Bone Marrow Mesenchymal Stem Cells (BMSCs) Enhance Endometrial Stromal Cell Migration and Epithelial-Mesenchymal Transition in Adenomyosis Through Upregulation of Neuropilin 1

Hormone support (estrogen and progesterone) is a key factor in decidualization and embryo implantation. Elevated levels of estrogen lead to luteal phase defects through Neuropilin 1, a membranecytoskeleton junction protein. This study aimed to explore the effect of BMSCs on endometrial stromal cells (ESCs) in adenomyosis. ESCs obtained from patients with adenomyosis were cocultured with BMSCs in the absence of presence of Neuropilin 1 inhibitor followed by analysis of expression of decidualization-related genes by RT-qPCR and western blot, cell viability by MTT assay, cell invasion and migration by Transwell assay, oxidative stress factors by ROS kit. Treatment with Neuropilin 1 inhibitor significantly decreased ESC proliferation and invasion, blocked epithelialmesenchymal transition (EMT) process, and restrained decidualization with a downregulation of decidualization-related genes. Furthermore, inhibition of Neuropilin 1 exerted effects through estrogen regulation. However, co-culture with BMSCs restored ESC activity by promoting Neuropilin expression and enhanced intrauterine ESC decidualization. In conclusion, Neuropilin 1 inhibitor restrains decidualization through estrogen regulation which can be abrogated by estrogen receptor antagonists. BMSCs restore the damaged ESC decidualization through increasing Neuropilin 1 expression, which provides new insights into the adverse effect of Neuropilin 1 on human ESCs, suggesting that BMSC is a potential therapeutic drug candidate for adenomyosis.

Shp2 in uterine stromal cells critically regulates on time embryo implantation and stromal decidualization by multiple pathways during early pregnancy

Approximately 75% of failed pregnancies are considered to be due to embryo implantation failure or defects. Nevertheless, the explicit signaling mechanisms governing this process have not yet been elucidated. Here, we found that conditional deletion of the Shp2 gene in mouse uterine stromal cells deferred embryo implantation and inhibited the decidualization of stromal cells, which led to embryonic developmental delay and to the death of numerous embryos mid-gestation, ultimately reducing female fertility. The absence of Shp2 in stromal cells increased the proliferation of endometrial epithelial cells, thereby disturbing endometrial epithelial remodeling. However, Shp2 deletion impaired the proliferation and polyploidization of stromal cells, which are distinct characteristics of decidualization. In human endometrial stromal cells (hESCs), Shp2 expression gradually increased during the decidualization process. Knockout of Shp2 blocked the decidual differentiation of hESCs, while Shp2 overexpression had the opposite effect. Shp2 knockout inhibited the proliferation of hESCs during decidualization. Whole gene expression profiling analysis of hESCs during the decidualization process showed that Shp2 deficiency disrupted many signaling transduction pathways and gene expression. Analyses of hESCs and mouse uterine tissues confirmed that the signaling pathways extracellular regulated protein kinases (ERK), protein kinase B (AKT), signal transducer and activator of transcription 3 (STAT3) and their downstream transcription factors CCAAT/enhancer binding protein β (C/EBPβ) and Forkhead box transcription factor O1 (FOXO-1) were involved in the Shp2 regulation of decidualization. In summary, these results demonstrate that Shp2 plays a crucial role in stromal decidualization by mediating and coordinating multiple signaling pathways in uterine stromal cells. Our discovery possibly provides a novel key regulator of embryo implantation and novel therapeutic target for pregnancy failure.

SCM-198 Prevents Endometriosis By Reversing Low Autophagy Levels Of Endometriotic Stromal Cells Via Inhibiting TNF-Α-Aromatase-Estrogen-Erα Pathway And Promoting PR Expression

Background: Endometriosis (EMS), an estrogen-dependent disease, is characterized by dysregulated inflammation and increased estrogen in ectopic lesions. However, the crosstalk and pathogenic mechanism of inflammation and estrogen has not been fully explored. SCM-198 is the synthetic form of leonurine with multiple pharmacological activities. Whether SCM-198 could inhibit the progress of EMS by regulating inflammation and estrogen signaling remains unknown. Methods: The therapeutic effects and potential mechanisms of SCM-198 on EMS were analyzed by establishing EMS mice models and performing RNA-seq assay. ELISA was performed to detect estrogen and TNF-α concentration in normal endometrial stromal cells (nESCs) and ectopic endometrial stromal cells (eESCs), with or without SCM-198 treatment. Western blotting, RNA silencing and plasmid overexpression were utilized to analyze the relationship among inflammation, endocrine and autophagy as well as the regulation of SCM-198 on inflammation-endocrine-autophagy axis. Results: Increased estrogen-ERα signaling and decreased PR expression co-led to the hypo-autophagy state in eESCs, which further inhibited the apoptosis of eESCs. Highly expressed TNF-α in eESCs enhanced low-autophagy mediated anti-apoptosis effect by activating aromatase-estrogen-ERα signaling. SCM-198 inhibited the growth of ectopic lesions in EMS mouse model and promoted the apoptosis of eESCs both in vivo and in vitro. The apoptosis effect of SCM-198 on eESCs were realized by upregulating the autophagy level via inhibiting TNF-α activated aromatase-estrogen-ERα signaling and increasing PR expression. Conclusion: Inflammation facilitated the progress of EMS by disturbing estrogen regulatory axis. SCM-198 restrained the growth of EMS by regulating inflammation-endocrine-autophagy axis.

