A method for isolating and culturing ectopic epithelial and stromal cells to study human adenomyosis.

PURPOSE: Although adenomyosis is a common and benign gynecological disease, the specific pathogenesis of this condition is yet to be fully elucidated. It is difficult to culture primary cells of the ectopic endometrial epithelia and stroma from human adenomyosis lesions. Most of the previous of studies on adenomyosis were based on primary eutopic endometrium cells. However, as yet, no efficient protocols have been developed for the isolation, culture or purification of primary ectopic epithelial and stromal cells from human adenomyosis lesions. Therefore, the present study aimed to develop an efficient protocol for the isolation and culture of primary ectopic epithelial and stromal cells from human adenomyosis lesions. METHODS: In the present study, we aimed to obtain ectopic endometrium tissue from human adenomyosis foci and use a simple and operable type I collagenase digestion method for primary culture. Cells were isolated by sterile cell strainer filtration and flow cytometry was performed to identify, purify, and evaluate the viability of isolated ectopic endometrial cells. RESULTS: Using our method, we successfully isolated and cultured highly purified and active ectopic endometrial epithelial and stromal cells from human adenomyosis foci. Ep-CAM was expressed in ectopic epithelial cells of human adenomyosis with a purity of 93.74% and a viability of 80.58%. In addition, CD10 were robustly expressed by ectopic stromal cells in human adenomyosis. Cellular purity and viability were determined to be 96.37 and 93.49%, respectively. CONCLUSION: Our method provides a new experimental model for studying the molecular pathogenesis of human adenomyosis.

The role of circRNA polyribonucleotide nucleoside transferase 1 on Gestational Diabetes Mellitus.

This study aimed to focus on the mechanism of circRNA polyribonucleotide nucleoside transferase 1 (circ-PNPT1)-mediated miR-889-3p/PAK1 on gestational diabetes mellitus (GDM). Placental tissues from normal pregnancy and GDM patients were collected to detect the levels of circ-PNPT1, miR-889-3p, and PAK1. The high glucose-induced human trophoblast cells HTR-8/SVneo were adopted to stimulate the GDM model in vitro (HG group) and were transfected with lentivirus to silence circ-PNPT1 (si-circ-PNPT1 group) and mimic to overexpress miR-889-3p (miR-889-3p group). Cell proliferation, apoptosis, migration, and invasion were detected by CKK-8, flow cytometry, Transwell, and scratch assay, respectively. The results showed that the expressions of circ-PNPT1 and PAK1 in the GDM patients were up-regulated, and miR-889-3p was down-regulated (P< 0.05). Compared with cells in the control group, the circ-PNPT1 and PAK1 in the HG group were up-regulated, and miR-889-3p was down-regulated (P< 0.05). The cell proliferation, migration, and invasion abilities were weakened, and the apoptosis rate increased (P< 0.05). E-cadherin protein was elevated, and the N-cadherin and Vimentin decreased (P< 0.05). Compared with the HG group, the expressions of circ-PNPT1 and PAK1 in the other two groups decreased, and miR-889-3p increased (P< 0.05). The cell proliferation, migration, and invasion were enhanced, and the apoptosis rate decreased (P< 0.05). E-cadherin, N-cadherin, and Vimentin decreased (P< 0.05). There were targeted binding sites for miR-889-3p with circ-PNPT1 and PAK1, indicating circ-PNPT1 promoted HG-induced trophoblast dysfunction through the miR-889-3p/PAK1 axis.

Recepteur d'origine nantais contributes to the development of endometriosis via promoting epithelial-mesenchymal transition of a endometrial epithelial cells.

Endometriosis is a benign, chronic inflammatory disease that commonly occurs in reproductive-aged women. Epithelial-mesenchymal transition (EMT) of endometrial epithelial cells plays an important role in the development of endometriosis. Recepteur d'origine nantais (RON), a receptor tyrosine kinase, has been reported to promote EMT and progression in tumours. However, whether and how RON mediates the EMT and endometriosis development is not known. Here, we found that RON activation could improve the migratory and invasive capabilities, change cellular morphologies, and decrease expression of E-cadherin and increase expression of N-cadherin in endometrial epithelial cells. Inhibition or knockdown of RON expression suppressed the migration and invasion of endometrial epithelial cells. Our studies also indicated that RON played its part in endometrial epithelial cells through protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways. Treatment with a RON inhibitor could decrease the number of ectopic lesions in a mouse model of endometriosis and mediate expression of EMT markers in endometriotic lesions. These data suggest that RON contributed to endometriosis development by promoting EMT of endometrial epithelial cells. Therefore, RON may be a new therapeutic target for endometriosis.

