α, PGE2, IL-1ß, TNF-α, IL-6, AK1B1 and AK1C3 enzymes produced by women during the menstrual period were compared after exposition or not to TLJ or meloxicam. The results showed that TLJ induces an increase in the contraction of myoblasts and the PGF2α supernatant level. Regarding in vivo analysis, a higher concentration of PGF2α and an unaltered PGE2 level was also found in the menstrual blood of women treated with TLJ, in contrast with a lower level of PGE2 and PGF2α observed in the meloxicam group. Concerning cytokines, only menstrual TNF-α levels decrease after treatment with TLJ or meloxicam. In conclusion, TLJ may favor the control of menstruation events via a PGF2α mediated muscle contractile response.

Physiologic Uterine Uptake of Radioiodine During Menstruation Demonstrated by SPECT/CT.

A 35-year-old woman with papillary thyroid cancer underwent I therapy after thyroidectomy. Post-therapy whole body scan revealed increased activity in the pelvis, in addition to the activity in the neck. On SPECT/CT images, the radioactivity in the pelvis was localized in the rectum and cervix. Further inquiry discovered that the patient was menstruating. We concluded that abnormal radioiodine uptake in menstrual uterus might be an exceptional finding mimicking a metastasis.

Activation of the Hippo/TAZ pathway is required for menstrual stem cells to suppress myofibroblast and inhibit transforming growth factor ß signaling in human endometrial stromal cells.

STUDY QUESTION: Can menstrual stem cells (MenSCs) inhibit myofibroblast differentiation and reverse transforming growth factor ß (TGFß)-mediated activation of myofibroblast phenotypes in human endometrial stromal cells (ESCs)? SUMMARY ANSWER: MenSCs suppressed endometrial myofibroblast differentiation and reversed TGFß-mediated activation of myofibroblast phenotypes, which might be associated with activation of the Hippo/TAZ pathway. WHAT IS KNOWN ALREADY: The potential effect of MenSCs as a cell therapy include attenuation of intrauterine adhesions, but the underlying mechanisms by which MenSCs exerts these effects are not entirely understood. STUDY DESIGN, SIZE, DURATION: We evaluated the antagonistic effects of MenSCs on myofibroblast differentiation as well as the broader effect of the Hippo/TAZ signaling pathway on TGFß-mediated induction of myofibroblast gene expression. The study design was based on a cohort of clinical proliferative phase endometrial samples obtained from three healthy premenopausal females with regular menstrual cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: ESCs were cocultured with MenSCs or in MenSC-conditioned medium. Fibrotic markers (αSMA, collagen I, CTGF and fibronectin) as well as proliferation and wound-healing abilities were evaluated. Components of the Hippo/TAZ pathway (TAZ, p-TAZ, MOB1, p-MOB1, LATS1 and p-LATS1) were also investigated. Cell Counting Kit 8, wound healing assay, real-time PCR, western blotting, immunofluorescence and shRNA knockdown approaches were used to validate the findings. MAIN RESULTS AND THE ROLE OF CHANCE: MenSCs inhibited myofibroblast activation, resulting in more rapid proliferation of ESCs. MenSCs downregulated the expression of myofibroblast markers αSMA and collagen I and promoted endometrial wound healing. Coculture with MenSCs also attenuated the TGFß-mediated increase in expression of fibrotic marker genes αSMA, collagen I, CTGF and fibronectin, and restored the wound-healing ability inhibited by TGFß. MenSCs induced Hippo/TAZ pathway activation, resulting in nuclear export and cytoplasmic retention of TAZ. TAZ inhibition was demonstrated to have similar effects even in the absence of MenSCs, and inhibition of TAZ was sufficient to attenuate TGFß-mediated myofibroblast activation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study included only in vitro experiments. Thus, additional data from in vivo experiments are needed in a future study. WIDER IMPLICATIONS OF THE FINDINGS: The Hippo/TAZ pathway may be an important therapeutic target for endometrial fibrosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (No. 81601236) and Zhejiang Provincial Natural Science Foundation of China (LY19H040009). None of the authors has any competing interests to declare.

Menstrual fluid factors facilitate tissue repair: identification and functional action in endometrial and skin repair.

