Single-cell transcriptome analysis uncovers the molecular and cellular characteristics of thin endometrium.

Infertility is a social and medical problem around the world and the incidence continues to rise. Thin endometrium (TE) is a great challenge of infertility treatment, even by in vitro fertilization and embryo transfer. It is widely believed that TE impairs endometrium receptivity. However, only a few studies have explained the molecular mechanism. Herein, in order to reveal the possible mechanism, we sampled endometrium from a TE patient and a control volunteer and got a transcriptomic atlas of 18 775 individual cells which was constructed using single-cell RNA sequencing, and seven cell types have been identified. The cells were acquired during proliferative and secretory phases, respectively. The proportion of epithelial cells and stromal cells showed a significant difference between the TE group and the control group. In addition, differential expressed genes (DEGs) in diverse cell types were revealed, the enriched pathways of DEGs were found closely related to the protein synthesis in TE of both proliferative and secretory phases. Some DEGs can influence cell-type ratio and impaired endometrial receptivity in TE. Furthermore, divergent expression of estrogen receptors 1 and progesterone receptors in stromal and epithelial cells were compared in the TE sample from the control. The cellular and molecular heterogeneity found in this study provided valuable information for disclosing the mechanisms of impaired receptivity in TE.

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.

Human menstrual blood-derived stem cells promote the repair of impaired endometrial stromal cells by activating the p38 MAPK and AKT signaling pathways.

Multiple studies have confirmed that human menstrual blood-derived stem cells (MenSCs) have potential applications in regenerative medicine or cell therapy. However, the contribution of MenSCs to endometrial repair is currently unknown. We evaluated the protective effects of MenSCs on impaired endometrial stromal cells (ESCs), as well as the signaling pathways involved in this process. Mifepristone was used to damage human ESCs, which were subsequently cocultured with MenSCs. The proliferation, apoptosis, and migration of ESCs were assessed, together with the expression of related signaling proteins including total p38 mitogen-activated protein kinase, P-p38, total protein kinase B (AKT), P-AKT, ß-catenin, and vascular endothelial growth factor (VEGF). MenSCs significantly recovered the proliferation and migration ability of impaired ESCs, inhibited ESC apoptosis, and upregulated protein expression of P-AKT, P-p38, VEGF, and ß-catenin. Our findings suggest that MenSC-based therapies could be promising strategies for the treatment of endometrial injury, and that AKT and p38 signaling pathways may be involved in this process.

Endometrial stem cells repair injured endometrium and induce angiogenesis via AKT and ERK pathways.

Intrauterine adhesions are common acquired endometrial syndromes secondary to endometrial injury, with limited effective therapies. Recently, several studies have reported that bone marrow stem cells (BMSCs) could repair injured endometrium in animal experiments. However, the role of stem cells in endometrial injury repair and its therapeutic mechanisms remain unclear. Here, we established mouse endometrial injury model and examined the benefit of human endometrial mesenchymal stem cells derived from menstrual blood (MenSCs) in restoration of injured endometrium. Injured endometrium exhibited significantly accelerated restoration at Day 7 after MenSCs transplantation, with increased endometrial thickness and microvessel density. Moreover, the fertility of mice with injured endometrium was improved, with higher conception rate (53.57% vs 14.29%, P = 0.014) and larger embryo number (3.1 ± 0.6 vs 0.9 ± 0.7, P = 0.030) in MenSCs group than control group, while no difference was found in undamaged horns between two groups. Conditioned medium from MenSCs (MenSCs-CM) could decrease H2O2-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and promote proliferation, migration and angiogenesis. Angiogenesis effect of MenSCs-CM was also confirmed in Matrigel plug assay in mice. Furthermore, we discovered that MenSCs-CM could activate AKT and ERK pathways and induce the overexpression of eNOS, VEGFA, VEGFR1, VEGFR2 and TIE2 in HUVECs, which are critical in MenSCs-CM-induced angiogenesis. Angiogenesis induced by MenSCs-CM could be reversed by inhibitors of AKT and/or ERK. Taken together, we concluded that MenSCs could restore injured endometrium and improve the fertility of the endometrial injury mice, which was partially attributed to angiogenesis induced by MenSCs.

Endometriotic mesenchymal stem cells significantly promote fibrogenesis in ovarian endometrioma through the Wnt/ß-catenin pathway by paracrine production of TGF-ß1 and Wnt1.

