What happens to the gut microbiota during development of murine endometriosis? Mice with the persistence of endometrial lesions for 42 days develop a distinct composition of gut microbiota. Disorders in the immune system play fundamental roles in changing the intestinal microbiota. No study has used high-throughput DNA sequencing to show how endometriosis changes the gut microbiota, although endometriosis is accompanied by abnormal cytokine expression and immune cell dysfunction. This study includes a prospective and randomized experiment on an animal endometriosis model induced via the intraperitoneal injection of endometrial tissues. The mice were divided into endometriosis and mock groups and were sacrificed at four different time points for model confirmation and fecal sample collection. To detect gut microbiota, 16S ribosomal-RNA gene sequencing was performed. Alpha diversity was used to analyze the complexity and species diversity of the samples through six indices. Beta diversity analysis was utilized to evaluate the differences in species complexity. Principal coordinate analysis and unweighted pair-group method with arithmetic means clustering were performed to determine the clustering features. The microbial features differentiating the fecal microbiota were characterized by linear discriminant analysis effect size method. The endometriosis and mock mice shared similar diversity and richness of gut microbiota. However, different compositions of gut microbiota were detected 42 days after the modeling. Among the discriminative concrete features, the Firmicutes/Bacteroidetes ratio was elevated in mice with endometriosis, indicating that endometriosis may induce dysbiosis. Bifidobacterium, which is known as a commonly used probiotic, was also increased in mice with endometriosis. N/A. More control groups should be further studied to clarify the specificity of the dysbiosis induced by endometriosis. This study was performed only on mice. Thus, additional data acquired from patients with endometriosis are needed in future research. We only detected the changes of gut microbiota at 42 days after the modeling, while the long-term effect of endometriosis on gut microbiota remains poorly understood. Moreover, we only revealed a single effect of endometriosis on gut microbiota. This study provided the first comprehensive data on the association of endometriosis and gut microbiota from high-throughput sequencing technology. The gut microbiota changed with the development of endometriosis in a murine model. The communication between the host and the gut microbiota is bidirectional, and further studies should be performed to clarify their relationship. This research was supported by Grant (81571417) from the National Science Foundation of China and Grant (2015GSF118092) from the Technology Development Plan of Shandong Province. The authors report no conflict of interest.

Background. A reliable noninvasive biomarker is not yet available for endometriosis diagnosis. Novel biomarkers for the diagnosis of endometriosis are urgently needed. The molecular constituents of exosomes, especially exosomal microRNAs (miRNAs), have considerable potential as novel biomarkers for clinical diagnosis. This study is aimed at exploring aberrant exosomal miRNA profiles by using miRNA microarray and at providing more accurate molecular biomarkers of endometriosis. Methods. Exosomes were isolated from the serum of patients with endometriosis and negative controls and identified by electron microscopy, nanoparticle tracking analysis, and Western blot. Exosomal miRNAs were profiled by miRNA microarrays. The expression of selective serum exosomal miRNA was validated by qRT-PCR. Receiver operating characteristic (ROC) curves were established to explore the diagnostic value of selective miRNAs. Finally, GO annotation and KEGG pathway enrichment analyses were used to display possible functions associated with the two miRNAs. Results. A total of 24 miRNAs showed differential levels of enrichment with P<0.05 and log2foldchange>1 by miRNA microarrays. Among the six selective miRNAs (i.e., miR-134-5p, miR-197-5p, miR-22-3p, miR-320a, miR-494-3p, and miR-939-5p), qRT-PCR analysis revealed that miR-22-3p and miR-320a were significantly upregulated in serum exosomes from patients with endometriosis compared with negative individuals. ROC curve revealed that the serum exosomal miR-22-3p and miR-320a yielded the area under the curve values of 0.855 and 0.827, respectively. Conclusion. Our results demonstrated that exosomal miR-22-3p and miR-320a were significantly increased in the sera of patients with endometriosis. The two miRNAs may be useful potential biomarkers for endometriosis diagnosis.

Do exosomes play a role in the pathogenesis of endometriosis in a murine model? Exosomes from endometriosis (EMS) can alternatively activate macrophages and thus contribute to the development of lesions in mice. The pathogenesis of endometriosis, an inflammatory disease, possibly involves peritoneal macrophages. Exosomes are recognized as a new communicator among cells and a key modulator in several inflammatory diseases. We performed in vitr and in vivo experiments to demonstrate the role of exosomes in modulating macrophages. RAW264.7 cells (macrophages) were used to examine the effects of exosomes on macrophages in vitro. An experiment was also conducted in vivo, as follows. Fifty C57BL/6 female mice were randomly allocated to five control and five experimental groups (n = 5/group). The experimental group was injected i.p. with EMS-exosomes derived from eutopic stromal cells, starting on Day-7 then every day for 1 week. The control group received CON-exosomes from mice without endometriosis. Peritoneal macrophages were assessed over the next 6 days. On Day 0, all mice were injected i.p. with endometrium to establish the endometriosis model. On Day 14, all mice were sacrificed, ectopic lesions were counted and measured. Exosomes were isolated from endometrial stromal cells (ESCs) by ultracentrifugation and characterized through transmission electron microscopy, nanoparticle tracking analysis and western blot. After treatment with exosomes, the polarization and phagocytic ability of the macrophages were detected by flow cytometry analysis, immunofluorescent staining and RT-PCR. C57BL/6 mice were utilized to establish an endometriosis model by i.p. injection of endometrial segments. After treatment with EMS-exosomes, the macrophages were polarized into an M2-like phenotype and their phagocytic ability decreased (P < 0.05 versus treatment with CON-exosomes). The total weight and volume of the lesions in mice treated with EMS-exosomes significantly increased compared with those in mice treated with CON-exosomes (P < 0.05). The infiltration of M2-like macrophages was enhanced in the EMS-exosome group (P < 0.001 versus treatment with CON-exosomes). N/A Detection of endometriosis following exosome treatment was only performed in a murine endometriosis model. Clinical data and additional mechanism studies must be conducted to understand the role of exosomes in the pathogenesis of endometriosis. This study emphasizes the importance of EMS-exosomes in the pathogenesis of endometriosis. Further investigations on the exosome signaling pathways may contribute to the development of effective treatments for endometriosis. This research was supported by grants (Nos. 81571417 and 81771552) from the National Science Foundation of China. The authors report no conflict of interest.