Lack of evidence for functional luteinising hormone chorionic gonadotropin receptors in non-pregnant human endometrial stromal cells

The human luteinising hormone chorionic gonadotropin receptor (LHCGR) is a G-protein coupled receptor activated by both human chorionic gonadotropin (hCG) and luteinizing hormone (LH), two structurally related gonadotropins with essential roles in ovulation and maintenance of the corpus luteum. LHCGR expression predominates in ovarian tissues where it elicits functional responses through cyclic adenosine mononucleotide (cAMP), Ca2+ and extracellular signal-regulated kinase (ERK) signalling. LHCGR has also been localized to the human endometrium, with purported roles in decidualization and implantation. However, these observations are contentious. In this investigation, transcripts encoding LHCGR were undetectable in bulk RNA sequencing datasets from whole cycling endometrial tissue and cultured human endometrial stromal cells (EnSC). However, analysis of single-cell RNA sequencing data revealed cell-to-cell transcriptional heterogeneity and identified a small subpopulation of stromal cells with discernible LHCGR transcripts. In HEK-293 cells expressing recombinant LHCGR, both hCG and LH elicited robust cAMP, Ca2+ and ERK signals that were absent in wild type HEK-293 cells. However, none of these responses were recapitulated in primary EnSC cultures. In addition, proliferation, viability and decidual transformation of EnSC were refractory to both hCG and LH, irrespective of treatment to induce differentiation. Although we challenge the assertion that LHCGR is expressed at a functionally active level in the human endometrium, the discovery of a discrete subpopulation of EnSC that express LHCGR transcripts may plausibly account for the conflicting evidence in the literature.

Kefir as a Prevention of Arsenic-mediated Toxicity in Uterine Female Rats

Background: kefir is a fermented milk product that demonstrates numerous health benefits including antioxidant and immunomodulatory. Aim: to study the protective effect kefir on the expression of estrogen receptor alpha (ERα) in endometrial stromal cells and endometrial thickness on female rats that were exposed to arsenic. Methods: twenty-five female Wistar rats (Rattus norvegicus) were divided into five groups (CRL, As, T1, T2, T3). Control group (given a normal diet), As group (given the normal diet and exposed to arsenic trioxide 2 mg/kgBW/day). The T1; T2; T3 were exposed to arsenic trioxide 2 mg/kgBW/day and treated with different doses of kefir (1.25; 2.5; and 5 mL/kgBW/day, respectively) for 35 days. The rats of group As treated with arsenic trioxide only and group CRL served as control with normal feed in water. Cytological samples were taken after 35 days of treatment and examined every day to see the rat oestrus phase, and the proestrus phase of the oestrous cycle was chosen for termination. Uterine tissue fixed in 10% neutral buffered formalin for tissue preparation. ERα expression in endometrial stromal cells was analized using immunohistochemistry method, endometrial thickness was observed using histopathological methods. Results: significant reduction of ERα expression in endometrial stromal cells and endometrial thickness in female rats exposed to arsenic were observed in groups on treated rats (p ≤ 0.000; 0.009, respectively). Conclusion: the administration of kefir in female Wistar rats exposed to arsenic had shown significantly differences on ERα expressions and endometrial thickness. The smallest dose of kefir (1.25 mL/kgBW/day) could increase ERα expression and endometrial thickness in female Wistar rats with arsenic exposure. Therefore kefir has protective effect related to female reproductive system.

Regulation and Function of Laminin A5 during Mouse and Human Decidualization

Decidualization is essential to the establishment of pregnancy in rodents and primates. Laminin A5 (encoding by Laminin α5) is a member of the laminin family, which is mainly expressed in the basement membranes. Although laminins regulate cellular phenotype maintenance, adhesion, migration, growth, and differentiation, the expression, function, and regulation of laminin A5 during early pregnancy are still unknown. Therefore, we investigated the expression and role of laminin A5 during mouse and human decidualization. Laminin A5 is highly expressed in mouse decidua and artificially induced deciduoma. Laminin A5 is significantly increased under in vitro decidualization. Laminin A5 knockdown significantly inhibits the expression of Prl8a2, a marker for mouse decidualization. Progesterone stimulates the expression of laminin A5 in ovariectomized mouse uterus and cultured mouse stromal cells. We also show that progesterone regulates laminin A5 through the PKA-CREB-C/EBPβ pathway. Laminin A5 is also highly expressed in human pregnant decidua and cultured human endometrial stromal cells during in vitro decidualization. Laminin A5 knockdown by siRNA inhibits human in vitro decidualization. Collectively, our study reveals that laminin A5 may play a pivotal role during mouse and human decidualization via the PKA-CREB-C/EBPβ pathway.