Repair after damage is essential for tissue homeostasis. Postmenstrual endometrial repair is a cyclical manifestation of rapid, scar-free, tissue repair taking ∼3-5 d. Skin repair after wounding is slower (∼2 wk). In the case of chronic wounds, it takes months to years to restore integrity. Herein, the unique "rapid-repair" endometrial environment is translated to the "slower repair" skin environment. Menstrual fluid (MF), the milieu of postmenstrual endometrial repair, facilitates healing of endometrial and keratinocyte "wounds" in vitro, promoting cellular adhesion and migration, stimulates keratinocyte migration in an ex vivo human skin reconstruct model, and promotes re-epithelialization in an in vivo porcine wound model. Proteomic analysis of MF identified a large number of proteins: migration inhibitory factor, neutrophil gelatinase-associated lipocalin, follistatin like-1, chemokine ligand-20, and secretory leukocyte protease inhibitor were selected for further investigation. Functionally, they promote repair of endometrial and keratinocyte wounds by promoting migration. Translation of these and other MF factors into a migration-inducing treatment paradigm could provide novel treatments for tissue repair.-Evans, J., Infusini, G., McGovern, J., Cuttle, L., Webb, A., Nebl, T., Milla, L., Kimble, R., Kempf, M., Andrews, C. J., Leavesley, D., Salamonsen, L. A. Menstrual fluid factors facilitate tissue repair: identification and functional action in endometrial and skin repair.

Characterization of human-like menstruation in the spiny mouse: comparative studies with the human and induced mouse model.

STUDY QUESTION: Is the newly discovered menstruating rodent, the spiny mouse, a valid model for studying endometrial morphology and menstruation? SUMMARY ANSWER: Our study is the first to demonstrate a primate-like pattern of natural menstruation in a rodent, with decidualization, spiral arteriole remodeling and piece-meal endometrial shedding. WHAT IS KNOWN ALREADY: The spiny mouse has a naturally occurring menstrual cycle. This unique feature has the potential to reduce the heavy reliance on primates and provide a more appropriate small animal model for menstrual physiology research. STUDY DESIGN, SIZE, DURATION: This study compares morphological changes in the endometrium during early, mid and late menstruation of the spiny mouse (n = 39), human (n = 9) and the induced mouse model of menstruation (n = 17). PARTICIPANTS/MATERIALS, SETTING, METHODS: We assessed tissue morphology with hematoxylin and eosin and erythrocyte patterns with Mallory's trichrome. We conducted staining for neutrophil gelatinase associated lipocalin (NGAL), cytokeratin and interleukin-11 (IL-11) in all species. We used double immunofluorescence staining for vascular endothelial growth factor and alpha-smooth muscle actin to detect vasculature remodeling and western immunoblot to detect interleukin-8 (IL-8) and macrophage migration inhibitory factor (MIF) in the menstrual fluid of spiny mice. MAIN RESULTS AND THE ROLE OF CHANCE: Menstruation occurs in the spiny mouse over a 72-h period, with heaviest menstrual breakdown occurring 24 h after initial observation of red blood cells in the vaginal cytology. During menstruation, the endometrium of the spiny mouse appeared to resemble human menstrual shedding with focal epithelial breakdown observed in conjunction with lysis of underlying stroma and detection of IL-8 and MIF in menstrual fluid. The mouse exhibits extensive decidualization prior to induced menses, with transformation of the entire uterine horn and cytokeratin expression absent until initiation of repair. Decidualization occurred spontaneously and was less marked in the spiny mouse, where epithelial integrity remained intact. In all species, the decidua was positive for IL-11 secretion and neutrophil recruitment was similar in each. Spiral arteriole formation was confirmed in the spiny mouse. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study comparing primarily morphological traits between the spiny mouse, the mouse and the human. Reagents specific to the spiny mouse were limited and resources for global use of this novel species are few. WIDER IMPLICATIONS OF THE FINDINGS: Our work supports the spiny mouse as a viable model, sharing many attributes of physiological menstruation with humans. The strength of a natural as opposed to an artificial model is validated through the striking similarities observed between the spiny mouse and human in uterine breakdown. The spiny mouse may be highly useful in large-scale investigations of menstruation and menstrual disorders. STUDY FUNDING/COMPETING INTEREST(S): N.B. and S.R. are each recipients of a Research Training Program scholarship supported by Monash University. This work was supported by the Victorian Government Operational Infrastructure and laboratory funds to H.D. The authors declare no competing interests.