Background: Endometriosis is a common nonmalignant gynecological disorder that affects 10–15% women of reproductive age and causes several symptoms that result in decreased quality of life and a huge social burden. In recent decades, extracellular vesicles (EVs) have gained attention as a potential therapeutic tool; however, the therapeutic effects of EVs against endometriosis have not been reported. Accordingly, in this study, we investigated the feasibility of nanovesicles (NVs) derived from M1 macrophages (M1NVs) in treating endometriosis. Methods: M1NVs were prepared by serial extrusion. Co-culture assays were performed to investigate changes in tube formation and migration/invasion of eutopic endometrial stroma cells (ESCs) obtained from patients with endometriosis (EM-ESCs). A mouse model of endometriosis was established, and mice were treated with phosphate-buffered saline, M0NVs, or M1NVs to evaluate the efficacy and safety of M1NV for treating endometriosis. Results: M1NVs directly or indirectly inhibited the migration and invasion of EM-ESCs and reduced tube formation. In the mouse model, M1NVs suppressed the development of endometriosis through reprogramming of M2 macrophages, without causing damage to the organs. Conclusions: M1NVs inhibit the development of endometriosis directly, or through repolarizing macrophages from M2 to M1 phenotype. Hence, administration of M1NVs may represent a novel method for the treatment of endometriosis.

Adenomyosis is a nonneoplastic condition characterized by the benign invasion of ectopic endometrium into the myometrium. Macrophages play significant roles in epithelial–mesenchymal transition (EMT) and adenomyosis. An EMT associated with adenomyosis has been extensively studied. This study investigated the process by which the interaction of macrophages with endometrial cells induces EMT in Ishikawa cells and epithelial cells of adenomyosis. Specimens were collected after hysterectomy or resection of adenomyosis lesions from women with adenomyosis and curettage from women without adenomyosis or endometriosis. Immunohistochemistry and immunofluorescent staining demonstrated that CD68-positive macrophages aggregated in adenomyosis lesions, along with the increased protein expressions of n-cadherin, vimentin, and S100A4. By contrast, the protein expressions of e-cadherin and CK7 were decreased. After the primary endometrium cells were cocultured with THP-1-derived macrophages, the protein expression levels of n-cadherin, vimentin, and S100A4 of endometrium cells were increased, whereas the protein expression levels of e-cadherin and CK7 were decreased. The proportion of alternatively activated (M2) macrophages derived from THP-1 macrophages was also increased. The M2 macrophages elicited a bidirectional effect on Ishikawa cells by inducing EMT-like or mesenchymal–epithelial transition-like processes. The apoptotic rate of the Ishikawa cells cocultured with macrophages was increased, whereas their cell proliferation rate was decreased. Transmission electron microscopy indicated that the number of intercellular junctions of the cocultured Ishikawa cells was reduced. Microarray-based gene expression analysis revealed that transforming growth factor-β1/Smad3 and interleukin-6/JAK2/STAT3 signaling pathways were upregulated. Therefore, macrophages can induce EMT-like processes in adenomyosis and undergo polarization to M2.

Endometriosis is a common multifactorial gynecological disorder defined as the proliferation of endometrial tissue outside of the uterine cavity. Neuroangiogenesis (co-recruitment of nerves and blood vessels) is believed to play an integral part in the establishment and growth of endometriotic lesions. We hypothesized that exosomes derived from abnormal endometrium may serve as the second identifier of endometriosis and play an important role in the development of endometriosis by regulating neuroangiogenesis. Primary human endometrial stromal cells (ESCs) were isolated from eutopic endometrium (EmESC, n = 22) with endometriosis and normal endometrium (CoESC, n = 6). Exosomes were isolated from ESCs using “standard” ultracentrifugation method, and the characterization of exosomes were identified through transmission electron microscopy, nanoparticle tracking analysis, and western blot. The role of exosomes in regulating neuroangiogenesis was determined through in vitro tube formation assay, neurite outgrowth assay, and dorsal root ganglion (DRG) neuron apoptosis analysis. The data showed that EmESCs could secrete exosomes with a diameter of approximately 100 nm and a biconcave morphological feature; they were internalized by human umbilical vein endothelial cells (HUVECs) and DRG neurons and enhanced neuroangiogenic effects. We further validated the role of exosomes through blocking experiments. We found that when the exosome secretion was blocked, the pro-neuroangiogenesis effects were decreased. In conclusion, these data suggested that exosomes may play a key role in endometriosis by promoting neuroangiogenesis.