Decreased Indian hedgehog signaling activates autophagy in endometriosis and adenomyosis.

Indian hedgehog (Ihh) signaling regulates endometrial receptivity and is an indispensable mediator of embryonic implantation. Hedgehog signaling is known to regulate autophagy, and aberrant regulation of autophagy is critically implicated in the pathogenesis of endometriosis and adenomyosis. However, potential dysregulation of Ihh signaling and its role in autophagy modulation in these diseases remain obscure. In this study, we found that components of Ihh signaling were significantly decreased, whereas the autophagy marker protein, LC3BII, was significantly increased in endometrial tissues of women with endometriosis or adenomyosis. Inhibition of Ihh signaling with the small-molecule inhibitor GANT61 or Gli1 silencing in primary endometrial stromal cells increased autophagic activity, as measured by LC3 turnover assay and tandem mCherry-eGFP-LC3B fluorescence microscopy. Furthermore, we observed that GANT61 treatment significantly attenuated hydrogen peroxide-induced cell death, whereas disruption of autophagy with chloroquine diminished this effect. Collectively, these findings reveal that Ihh signaling is suppressed in endometrial tissues of patients with endometriosis or adenomyosis. This abnormal decrease may contribute to endometrial autophagy activation, which may promote aberrant survival of endometrial cells in ectopic sites in these two gynecological diseases.

[Clinic research for induced labour in midpregnancy women with central placenta previa].

OBJECTIVE: To investigate the efficiency of uterine arterial embolization in induced labour among pregnant women with placenta previa. METHODS: All pregnant women (20 cases) with placenta previa who received induced labour in Taizhou First People's Hospital between 2009 and 2014 were included in this retrospective study. All cases were given rivanol induction combined with uterine arterial embolization and mifepristone.The antepartum and postpartum history were reviewed. RESULTS: 16 cases out of 20 delivered vaginally with small amount of bleeding. The other 4 cases got emergency cesarean delivery because of uncontrolled vaginal bleeding. No case needed hysterectomy.The average interval between the procedure and menstruation recover was 42 days. CONCLUSIONS: Uterine arterial embolization improves the efficiency of induced labour and shows advantages to postpartum haemorrhage and prevent infertility. Uterine arterial embolization is recommended as a effective management for pregnancies with placenta previa state.

CCL18 promotes endometriosis by increasing endometrial cell migration and neuroangiogenesis.

Endometriosis is an estrogen-dependent inflammatory gynecological disease whose pathogenesis is unclear. C-C motif chemokine ligand 18 (CCL18), a chemokine, is involved in several inflammatory diseases. In this study, we aimed to investigate the role of CCL18 in endometriosis and its underlying mechanisms. Human endometrium and peritoneal fluid were obtained from women with and without endometriosis for molecular studies. The expression level of CCL18 in each tissue sample was examined by RNA sequencing analysis, quantitative PCR analysis and immunohistochemistry staining. The effects of CCL18 on cell migration, tube formation and neurite growth were investigated in vitro using primary endometrial cells, human umbilical vein endothelial cells (HUVECs) and dorsal root ganglion (DRG) neurons, respectively. Moreover, the development of endometriosis in mice was studied in vivo by blocking CCL18. CCL18 was shown to be overexpressed in endometrial foci and peritoneal fluid in women with endometriosis and was positively correlated with endometriosis pain. In vitro, CCL18 promoted the migration of ectopic endometrial cells, tube formation of HUVECs, and nerve outgrowth of DRG neurons. More importantly, inhibition of CCL18 significantly suppressed lesion development, angiogenesis, and nerve infiltration in a mouse model of endometriosis. In conclusion, CCL18 may play a role in the progression of endometriosis by increasing endometrial cell migration and promoting neuroangiogenesis.

Treatment of infected placenta accreta in the uterine horn by transabdominal temporary occlusion of internal iliac arteries: A case report and literature review.

RATIONALE: This case report aims to describe the treatment of infected placenta accreta in the uterine horn by transabdominal temporary occlusion of internal iliac arteries. PATIENT CONCERNS: A 29-year-old female patient had a history of retained placenta for 28 days after labor induction in the second trimester of pregnancy because of fetal malformation. DIAGNOSES: Placenta accreta in the uterine horn was diagnosed by 3-dimensional ultrasound and magnetic resonance imaging, and the diagnosis was confirmed during the operation. INTERVENTIONS: Laparotomy was performed to remove the placenta and repair the uterine defect after temporary occlusion of both internal iliac arteries. OUTCOMES: Body temperature and inflammatory markers were elevated at admission but returned to normal on the second day after surgery. Normal menstruation resumed approximately 1 month postoperatively. Ultrasound examination showed that the shape of the uterine cavity was normal. No postoperative complications were observed. LESSONS: Temporary occlusion of the internal iliac artery can help effectively manage infected placenta accreta in the uterine horn.