STUDY QUESTION: Are endometriotic mesenchymal stem cells (Ecto-MSCs) involved in the fibrosis of ovarian endometrioma? SUMMARY ANSWER: Ecto-MSCs enhanced the fibrotic behavior of stromal cells in ovarian endometrioma through the Wnt/ß-catenin pathway by paracrine production of transforming growth factor-ß1 (TGF-ß1) and Wnt1. WHAT IS KNOWN ALREADY: Endometriosis is characterized by ectopic outgrowth of endometrial stroma and glands surrounded by dense fibrous tissues. The pathogenesis of endometriosis, especially ovarian endometrioma-associated fibrosis, is still unknown. STUDY DESIGN, SIZE, DURATION: We analyzed endometrial samples from 15 patients of reproductive age with ovarian endometrioma and normal menstrual cycles. A total of 54 nude mice received a single injection of proliferative endometrial fragments from 14 individuals without endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Conditioned medium (CM) was collected from endometrial mesenchymal stem cells (Euto-MSCs) and Ecto-MSCs. The effects of CM on cell proliferation, migration, invasion and collagen gel contraction of endometrial and endometriotic stromal cells (Euto- and Ecto-ESCs) in ovarian endometrioma were evaluated by cell counting kit-8, transwell and collagen gel contraction assays. Effects of CM on fibrotic markers' expression [including α-smooth muscle actin, Type I collagen, connective tissue growth factor and fibronectin (FN)] in Euto- and Ecto-ESCs were determined by real-time reverse-transcription-polymerase chain reaction and western blotting. Additionally, fibrogenic effects of Ecto-MSC CM treatment on endometriotic implants were analyzed using a xenograft model of endometriosis in immunodeficient nude mice. MAIN RESULTS AND THE ROLE OF CHANCE: Our results demonstrated that Ecto-MSC CM significantly promoted cell proliferation, migration, invasion and collagen gel contraction of Euto- and Ecto-ESCs from patients with ovarian endometrioma compared with control and Euto-MSC CM. Expression levels of fibrotic markers in Euto- and Ecto-ESCs were dramatically elevated after treatment with Ecto-MSC CM. Ecto-MSCs secreted higher levels of TGF-ß1 and Wnt1 compared with Euto-MSCs. Furthermore, both TGF-ß1 and Wnt1 significantly increased expression of fibrotic markers in Euto- and Ecto-ESCs, which was reversed by an anti-TGF-ß1 antibody or Wnt1 negative regulator, Dickkopf-related protein 1 (Dkk1). Mechanistic studies demonstrated that Wnt/ß-catenin signaling pathways in stromal cells were activated by Ecto-MSC CM. Animal experiments showed that TGF-ß1 and Wnt1 as well as Ecto-MSC CM markedly increased the expression of FN and collagen I, which enhanced the progression of fibrosis in endometriosis. LIMITATIONS, REASONS FOR CAUTION: To our knowledge, this is the first study to identify the role of Ecto-MSCs in the pathogenesis of fibrosis in ovarian endometrioma. However, numerous other growth factors and cell types may also be involved in the pathogenesis. Therefore, further studies are required to elucidate the paracrine effects of cells in ovarian endometrioma. WIDER IMPLICATIONS OF THE FINDINGS: Ecto-MSCs may be involved in the pathogenesis of fibrosis in ovarian endometrioma. STUDY FUNDING/COMPETING INTERESTS: This study was supported in part by the National Natural Science Foundation of China (81471505 and 81270657). No competing interests are declared.

[Establishment of mouse endometrial injury model by electrocoagulation].

OBJECTIVE: To establish the murine model of moderate endometrial injury. METHODS: Electrocoagulation was applied to induce endometrial injury of ICR mice with 0.5 watts power while contralateral uterine cavity acted as control without electrocoagulation. The endometrial histomorphology was observed in 7 days later by microscopy and fetal number of each lateral uterus assessed at 17.5 days after pregnancy. RESULTS: At 7 days post-electrocoagulation, the average endometrial thickness of operating side was significantly thinner than that of control side (1.14 ± 0.08 vs 1.88 ± 0.15 mm, P < 0.05). The density of endometrial glands of operating side was significantly lower than that of control side (20 ± 2 vs 32 ± 3 per 100x field, P < 0.05). After pregnancy, the average number of embryos at operating side decreased by 63.1% compared with control (3 ± 2 vs 8 ± 2, P < 0.01). CONCLUSION: The established model of endometrial electrocoagulation injury shows morphologic changes and decreased fertile ability. It has potential uses for animal studies of endometrial injury treatment.