A missing piece: the spiny mouse and the puzzle of menstruating species.

We recently discovered the first known menstruating rodent. With the exception of four bats and the elephant shrew, the common spiny mouse (Acomys cahirinus) is the only species outside the primate order to exhibit menses. There are few widely accepted theories on why menstruation developed as the preferred reproductive strategy of these select mammals, all of which reference the evolution of spontaneous decidualisation prior to menstrual shedding. Though menstruating species share several reproductive traits, there has been no identifiable feature unique to menstruating species. Such a feature might suggest why spontaneous decidualisation, and thus menstruation, evolved in these species. We propose that a ≥3-fold increase in progesterone during the luteal phase of the reproductive cycle is a unique characteristic linking menstruating species. We discuss spontaneous decidualisation as a consequence of high progesterone, and the potential role of prolactin in screening for defective embryos in these species to aid in minimising implantation of abnormal embryos. We further explore the possible impact of nutrition in selecting species to undergo spontaneous decidualisation and subsequent menstruation. We summarise the current knowledge of menstruation, discuss current pre-clinical models of menstruation and how the spiny mouse may benefit advancing our understanding of this rare biological phenomenon.

Does human endometrial LGR5 gene expression suggest the existence of another hormonally regulated epithelial stem cell niche?

STUDY QUESTION: Is human endometrial leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) gene expression limited to the postulated epithelial stem cell niche, stratum basalis glands, and is it hormonally regulated? SUMMARY ANSWER: LGR5 expressing cells are not limited to the postulated stem cell niche but LGR5 expression is hormonally regulated. WHAT IS KNOWN ALREADY: The human endometrium is a highly regenerative tissue; however, endometrial epithelial stem cell markers are yet to be confirmed. LGR5 is a marker of stem cells in various epithelia. STUDY DESIGN, SIZE, DURATION: The study was conducted at a University Research Institute. Endometrial samples from 50 healthy women undergoing benign gynaecological surgery with no endometrial pathology at the Liverpool Women's hospital were included and analysed in the following six sub-categories; proliferative, secretory phases of menstrual cycle, postmenopausal, those using oral and local progestagens and samples for in vitro explant culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we used the gold standard method, in situ hybridisation (ISH) along with qPCR and a systems biology approach to study the location of LGR5 gene expression in full thickness human endometrium and Fallopian tubes. The progesterone regulation of endometrial LGR5 was examined in vivo and in short-term cultured endometrial tissue explants in vitro. LGR5 expression was correlated with epithelial proliferation (Ki67), and expression of previously reported epithelia progenitor markers (SOX9 and SSEA-1) immunohistochemistry (IHC). MAIN RESULTS AND THE ROLE OF CHANCE: LGR5 gene expression was significantly higher in the endometrial luminal epithelium than in all other epithelial compartments in the healthy human endometrium, including the endometrial stratum basalis (P < 0.05). The strongest SSEA-1 and SOX9 staining was observed in the stratum basalis glands, but the general trend of SOX9 and SSEA-1 expression followed the same cyclical pattern of expression as LGR5. Stratum functionalis epithelial Ki67-LI and LGR5 expression levels correlated significantly (r = 0.74, P = 0.01), however, they did not correlate in luminal and stratum basalis epithelium (r = 0.5 and 0.13, respectively). Endometrial LGR5 demonstrates a dynamic spatiotemporal expression pattern, suggesting hormonal regulation. Oral and local progestogens significantly reduced endometrial LGR5 mRNA levels compared with women not on hormonal treatment (P < 0.01). Our data were in agreement with in silico analysis of published endometrial microarrays. LARGE SCALE DATA: We did not generate our own large scale data but interrogated publically available large scale data sets. LIMITATIONS, REASONS FOR CAUTION: In the absence of reliable antibodies for human LGR5 protein and validated lineage markers for the various epithelial populations that potentially exist within the endometrium, our study does not formally characterise or examine the functional ability of the resident LGR5+ cells as multipotent. WIDER IMPLICATIONS OF THE FINDINGS: These data will facilitate future lineage tracing studies in the human endometrial epithelium; to identify the location of stem cells and further complement the in vitro functional studies, to confirm if the LGR5 expressing epithelial cells indeed represent the epithelial stem cell population. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the Wellbeing of Women project grant (RTF510) and Cancer Research UK (A14895). None of the authors have any conflicts of interest to disclose.