Effect of Vaginoscopy versus Conventional Hysteroscopy on Pain, Complications, and Patient Satisfaction in Patients with Endometrial Polyps.

Background: Hysteroscopy is considered the gold standard for diagnosing intrauterine pathology. Traditional hysteroscopy requires the placement of a vaginal speculum and cervical forceps, which are large in diameter, causing discomfort and pain to the patient and even causing vagal reflexes. Aims: To investigate the impact and clinical value of vaginoscopy versus conventional hysteroscopy on pain, complications, and patient satisfaction in patients with endometrial polyps and to analyse the advantages of clinical application of vaginoscopy examination. Materials and Methods: One hundred and twenty-five patients with endometrial polyps treated in our hospital from May 2021 to December 2021 were selected for this study and divided into 52 cases in the hysteroscopy group and 73 cases in the vaginoscopy group according to the random remainder grouping method. Conventional hysteroscopy was used, and in the vaginoscopy group, vaginoscopy was performed. The impact of pain, complications, patient satisfaction, and clinical value of the two groups was observed and compared. Results: The time taken for the examination varied between the different hysteroscopic methods, with the hysteroscopy group taking the longest time compared to the vaginoscopy group (P < 0.01). The VAS scores immediately after the examination and 30 minutes after the examination were both significantly higher in the hysteroscopy group than in the vaginoscopy group (P < 0.01). The difference in NPY, PGE2, and 5-HT after the pain-causing mediator intervention was significantly better in the vaginoscopy group than in the hysteroscopy group. The difference in the incidence of complications such as abortion syndrome, cervical laceration, uterine perforation, and haemorrhage after treatment was significantly lower in the vaginoscopy group than in the hysteroscopy group. In the vaginoscopy group, the satisfaction rate was 91% significantly higher than that of the hysteroscopy group (P < 0.05). Conclusion: The vaginoscopy technique shortens the examination and treatment time, reduces patient pain, improves patient compliance, reduces the use of preintervention drugs and anaesthetics, and reduces complications.

Iron overload inhibits cell proliferation and promotes autophagy via PARP1/SIRT1 signaling in endometriosis and adenomyosis.

Emerging evidence suggests that excess iron accumulates in endometriotic and adenomyotic lesions. However, the role iron overload plays in the pathogenesis of endometriosis or adenomyosis remains unknown. Primary human eutopic endometrial stromal cells (EuESCs) from endometriosis or adenomyosis patients were used as the in vitro model of endometriosis or adenomyosis in this study. We found that iron, manifesting as ferric ammonium citrate (FAC; 0.05-4.8 mM), significantly inhibited cell growth, induced oxidative stress through the Fenton reaction, and functionally activated autophagy in EuESCs, as measured by 5-ethynyl-2'-deoxyuridine incorporation assay, MitoSOX™ Red staining, LC3 turnover assay, and tandem mCherry-eGFP-LC3B fluorescence microscopy. Immunohistochemistry analysis of Ki67 expression in proliferative-phase endometrial tissues revealed that cell proliferation in ectopic tissues was dramatically compromised, suggesting that iron overload may play a role in cell growth inhibition in vivo. We observed that autophagy may alleviate the FAC-induced inhibition of endometrial stromal cell proliferation. Furthermore, sequential FAC (0.8 mM, 24 h) and hydrogen peroxide (H2O2; 300 µM, 2 h) treatment successfully induced the Fenton reaction in EuESCs and caused extensive apoptosis, whereas the disruption of autophagy by the knockdown of BECN1 further aggravated cell death. MitoSOX™ Red staining showed that autophagy may promote the survival of EuESCs by decreasing of the Fenton reaction-induced reactive oxygen species generation. In addition, we observed that the Fenton reaction-induced oxidative stress significantly suppressed iron overload-induced autophagy. Moreover, we found that FAC treatment impaired poly(ADP-ribose)-polymerase 1 (PARP1) expression while simultaneously upregulating SIRT1 expression in EuESCs. Our data further showed that PARP1 expression decreased in endometriotic lesions, which may partially result from iron overload. We also found that PARP1 inhibition aggravated iron overload-induced cell growth suppression, and was implicated in iron overload-induced autophagy. In addition, SIRT1 silencing alleviated iron overload-induced PARP1 downregulation and autophagy activation. Overall, our data suggest that iron overload in endometrial stromal cells of endometriotic or adenomyotic lesions may be involved in the inhibition of cell proliferation, simultaneously with the activation of protective autophagy via PARP1/SIRT1 signaling.