The Role of Decidual Subpopulations in Implantation, Menstruation and Miscarriage

In each menstrual cycle, the endometrium becomes receptive to embryo implantation while preparing for tissue breakdown and repair. Both pregnancy and menstruation are dependent on spontaneous decidualization of endometrial stromal cells, a progesterone-dependent process that follows rapid, oestrogen-dependent proliferation. During the implantation window, stromal cells mount an acute stress response, which leads to the emergence of functionally distinct decidual subsets, reflecting the level of replication stress incurred during the preceding proliferative phase. Progesterone-dependent, anti-inflammatory decidual cells (DeC) form a robust matrix that accommodates the conceptus whereas pro-inflammatory, progesterone-resistant stressed and senescent decidual cells (senDeC) control tissue remodelling and breakdown. To execute these functions, each decidual subset engages innate immune cells: DeC partner with uterine natural killer (uNK) cells to eliminate senDeC, while senDeC co-opt neutrophils and macrophages to assist with tissue breakdown and repair. Thus, successful transformation of cycling endometrium into the decidua of pregnancy not only requires continuous progesterone signalling but dominance of DeC over senDeC, aided by recruitment and differentiation of circulating NK cells and bone marrow-derived decidual progenitors. We discuss how the frequency of cycles resulting in imbalanced decidual subpopulations may determine the recurrence risk of miscarriage and highlight emerging therapeutic strategies.

RNA-driven JAZF1-SUZ12 gene fusion in human endometrial stromal cells

Oncogenic fusion genes as the result of chromosomal rearrangements are important for understanding genome instability in cancer cells and developing useful cancer therapies. To date, the mechanisms that create such oncogenic fusion genes are poorly understood. Previously we reported an unappreciated RNA-driven mechanism in human prostate cells in which the expression of chimeric RNA induces specified gene fusions in a sequence-dependent manner. One fundamental question yet to be addressed is whether such RNA-driven gene fusion mechanism is generalizable, or rather, a special case restricted to prostate cells. In this report, we demonstrated that the expression of designed chimeric RNAs in human endometrial stromal cells leads to the formation of JAZF1-SUZ12, a cancer fusion gene commonly found in low-grade endometrial stromal sarcomas. The process is specified by the sequence of chimeric RNA involved and inhibited by estrogen or progesterone. Furthermore, it is the antisense rather than sense chimeric RNAs that effectively drive JAZF1-SUZ12 gene fusion. The induced fusion gene is validated both at the RNA and the genomic DNA level. The ability of designed chimeric RNAs to drive and recapitulate the formation of JAZF1-SUZ12 gene fusion in endometrial cells represents another independent case of RNA-driven gene fusion, suggesting that RNA-driven genomic recombination is a permissible mechanism in mammalian cells. The results could have fundamental implications in the role of RNA in genome stability, and provide important insight in early disease mechanisms related to the formation of cancer fusion genes.

Kefir as a Prevention of Arsenic-mediated Toxicity in Uterine Female Rats

Background: kefir is a fermented milk product that demonstrates numerous health benefits including antioxidant and immunomodulatory. Aim: to study the protective effect kefir on the expression of estrogen receptor alpha (ERα) in endometrial stromal cells and endometrial thickness on female rats that were exposed to arsenic. Methods: twenty-five female Wistar rats (Rattus norvegicus) were divided into five groups (CRL, As, T1, T2, T3). Control group (given a normal diet), As group (given the normal diet and exposed to arsenic trioxide 2 mg/kgBW/day). The T1; T2; T3 were exposed to arsenic trioxide 2 mg/kgBW/day and treated with different doses of kefir (1.25; 2.5; and 5 mL/kgBW/day, respectively) for 35 days. The rats of group As treated with arsenic trioxide only and group CRL served as control with normal feed in water. Cytological samples were taken after 35 days of treatment and examined every day to see the rat oestrus phase, and the proestrus phase of the oestrous cycle was chosen for termination. Uterine tissue fixed in 10% neutral buffered formalin for tissue preparation. ERα expression in endometrial stromal cells was analized using immunohistochemistry method, endometrial thickness was observed using histopathological methods. Results: significant reduction of ERα expression in endometrial stromal cells and endometrial thickness in female rats exposed to arsenic were observed in groups on treated rats (p ≤ 0.000; 0.009, respectively). Conclusion: the administration of kefir in female Wistar rats exposed to arsenic had shown significantly differences on ERα expressions and endometrial thickness. The smallest dose of kefir (1.25 mL/kgBW/day) could increase ERα expression and endometrial thickness in female Wistar rats with arsenic exposure. Therefore kefir has protective effect related to female reproductive system.