Hypoxia and hypoxia inducible factor-1α are required for normal endometrial repair during menstruation.

Heavy menstrual bleeding (HMB) is common and debilitating, and often requires surgery due to hormonal side effects from medical therapies. Here we show that transient, physiological hypoxia occurs in the menstrual endometrium to stabilise hypoxia inducible factor 1 (HIF-1) and drive repair of the denuded surface. We report that women with HMB have decreased endometrial HIF-1α during menstruation and prolonged menstrual bleeding. In a mouse model of simulated menses, physiological endometrial hypoxia occurs during bleeding. Maintenance of mice under hyperoxia during menses decreases HIF-1α induction and delays endometrial repair. The same effects are observed upon genetic or pharmacological reduction of endometrial HIF-1α. Conversely, artificial induction of hypoxia by pharmacological stabilisation of HIF-1α rescues the delayed endometrial repair in hypoxia-deficient mice. These data reveal a role for HIF-1 in the endometrium and suggest its pharmacological stabilisation during menses offers an effective, non-hormonal treatment for women with HMB.

Expression of vascular endothelial growth factor A isoforms is dysregulated in women with endometriosis.

Angiogenesis is a critical step in the development of ectopic lesions during endometriosis. Although total vascular endothelial growth factor (VEGF) A is elevated in the peritoneal fluid of women with endometriosis, there are contradictory reports on how levels of total endometrial VEGFA are altered in this disease. Furthermore, limited research is available on different VEGFA isoforms in women with endometriosis. Thus, the aim of the present study was to analyse levels of various VEGFA isoforms in women with and without endometriosis at different stages of the menstrual cycle. Quantitative polymerase chain reaction analysis showed that total VEGFA was highest during menstruation in endometriosis compared with controls (P=0.0373). VEGF121 and VEGF189 were similarly highest during menstruation in endometriosis compared with controls (P=0.0165 and 0.0154 respectively). The present study is also the first to identify the natural expression of VEGF111 in human tissue, which is also highest during menstruation in endometriosis (P=0.0464). This discovery of the natural production of VEGF111 in human endometrium, as well as the upregulation of VEGFA isoforms during menstruation in endometriosis, may shed further light on the development and progression of the disease, and improve our understanding of the regulation of endometrial angiogenesis.

Steroids Regulate CXCL4 in the Human Endometrium During Menstruation to Enable Efficient Endometrial Repair.

Context: Repair of the endometrial surface at menstruation must be efficient to minimize blood loss and optimize reproductive function. The mechanism and regulation of endometrial repair remain undefined. Objective: To determine the presence/regulation of CXCL4 in the human endometrium as a putative repair factor at menses. Patients/Setting: Endometrial tissue was collected throughout the menstrual cycle from healthy women attending the gynecology department. Menstrual blood loss was objectively measured in a subset, and heavy menstrual bleeding (HMB) was defined as >80 mL per cycle. Monocytes were isolated from peripheral blood. Design: CXCL4 messenger RNA (mRNA) and protein were identified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The function/regulation of endometrial CXCL4 was explored by in vitro cell culture. Results: CXCL4 mRNA concentrations were significantly increased during menstruation. Intense staining for CXCL4 was detected in late secretory and menstrual tissue, localized to stromal, epithelial and endothelial cells. Colocalization identified positive staining in CD68+ macrophages. Treatment of human endometrial stromal and endothelial cells (hESCs and HEECs, respectively) with steroids revealed differential regulation of CXCL4. Progesterone withdrawal resulted in significant increases in CXCL4 mRNA and protein in hESCs, whereas cortisol significantly increased CXCL4 in HEECs. In women with HMB, CXCL4 was reduced in endothelial cells during the menstrual phase compared with women with normal menstrual bleeding. Cortisol-exposed macrophages displayed increased chemotaxis toward CXCL4 compared with macrophages incubated with estrogen or progesterone. Conclusions: These data implicate CXCL4 in endometrial repair after menses. Reduced cortisol at the time of menses may contribute to delayed endometrial repair and HMB, in part by mechanisms involving aberrant expression of CXCL4.