Cell subtypes and immune dysfunction in peritoneal fluid of endometriosis revealed by single-cell RNA-sequencing.

BACKGROUND: Endometriosis is a refractory and recurrent disease and it affects nearly 10% of reproductive-aged women and 40% of infertile patients. The commonly accepted theory for endometriosis is retrograde menstruation where endometrial tissues invade into peritoneal cavity and fail to be cleared due to immune dysfunction. Therefore, the comprehensive understanding of immunologic microenvironment of peritoneal cavity deserves further investigation for the previous studies mainly focus on one or several immune cells. RESULTS: High-quality transcriptomes were from peritoneal fluid samples of patients with endometriosis and control, and firstly subjected to 10 × genomics single-cell RNA-sequencing. We acquired the single-cell transcriptomes of 10,280 cells from endometriosis sample and 7250 cells from control sample with an average of approximately 63,000 reads per cell. A comprehensive map of overall cells in peritoneal fluid was first exhibited. We unveiled the heterogeneity of immune cells and discovered new cell subtypes including T cell receptor positive (TCR+) macrophages, proliferating macrophages and natural killer dendritic cells in peritoneal fluid, which was further verified by double immunofluorescence staining and flow cytometry. Pseudo-time analysis showed that the response of macrophages to the menstrual debris might follow the certain differentiation trajectory after endometrial tissues invaded into the peritoneal cavity, that is, from antigen presentation to pro-inflammation, then to chemotaxis and phagocytosis. Our analyses also mirrored the dysfunctions of immune cells including decreased phagocytosis and cytotoxic activity and elevated pro-inflammatory and chemotactic effects in endometriosis. CONCLUSION: TCR+ macrophages, proliferating macrophages and natural killer dendritic cells are firstly reported in human peritoneal fluid. Our results also revealed that immune dysfunction happens in peritoneal fluid of endometriosis, which may be responsible for the residues of invaded menstrual debris. It provided a large-scale and high-dimensional characterization of peritoneal microenvironment and offered a useful resource for future development of immunotherapy.

Possible involvement of crosstalk between endometrial cells and mast cells in the development of endometriosis via CCL8/CCR1.

BACKGROUND: The density and the activity of mast cells are associated with endometriosis. However, the role of mast cells on the pathogenesis of endometriosis remains unclear. Our study aims to investigate whether endometrial cells interact with mast cells and the involvement of their crosstalk in the development of endometriosis. METHODS: The transwell assay was applied to investigate the effect of mast cells on the migratory ability of human primary endometrial cells. Mast cells were cocultured with endometrial epithelial and stromal cells respectively and total RNAs were isolated and subjected to mRNA sequencing. Next, the transwell assay, CCK-8, and tube formation were applied to study the role of CCL8 on the endometrial and endothelial cells in vitro. The mouse model was also established to confirm the role of CCL8 in the development and angiogenesis of endometriosis. RESULTS: CCL8 was up-regulated in mast cells when cocultured with endometrial cells. CCL8 was highly expressed in the ectopic endometrium and the serum of patients with endometriosis. CCL8 promoted the migratory ability of endometrial epithelial and stromal cells and increased the proliferation, migration, and tube formation of endothelial cells. CCR1, the receptor of CCL8, was over-expressed in the ectopic endometrium and colocalized with blood vessels in ovarian endometriomas. The inhibition of CCR1 suppressed the development and angiogenesis of endometriosis in vivo. CONCLUSION: The crosstalk between endometrial cells and mast cells in the development of endometriosis via CCL8/CCR1 was demonstrated, thereby providing a new treatment strategy for endometriosis.

Macrophage-derived netrin-1 is critical for neuroangiogenesis in endometriosis.

Netrin-1 is an extracellular guidance cue of neuronal navigation, mediated through interaction with its main receptors, and is known to be crucial in the development of multiple chronic inflammatory diseases. However, the expression pattern and mechanism of netrin-1 in endometriosis are currently undefined. Here we report that netrin-1 expression peaked in peritoneal macrophages found in endometriosis. Netrin-1 induced angiogenesis in ovarian endometriomas through interaction with CD146 in vascular endothelial cells. Through another receptor, neogenin, netrin-1 promoted neurite growth and sensitization in endometriosis through the up-regulation of MAP4, TAU, and CGRP. Targeted knockdown of neogenin in dorsal root ganglion (DRG) nerve cells compromised its response to netrin-1 through inhibiting phosphorylation of ERK1/2. The inhibition of netrin-1 using a neutralizing antibody reduced vascular and nerve infiltration in rat endometriotic lesions. In summary, our results suggest that netrin-1 is an important factor that promotes neuroangiogenesis in endometriosis.