Reduced Transforming Growth Factor-ß Activity in the Endometrium of Women With Heavy Menstrual Bleeding.

Context: Heavy menstrual bleeding (HMB) is common and incapacitating. Aberrant menstrual endometrial repair may result in HMB. The transforming growth factor (TGF)-ß superfamily contributes to tissue repair, but its role in HMB is unknown. Objective: We hypothesized that TGF-ß1 is important for endometrial repair, and women with HMB have aberrant TGF-ß1 activity at menses. Participants/Setting: Endometrial biopsies were collected from women, and menstrual blood loss objectively measured [HMB >80 mL/cycle; normal menstrual bleeding (NMB) <80 mL]. Design: Immunohistochemistry and reverse transcription polymerase chain reaction examined endometrial TGF-ß1 ligand, receptors, and downstream SMADs in women with NMB and HMB. The function and regulation of TGF-ß1 were examined using cell culture. Results: TGFB1 mRNA was maximal immediately prior to menses, but no differences detected between women with NMB and HMB at any cycle stage. Histoscoring of TGFB1 revealed reduced staining in the stroma during menses in women with HMB (P < 0.05). There were no significant differences in TGFBR1/2 or TGFBR1/2 immunostaining. Cortisol increased activation of TGFB1 in the supernatant of human endometrial stromal cells (HES; P < 0.05) via thrombospondin-1. Endometrial SMAD2 and SMAD3 were lower in women with HMB during menstruation (P < 0.05), and decreased phosphorylated SMAD2/3 immunostaining was seen in glandular epithelial cells during the late secretory phase (P < 0.05). Wound scratch assays revealed increased repair in HES cells treated with TGF-ß1 versus control (P < 0.05). Conclusions: Women with HMB had decreased TGF-ß1 and SMADs perimenstrually. Cortisol activated latent TGF-ß1 to enhance endometrial stromal cell repair. Decreased TGF-ß1 activity may hinder repair of the denuded menstrual endometrium, resulting in HMB.

The Presence of Endometrial Cells in Peritoneal Fluid of Women With and Without Endometriosis.

To reinforce Sampson's theory of retrograde menstruation in the pathogenesis of endometriosis, proof should be provided that during menstruation endometrial cells are present in peritoneal fluid (PF). We hypothesize that the prevalence of PF samples containing endometrial cells is higher in patients with endometriosis than in controls without endometriosis during menstruation. We selected from our biobank PF samples of 17 reproductive-age women with (n = 9) or without (n = 8) endometriosis who had received a diagnostic laparoscopy for investigation of pain/infertility. Peritoneal fluid had been collected during laparoscopy in the menstrual phase of the cycle, centrifuged, and the resulting pellet was stored at -80°C. About 5-µm sections of frozen PF pellets were stained using the Dako Envision Flex system with primary antibodies against epithelial cell adhesion molecule (Ep-CAM; endometrial epithelial cells), CD10 (endometrial stromal cells), prekeratin (epithelial/mesothelial cells), vimentin (endometrial/mesothelial/immune cells), calretinin (mesothelial cells), and CD68 (macrophages). The PF cells positive for Ep-CAM were detected in 5 of 9 patients with endometriosis and 6 of 8 controls ( P = .62). CD10 stained positively in 6 of the 9 patients with endometriosis and 3 of the 8 controls ( P = .35). Calretinin and prekeratin staining showed the presence of mesothelial cells in all pellets. Vimentin stained approximately 100% of the PF cells. CD68+ macrophages represented >50% of cells in all pellets. The prevalence of PF samples containing endometrial epithelial and stromal cells was not higher in patients with endometriosis than in controls without endometriosis during menstruation. Our findings question the relevance of endometrial cells in PF for the pathogenesis of endometriosis and support the importance of other mechanisms such as immune dysfunction and/or endometrial stem cells.